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https://openalex.org/W2936848727	https://hal-cea.archives-ouvertes.fr/cea-02143113/file/aa33870-18.pdf	English	null	<i>Herschel</i>-HOBYS study of the earliest phases of high-mass star formation in NGC 6357	Astronomy & astrophysics	2,019	cc-by	20,350	To cite this version: D. Russeil, M. Figueira, Annie Zavagno, F. Motte, N. Schneider, et al.. Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357. Astronomy and Astrophysics - A&A, 2019, 625, pp.A134. 10.1051/0004-6361/201833870. cea-02143113 Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 D. Russeil, M. Figueira, Annie Zavagno, F. Motte, N. Schneider, A. Men’shchikov, Sylvain Bontemps, P. André, L. D. Anderson, M. Benedettini, et al. Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357⋆,⋆⋆ Gobetti 101, 40129 B 5 15 School for Physical Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK 15 School for Physical Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK 16 RAL Space STFC Rutherford Appleton Laboratory Chilton Didcot Oxfordshire OX11 0QX UK Received 16 July 2018 / Accepted 2 April 2019 Received 16 July 2018 / Accepted 2 April 2019 Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4. which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357⋆,⋆⋆ D. Russeil1, M. Figueira1,2, A. Zavagno1, F. Motte3, N. Schneider4,5, A. Men’shchikov6, S. Bontemps5, P. André6, L. D. Anderson7,8,9, M. Benedettini10, P. Didelon6, J. Di Francesco10, D. Elia10, V. Könyves6,11, Q. Nguyen Luong12,13, T. Nony3, S. Pezzuto10, K. L. J. Rygl14, E. Schisano10, L. Spinoglio10, J. Tigé1, and G. J. White15,16 D. Russeil1, M. Figueira1,2, A. Zavagno1, F. Motte3, N. Schneider4,5, A. Men’shchikov6, S. Bontemps5, P. André6, L. D. Anderson7,8,9, M. Benedettini10, P. Didelon6, J. Di Francesco10, D. Elia10, V. Könyves6,11, Q. Nguyen Luong12,13, T. Nony3, S. Pezzuto10, K. L. J. Rygl14, E. Schisano10, L. Spinoglio10, J. Tigé1, and G. J. White15,16 1 CNRS, CNES, LAM, Aix-Marseille Univ., Marseille, France e-mail: delphine.russeil@lam.fr 1 CNRS, CNES, LAM, Aix-Marseille Univ., Marseille, France e-mail: delphine.russeil@lam.fr 2 National Centre for Nuclear Research, ul. Hoza 69, 00-681 Warszawa, Poland 3 2 National Centre for Nuclear Research, ul. Hoza 69, 00-681 Warszawa, Pola 3 p 4 I. Physik. Institut, University of Cologne, 50937 Cologne, Germany 5 4 I. Physik. Institut, University of Cologne, 50937 Cologne, Germany 5 5 Laboratoire d’Astrophysique de Bordeaux, CNRS/INSU, Université de Bordeaux, UMR 5804, France 6 5 Laboratoire d’Astrophysique de Bordeaux, CNRS/INSU, Université de 6 6 Laboratoire AIM, CEA/IRFU CNRS/INSU Université Paris Diderot, CEA-Saclay, 91191 Gif-sur-Yvette C 7 7 Department of Physics and Astronomy, West Virginia University, Morgantown WV 26506, USA 8 7 Department of Physics and Astronomy, West Virginia University, Morgantown WV 26506, USA 8 p y y g y g 8 Adjunct Astronomer at the Green Bank Observatory, PO Box 2, Green Bank WV 24944, USA 8 Adjunct Astronomer at the Green Bank Observatory, PO Box 2, Green Bank WV 24944, U j y, , , 9 Center for Gravitational Waves and Cosmology, West Virginia University, Chestnut Ridge Research Building, Morgantown, WV 26505, USA 10 INAF – IAPS, Via Fosso del Cavaliere 100, 00133 Rome, Italy 10 INAF – IAPS, Via Fosso del Cavaliere 100, 00133 Rome, Italy 11 Jeremiah Horrocks Institute, University of Central Lancashire, Preston PR1 2HE, UK 11 Jeremiah Horrocks Institute, University of Central Lancashire, Preston PR1 2HE, UK 12 12 Korea Astronomy and Space Science Institute, 776 Daedeok daero, Yuseoung, Daejeon 34055, Kor 13 y y 14 INAF – Istituto di Radioastronomia & Italian ALMA Regional Centre, via P. ABSTRACT Aims. To constrain models of high-mass star formation it is important to identify the massive dense cores (MDCs) that are able to form high-mass star(s). This is one of the purposes of the Herschel/HOBYS key programme. Here, we carry out the census and characterise of the properties of the MDCs population of the NGC 6357 H II region. Methods. Our study is based on the Herschel/PACS and SPIRE 70−500 µm images of NGC 6357 complemented with (sub-)millimetre and mid-infrared data. We followed the procedure established by the Herschel/HOBYS consortium to extract ∼0.1 pc massive dense cores using the getsources software. We estimated their physical parameters (temperatures, masses, luminosities) from spectral energy distribution (SED) ﬁtting. Results. We obtain a complete census of 23 massive dense cores, amongst which one is found to be IR-quiet and twelve are starless, representing very early stages of the star-formation process. Focussing on the starless MDCs, we have considered their evolutionary status, and suggest that only ﬁve of them are likely to form a high-mass star. Results. We obtain a complete census of 23 massive dense cores, amongst which one is found to be IR-quiet and twelve are starless, representing very early stages of the star-formation process. Focussing on the starless MDCs, we have considered their evolutionary status, and suggest that only ﬁve of them are likely to form a high-mass star. gg y y g Conclusions. We ﬁnd that, contrarily to the case in NGC 6334, the NGC 6357 region does not exhibit any ridge or hub features that are believed to be crucial to the massive star formation process. This study adds support for an empirical model in which massive dense cores and protostars simultaneously accrete mass from the surrounding ﬁlaments. In addition, the massive star formation in NGC 6357 seems to have stopped and the hottest stars in Pismis 24 have disrupted the ﬁlaments. HAL Id: cea-02143113 https://cea.hal.science/cea-02143113v1 Submitted on 29 May 2019 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. Astronomy & Astrophysics Astronomy & Astrophysics A&A 625, A134 (2019) https://doi.org/10.1051/0004-6361/201833870 © D. Russeil et al. 2019 Key words. stars: massive – stars: formation ⋆Full Table C1, Tables C2–C5, the reduced Herschel FITS images and the column density FITS image are only available at the CDS via anony- mous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http:// cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/625/A134 ⋆⋆Herschel is an ESA space observatory with science instruments provided by European-led Principal Investigator consortia and with important participation from NASA. 2.1. Herschel observations, data reduction, and column density images NGC 6357 has been observed by the Herschel space observatory with the PACS (Poglitsch et al. 2010) and SPIRE (Grifﬁn et al. 2010) instruments2 as part of the HOBYS (Motte et al. 2010) Key Programme (OBSIDs: 1342204847 and 1342204848). Data were taken in ﬁve bands: 70 and 160 µm for PACS and 250, 350, and 500 µm for SPIRE with FWHM resolutions of 5.9′′, 11.7′′, 18.2′′, 24.9′′, and 36.3′′, respectively. Observations were per- formed in parallel mode, using both instruments simultaneously, with a scanning speed of 20′′ s−1. The size of the observed ﬁeld is 1.7◦× 1.1◦, which corresponds to 52 pc × 34 pc at a distance of 1.75 kpc. Tigé et al. (2017), studying massive dense cores (MDCs) in the star-forming region NGC 6334, favour a scenario wherein ridges/hubs, MDCs and high-mass protostellar embryos form and grow simultaneously (see the review of Motte et al. 2018a). A few high-resolution studies have already been performed with (sub-)millimetre interferometers revealing that starless high- mass cores are very difﬁcult to ﬁnd (e.g. Duarte-Cabral et al. 2013; Tan et al. 2013; Nony et al. 2018). Even in NGC 6334 no high-mass pre-stellar cores (corresponding to the high-mass ana- logues of low-mass pre-stellar cores) were found (Louvet et al. 2019) supporting this. p Data were reduced using the Herschel Interactive Processing Environment (HIPE, Ott 2010)3 software, version 10.0.2751. Ver- sions 7.0 onwards contain a module which signiﬁcantly removes the stripping effects that have been observed in SPIRE maps produced with previous HIPE versions. SPIRE nominal and orthogonal maps were separately processed and subsequently combined and reduced for de-stripping, relative gains, and colour correction with HIPE. PACS maps were reduced with HIPE up to Level 1 and, from there up to their ﬁnal version (Level 3) using Scanamorphos v21.0 (Roussel 2013). ) pp g NGC 6334 has similar velocity and distance to NGC 6357 (the adopted distance is 1.75 kpc, Russeil et al. 2012) and since the extinction and the 1.2 mm emission morphology tend to indi- cate that they are connected by a ﬁlamentary structure (Russeil et al. 2010) we usually consider them as a “twin massive star- forming complex”. However, contrary to NGC 6357, NGC 6334 exhibits a dense molecular ridge and two hubs (Matthews et al. 2008; Tigé et al. 2017), meaning that it could have a differ- ent star-formation history. 1. Introduction and supernova events. While their stellar and supernova phases are optically bright, their earliest phase of evolution occurs in cold massive dense cores (MDCs with sizes and volume den- sities of ∼0.1 pc and >105 cm−3, respectively, see Motte et al. 2007), observable only in the far infra-red (FIR) and sub- millimetre (sub-mm) domains. Thanks to the development of FIR and sub-mm instrumentation, our understanding of high- mass star formation processes become clearer, suggesting a much more dynamical process than the formation of low-mass stars such as accretion streams, associated with converging ﬂows, and cloud hierarchical global collapse (Schneider et al. 2010; Csengeri et al. 2011; Peretto et al. 2014). In particular, High-mass stars (O-B3 type, >8 M⊙) are the ionising sources of H II regions. They impact the interstellar medium mainly via their UV radiation, the dynamical expansion of their H II region ⋆Full Table C1, Tables C2–C5, the reduced Herschel FITS images and the column density FITS image are only available at the CDS via anony- mous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http:// cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/625/A134 ⋆⋆Herschel is an ESA space observatory with science instruments provided by European-led Principal Investigator consortia and with important participation from NASA. A134, page 1 of 42 A134, page 1 of 42 A&A 625, A134 (2019) In this paper, we have focussed our study on NGC 6357 based on data (Herschel-HOBYS1 imaging survey) and an approach and method similar to that presented by Tigé et al. (2017) for the region NGC 6334. Our main goal is to identify and characterise MDCs in order to compare the massive star-formation processes in both regions. Clouds are hierarchical multi-scale structures, which are sub-divided into 1 pc clumps, ∼0.1 pc MDC, and ∼0.01 pc cores. Given the Herschel angular resolution (12′′ at 160 µm) and the homogenous 1–3 kpc distances of HOBYS clouds, the HOBYS key programme is dedicated to identify and characterise 0.1 pc MDCs. Since density is a better criterion than mass to evaluate whether a cloud structure has the ability to form high-mass stars we chose to focus on the densest cloud struc- tures. In this paper, we will then deﬁne MDCs as massive ∼0.03 to ∼0.3 pc cloud structures whose mass is deﬁned in Sect. 4.1. 352.50° 353.00° 353.50° Galactic Longitude +00.00° +00.50° +01.00° +01.50° Galactic Latitude 10 pc Fig. 1. 1 http://hobys-herschel.cea.fr 2 Instrument parameters and calibration are given in the PACS and SPIRE observers manuals. See http://Herschel.esac.esa.int/ Docs/PACS/html/pacs_om.html for PACS and http://Herschel. esac.esa.int/Docs/SPIRE/html/spire_handbook.html for SPIRE. 3 HIPE has been jointly developed by the Herschel Science Ground Segment Consortium, consisting of ESA, the NASA Herschel Science Center, and the HIFI, PACS, and SPIRE consortia. 1. Introduction Herschel-colour image of NGC 6357: 70 µm (red, resolution 5.9′′) and high-resolution column density (green, resolution 18.2′′). The coordinates are Galactic coordinates. The dashed yellow line outlines the Galactic latitude above which we assume the emission belongs to NGC 6357 and that we used to build the HOBYS catalogue. The cyan and magenta symbols indicate the position of the OB clusters Pismis 24 and AH03J1725-34.4, respectively. 352.50° 353.00° 353.50° Galactic Longitude +00.00° +00.50° +01.00° +01.50° Galactic Latitude 10 pc +00.50° p Combining the Herschel-HOBYS data with complementary images described in Sect. 2, we extract the dense cores and char- acterise their properties (Sect. 3). Section 4 presents a complete sample of MDCs with robust mass estimates. Finally, in Sect. 5, we discuss the MDCs properties in comparison with the ones belonging to NGC 6334. Conclusions are given in Sect. 6. +00.00° 353.00° 0° Galactic Longitude 352 353.00° 353.50° Galactic Longitude 353.00° 0° Galactic Longitude 2. Observations Fig. 1. Herschel-colour image of NGC 6357: 70 µm (red, resolution 5.9′′) and high-resolution column density (green, resolution 18.2′′). The coordinates are Galactic coordinates. The dashed yellow line outlines the Galactic latitude above which we assume the emission belongs to NGC 6357 and that we used to build the HOBYS catalogue. The cyan and magenta symbols indicate the position of the OB clusters Pismis 24 and AH03J1725-34.4, respectively. 2.1. Herschel observations, data reduction, and column density images 2.1. Herschel observations, data reduction, and column density images 1 http://hobys-herschel.cea.fr 2 Instrument parameters and calibration are given in the PACS and SPIRE observers manuals. See http://Herschel.esac.esa.int/ Docs/PACS/html/pacs_om.html for PACS and http://Herschel. esac.esa.int/Docs/SPIRE/html/spire_handbook.html for SPIRE. 3 HIPE has been jointly developed by the Herschel Science Ground Segment Consortium, consisting of ESA, the NASA Herschel Science Center, and the HIFI, PACS, and SPIRE consortia. 5 SIMBA/SEST was a former bolometer array of the SEST 15 m. 6 Detailed information and reduced images are available at https:// irsa.ipac.caltech.edu/data/SPITZER/docs/ spitzerdataarchives/ 7 see http://irsa.ipac.caltech.edu/Missions/wise.html 8 see http://irsa.ipac.caltech.edu/Missions/msx.html 2.1. Herschel observations, data reduction, and column density images Hα (arbitrary units), Spitzer 8 µm (MJy sr−1), Herschel/PACS 70 µm (Jy pixel−1) and Column density (cm−2) images of NGC 6357 overlain with column density iso-contours. The ﬁeld is oriented following the Galactic coordinates and its size is 1.32◦× 1.18◦. 870 µm, covered the NGC 6357 molecular cloud with 19.2′′ reso- lution. NGC 6357 was also covered with 24′′ angular resolution, by dedicated SIMBA/SEST5 1.2 mm observations presented by Muñoz et al. (2007) and Russeil et al. (2010). We also used mid-infrared wavelength images from Spitzer/IRAC and MIPS at 3.6−24 µm, as part of the GLIMPSE (Benjamin et al. 2003) and MIPSGAL (Carey et al. 2009) surveys6. The dust opacity law used (κ0 = 0.1 × (ν/1000 GHz)2 cm2 g−1) is similar to that of Hildebrand (1983) with β = 2 and assuming a gas-to-dust ratio of 100 (this dust opacity law is commonly adopted in the other HOBYS papers). p p p Figures 1 and 2 present Hα, Spitzer 8 µm, Herschel/PACS 70 µm and column density images of NGC 6357. While the 8 (PAHs) and 70 µm (emitted from warm dust) emissions show large and extended ﬁlaments and delineate cavities, the Hα (ionised gas) emission suggests that they are ﬁlled by ionised gas. The column density map appears to be more patchy here than in the case of NGC 6334. NGC 6357 was also covered with surveys made by the WISE7 and MSX8 space observatories (Wright et al. 2010; Egan et al. 2003). WISE provides full-sky images at four mid-infrared bands, notably at 22 µm with an angular resolution of 12′′. The Midcourse Space Experiment (MSX) surveyed the Galactic plane in four mid-infrared broad bands, including 21.3 µm, with 18.3′′ angular resolution. One should note that, due to its strong 4 Detailed information and reduced images are available at http:// www3.mpifr-bonn.mpg.de/div/atlasgal/ 2.1. Herschel observations, data reduction, and column density images The main characteristic of NGC 6357 (l = 353.4◦, b = +0.6◦) is its shell like morphology identiﬁed in Hα, and by its surrounding photo-dissociation region (PDR, as seen by its PAH-8 µm emission). Filled by hot gas (Cappa et al. 2011) the cavity is shaped by the ionising open cluster Pismis 24. A shell of fragmented molecular gas was also identiﬁed in CO emission (e.g. Massi et al. 1997) and Giannetti et al. (2012) show the presence of a velocity gradient in the region suggesting that the expansion of the ionised gas is pushing the molecular gas. In addition, at least four other H II regions (see Fig. 1 in Russeil et al. 2016), adjacent to the cavity, belong to NGC 6357, one of them being powered by the cluster AH03J1725-34.4 (Dias et al. 2002). Column density maps were built both at the 36.3′′ and 18.2′′ resolutions of SPIRE 500 and 250 µm data. The procedure used to construct the 36.3′′ resolution image uses the SED (Spectral Energy Distribution) ﬁtting method fully described in Hill et al. (2011, 2012). To build the high-resolution column density map a multi-scale decomposition of the imaging data was performed and described in detail in Appendix A of Palmeirim et al. (2013). A134, page 2 of 42 A134, page 2 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 AAO-UKST Hα Spitzer 8 µm Herschel-PACS 70 µm Column density g. 2. Hα (arbitrary units), Spitzer 8 µm (MJy sr−1), Herschel/PACS 70 µm (Jy pixel−1) and Column density (cm−2) images of NGC 6357 overla th column density iso-contours. The ﬁeld is oriented following the Galactic coordinates and its size is 1.32◦× 1.18◦. AAO-UKST Hα Spitzer 8 µm Spitzer 8 µm (MJy sr−1), Herschel/PACS 70 µm (Jy pixel−1) and Column density (cm−2) images of NGC 6357 overlain ntours. The ﬁeld is oriented following the Galactic coordinates and its size is 1.32◦× 1.18◦. Fig. 2. Hα (arbitrary units), Spitzer 8 µm (MJy sr−1), Herschel/PACS 70 µm (Jy pixel−1) and Column density (cm−2) im with column density iso-contours. The ﬁeld is oriented following the Galactic coordinates and its size is 1.32◦× 1.18◦. Fig. 2. 3.2. Compact source selection The Herschel-HOBYS imaging of NGC 6357 extends in Galactic latitude from approximately −0.4◦to +1.4◦(see Fig. 1). Russeil et al. (2016) note that sources below b ∼+0.1◦(the yellow dashed line in Fig. 1) are Galactic plane sources not related to NGC 6357. By masking this area, we collected 922 (out of the 1391 sources located on the whole ﬁeld) sources in the direction of NGC 6357 from the getsources catalogue. emission, the central part of NGC 6357 is saturated on both MIPS 24 µm and WISE 22 µm images. In order to keep only reliable ﬂux measurements and com- pact structures, and to be able to subsequently perform the SED ﬁtting, we applied additional source selection criteria as described and discussed in Tigé et al. (2017). We summarise these selection criteria below. In addition, we retrieved public JCMT-HARP-ACSIS9 reduced datacubes of NGC 6357 (proposal M14AU32, PI J. Wouterloot). During this run, the central part of NGC 6357 (31.8′ × 28.1′) was observed at 345.795 GHz (12CO(3–2)) and 330.587 GHz (13CO(3–2)) and automatically reduced using ORAC-DR (Jenness et al. 2015). The 12CO(3–2) and 13CO(3–2) lines datacubes have respectively 1884 and 1852 channels with a velocity resolution of 0.42 and 0.44 km s−1. The beam FWHM is 14′′ and the pixel scale is 7.27′′ pixel−1. In parallel, we col- lected the 93 MALT9010 datacubes pointing in the direction of NGC 6357. MALT90 (Jackson et al. 2013; Foster et al. 2013) is a survey of 2000 dense cores located in the Galactic plane led with the ATNF Mopra 22-m telescope. The database provides 3′×3′ datacubes for 16 lines simultaneously observed around 90 GHz with an angular and spectral resolution of 38′′ and 0.11 km s−1, respectively. We complemented the above data with ATNF Mopra 22-m telescope observations (on the ﬂy map- ping) of the lines HCO+ (89.188 GHz) and N2H+ (93.174 GHz) observed in 2008 (reduced with Livedata and Gridzilla codes). The angular and spectral resolution are 30′′ and 0.11 km s−1, respectively. – For each source and each band: – The signal-to-noise ratio (S/N) must be greater than two (limiting false detections while allowing the SEDs to be well constrained by many ﬂux measurements) for both the peak and integrated ﬂuxes12; – The deconvolved size must be less than 0.3 pc (to discard clumps); – The aspect ratio must be smaller than two (to discard elongated, ﬁlament-like features). 11 The detection signiﬁcance refers to a single-scale analogue to a clas- sical signal-to-noise ratio (S/N) (see Eq. (17) of Men’shchikov et al. 2012). 2.2. Ancillary data We complemented our Herschel-HOBYS observations with sub- millimetre and mid-infrared data (see Table 1). The ATLASGAL survey4 (APEX Telescope Large Area Survey of the GALaxy, Schuller et al. 2009), using the LABOCA/APEX camera at A134, page 3 of 42 A&A 625, A134 (2019) Table 1. Data description. Table 1. Data description. Tigé et al. 2017) helping getsources to identify the compact sources against the strong background emission. During this detection step getsources deﬁnes a catalogue of sources with a unique position. Data λ HPBW (µm) (′′) Herschel/PACS 70 5.9 160 11.7 and SPIRE 250 18.2 350 24.9 500 36.3 APEX/LABOCA 870 19.2 SEST/SIMBA 1200 24.0 Spitzer/IRAC 3.6 1.5 4.5 1.7 5.8 1.7 and MIPS 8 2.0 24 6.0 WISE 22 12.0 MSX 21 18.3 For the measurement step, we used the original (not temperature-corrected) Herschel maps from 70 to 500 µm plus the available sub-millimetre maps listed in Sect. 2.2, that is, the 870 µm LABOCA and 1.2 mm SIMBA images. At this step, getsources derives ﬂux measurements that are background subtracted and deblended from overlapping sources. The output source catalogue lists, among others, the monochromatic detec- tion signiﬁcance index11 (Sigmono), peak and integrated ﬂuxes (with errors), FWHM major and minor sizes, and the position angle of the elliptical footprint for each extracted source in each far-infrared to sub-millimetre band. ) 12 These criteria correspond to: S peak/σ > 2, S int/σ > 2, and Sigmono > 5. 9 http://www.cadc-ccda.hia-iha.nrc-cnrc.gc.ca/ 10 http://malt90.bu.edu/index.html 3.2. Compact source selection This ﬁrst step selection process discards 41% of the sources. They are mainly sources with low signal-to-noise ﬂuxes hence sources at the limit of the detection level in Herschel wavelengths and corresponding to dense cores with very low mass. We can note that only 12 (1.3%) sources have been discarded because of the size criterion. – For the SED ﬁtting we require a minimum of three reliable ﬂux measurements: – One at either Herschel-160 µm or Herschel-250 µm, which we call the reference wavelength; 3. Building the massive dense core catalogue 3.1. Compact source extraction g – A second Herschel ﬂux measurement at λ ≥250 µm; 3.3. Compact sources physical characterisation The SED ﬁtting was made for the 155 selected sources to deter- mine their mass and temperature (see Table C.1). To construct SEDs with ﬂuxes measured within a similar aperture, we applied the ﬂux scaling procedure and colour correction as described in Tigé et al. (2017). The study of the proﬁle of each core is beyond the scope of this paper. As a result, a ﬂux scaling was performed assuming that sources have a quasi-spherical radial density distribution following a ρ(r) ∼r−2 law as it is observed for protostellar envelopes (e.g. Beuther et al. 2002; Nguyen Luong et al. 2011a). This ﬂux scaling allows to reconcile Herschel ﬂuxes with higher resolution observations and has been explained at length in for example Nguyen Luong et al. (2011a) and Tigé et al. (2017). We stress that it has a rela- tively weak impact (at most 50% decrease in mass) on the results. The ﬁtted model is a modiﬁed black-body model with a dust emissivity spectral index, β, set to 2 and the 70 µm ﬂux is not used for the SED ﬁtting except when an extreme tem- perature (larger than 32 K) is found (Tigé et al. 2017). Indeed, Men’shchikov (2016) demonstrated that mass derivation with a free variable spectral index leads to very strong biases and erroneous masses. Fig. 3. Mass distribution of the 155 robust sources of NGC 6357. measurement to the last one in the 3.6 µm to 1.2 mm range) sam- pling the SED (LData) and using the trapezoid rule (requiring a linear interpolation in the ﬂux density versus frequency space). The data-points which are upper limits are not used in this ﬁt- ting process. In parallel, we calculated the ﬂux integration in the same way but under the full ﬁtted curve (LFit). Similarly, the sub- millimetre luminosity (Lsub−mm) is the ﬂux integration under the ﬁtted curve but only from 350 µm to 1.2 mm. The ﬂux uncertainties were evaluated as in Tigé et al. (2017) and the uncertainties produced by the ﬁtting routine (similar to Tigé et al. 2017) allow us to obtain the mass and temperature uncertainties (their mean value being 35 and 11%, respectively). 3.3. Compact sources physical characterisation The core’s mass and temperature are obtained by ﬁtting the equation: y µ When no mid-IR and near-IR counterparts were found LFit is a better evaluation of the true bolometric luminosity, then we adopted Lbol = max (LFit, LData) (in general LFit ≥LData). When mid-IR and/or near-IR counterparts are found the trapezoid inte- gration method is favoured and then Lbol = LData. However, we must keep in mind that ∼19% of the sources fall in saturated areas of the 22 and 24 µm images and only ﬁve sources have a compact MSX 21 µm counterpart. When the 21/22/24 µm ﬂux is missing this leads to an over-estimate of LData, relative to the true bolometric luminosity, by a factor of between two and four (Tigé 2014). In addition, LData must be considered with great caution when the source has near-IR counterparts but no 21/22/24 µm ﬂux and no 70 µm ﬂux. In this case the LData is unrealistically large because the linear interpolation in the trapezoid integra- tion method implies its strong over-estimation (by a factor of up to ten). S ν = Mass κνBν(Tdust) d2 , (1) (1) where S ν is the dust continuum emission, d is the distance to the Sun, and κν the dust mass opacity (the chosen value of κν is discussed in Tigé et al. 2017). The volume-averaged density is then calculated (as in Tigé et al. 2017) by ⟨nH2⟩= Mass 4 3πµmH(size/2)3 , (2) (2) where µ = 2.8 and the size is either the deconvolved equivalent FWHM measured at the reference wavelength or, for sources unresolved at 160 or 250 µm, it is deﬁned13 as 0.5 × HPBWRefλ. Cross-matching (cone search 7′′) with GLIMPSE 3.6−8 µm, MIPSGAL 24 µm, WISE 22 µm, and MSX 21 µm catalogues14 was performed to complement the SED in the mid-IR and near- IR ranges. In addition, counterpart with GLIMPSE sources with rising ﬂuxes between 3.6 and 8 µm are favoured, since the 1 − 10 µm SED portion is expected to be rising for both Class I and II young stellar objects (YSOs) (Whitney et al. 2004; Molinari et al. 2008). Due to its large uncertainty, Lbol is used no further in this paper and does not affect its conclusions. Table 2 summarises the main properties of the sample of 155 cores and the mass distribution is shown in Fig. 3. The mass distribution (Fig. 13 For unresolved sources at 160 µm or 250 µm, their upper limit size of 5.85′′ (resp. 9.1′′) leads to lower limits for their volume-averaged density. 14 3.1. Compact source extraction – A third ﬂux measurement taken at λ > 250 µm with either – A third ﬂux measurement taken at λ > 250 µm with either Herschel-SPIRE, APEX/LABOCA, or SEST/SIMBA. The compact sources were extracted using getsources (v1.140127; Men’shchikov et al. 2012; Men’shchikov 2013). Before running getsources, background subtracted and ﬂattened detection images were produced using getimages (Men’shchikov 2017). getsources ﬁrst performs the compact source detection, and then measures the ﬂuxes. Herschel-SPIRE, APEX/LABOCA, or SEST/SIMBA. From this second step selection process, 37% of the sources are discarded because they have no reliable ﬂuxes at Herschel- 160 µm and/or Herschel-250 µm. These sources are either PDR or elongated features. Indeed, dense cores should have SED peaking between 100 and 300 µm and should therefore have reliable ﬂux measurements at Herschel-160 µm and/or Herschel-250 µm. Finally, 5% are excluded because they are The detection module of getsources was run on the Herschel ﬂux maps as well as on the high-resolution col- umn density map. At this step the used PACS-160 µm and SPIRE-250 µm images are the temperature-corrected ones (see 12 These criteria correspond to: S peak/σ > 2, S int/σ > 2, and Sigmono > 5. A134, page 4 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 Table 2. Main physical properties of the 155 dense cores in NGC 6357 Min Median Max FWHMDec (pc) 0.05 0.08 0.29 ⟨Tdust⟩(K) 12.1 18.8 44.9 Lbol (L⊙) 4.5 203 1.2 104 Mass (M⊙) 0.5 22.2 385.8 ⟨nH2⟩(cm−3) 2.9 × 104 7.8 × 105 ≥1.1 × 107 0:5 1 2 5 10 20 50 100 200 500 Mass[M¯] 0 5 10 15 20 Number Fig. 3. Mass distribution of the 155 robust sources of NGC 6357. 70 µm-only sources. This gives us a sub-sample of 155 robust sources which fullﬁll these selection criteria. 70 µm-only sources. This gives us a sub-sample of 155 robust sources which fullﬁll these selection criteria. 14 The catalogues (GLIMPSE (I + II + 3D), MIPSGAL, WISE All-Sky Source, and MSXPSC v2.3) can be found via http://irsa.ipac. caltech.edu/applications/Gator/ 4. The complete sample of NGC 6357 MDCs This allowed us to compute the bolometric luminosity (Lbol). We ﬁrst computed the ﬂux density integration over the ﬁnite number of reliable data-points (from the ﬁrst available data-point 3.3. Compact sources physical characterisation 3) peaking at ∼30 M⊙, we adopted this value as completeness level. 13 For unresolved sources at 160 µm or 250 µm, their upper limit size of 5.85′′ (resp. 9.1′′) leads to lower limits for their volume-averaged density. 14 The catalogues (GLIMPSE (I + II + 3D), MIPSGAL, WISE All-Sky Source, and MSXPSC v2.3) can be found via http://irsa.ipac. caltech.edu/applications/Gator/ 4.3. Velocity structure and massive dense cores properties From the 12CO(3–2) integrated proﬁle a systemic velocity for the region of −3.5 km s−1 is measured, but several velocity compo- nents can be seen (Fig. 5). The −4 km s−1 component is well correlated with the column density, delineating the cavity that is clearly ﬁlled by ionised gas. In the direction of the cavity seen by Russeil et al. (2016), the ionised gas (Hα emission) shows a velocity of −2 km s−1 in the central part while the surroundings consist of an Hα semi-ring-like feature at −8 km s−1, to which the region G353.2+0.9 Giannetti et al. 2012 belongs. A few fea- tures are found around between −9 and −12 km s−1. They are pillar-like features at l,b = 353.16◦, +0.82◦(α, δ = 17h 24m 55s, −34◦13′′) pointing towards the cavity centre and the elongated feature at l,b = 353.09◦,+0.71◦(α, δ = 17h 25m 15s, −34◦22′). Both are located, in projection, inside the cavity and are seen in absorption on the Hα image suggesting they are at the front edge of the region. At l,b = 353.15◦,+0.67◦(α, δ = 17h 25m 35s, −34◦20′) this −9 km s−1 component is superimposed on the main −4 km s−1 emission. At +2 km s−1 the emission fol- lows the Hα emission around the region G353.2+0.9, tracing a For NGC 6357, we ﬁnally obtain a selection of 23 MDCs. Table 3 gives their main physical properties while their ﬂuxes, multi-wavelength images and SEDs are displayed in Appendixes C and D. Because the mass limit (75 M⊙) is well above the completeness level, we assume that we do not miss any MDC in NGC 6357. At a ﬁrst look, we note that MDCs in NGC 6357 are slightly larger and less dense and massive than those in NGC 6334. 4.1. Mass limit for massive dense cores Only one core exhibits a CH3OH maser and a strong and compact MSX 21 µm emission (it is in a saturated area on MIPSGAL 24 µm and WISE 22 µm images). In addition this core is classiﬁed as an “extended green object” (an object within an enhanced Spitzer 4.5 µm emission usualy attributed to shock exited H2 features tracing outﬂow, Cyganowski et al. 2008) by Chambers et al. (2014). Assuming the evolutionnary tracks from Molinari et al. (2008), the evolved nature of this core, with a present mass of 48 M⊙, could suggest an earlier mass around 140 M⊙consistent with the adopted mass limit. µ ( y) An MDC is qualiﬁed as “starless” MDC candidate if no compact 70 µm and no 21/22/24 µm emission are detected, in addition to be centrally concentrated15 If it is not centrally con- centrated the MDC is then qualiﬁed as an “undeﬁned cloud structure” (corresponding to unbound cloud structures). The qualiﬁcation of undeﬁned cloud structures was introduced by Tigé et al. (2017) and Rayner et al. (2017). These are cloud structures that are not centrally concentrated. To estimate the MDCs detection reliability we check whether they are also in the Hi-GAL (Molinari et al. 2016) source catalogue obtained with the CuTEx16 algorithm. The two MDCs (see Table 4) straddling two Hi-GAL sources are undeﬁned cloud structures. The ﬁnal sample of MDCs is plotted on Fig. 4. We note that they are located on high column density regions and they are mainly distributed along a strip close to l ∼353.1◦. 15 The core concentration on the different Herschel maps was evalu- ated by looking at the longitudinal and transverse cuts but also by an automated procedure described in Konyves et al. (in prep.). In short, this automated procedure checked directly in the maps whether a core is centrally peaked within its measured FWHM, by evaluating map values under concentric annular masks constructed inside and outside of the FWHM ellipse of a given source at the wavelengths of 70–500 µm, and also in the high-resolution column density map. 16 4.1. Mass limit for massive dense cores Tigé et al. (2017) established a lower mass limit of 75 M⊙to select the massive dense cores (MDCs) in NGC 6334. Due to a poor maser and compact H II region surveys coverage and strong saturation in mid-infrared images in NGC 6357 we cannot make a proper estimate of this threshold. Therefore, we adopted the same lower mass limit for the MDCs of NGC 6334. This also A134, page 5 of 42 A134, page 5 of 42 A&A 625, A134 (2019) Table 3. Main physical properties of the 23 MDCs in NGC 6357. Table 3. Main physical properties of the 23 MDCs in NGC 6357. Min Median Max FWHMDec (pc) 0.07 (0.05) 0.13 (0.08) 0.29 (“0.30”) ⟨Tdust⟩(K) 12.1 (9.5) 16.7 (16.7) 25.2 (40.1) Lbol (L⊙) 24 (10) 265 (320) 2.4 × 103 (8.7 × 104) Mass (M⊙) “75” (“75”) 102 (120) 386 (1020) ⟨nH2⟩(cm−3) 1.2 × 105 (1× 105) 1.3 × 106 (6× 106) ≥6.2 × 106 (≥7 × 107) Notes. Values in italic and into parenthesis are for NGC 6334, from Tigé et al. (2017). Values in quotes are lower or upper limits due to the selection process. nto parenthesis are for NGC 6334, from Tigé et al. (2017). Values in quotes are lower or upper limits due to the selection Notes. Values in italic and into parenthesis are for NGC 6334, from Tigé et al. (2017). Values in quotes are lower or process. Notes. Values in italic and into parenthesis are for NGC 6334, from Tigé et al. (2017). Values in quotes are lower or upper limits due to the selection process. Notes. Values in italic and into parenthesis are for NGC 6334, from Tigé et al. (2017). Values in quotes are lower or upper limits due to the selection process. allowed us to make an homogeneous comparison between the two regions. by looking for signposts of high-mass star forma- tion. Nevertheless, to validate this choice we look for signs of high-mass star formation by cross-correlating (cone search 10′′) the 155 robust sources with Class II CH3OH masers (Caswell et al. 2010; Urquhart et al. 2013) and radio centimeter UCH II regions (White et al. 2005; Condon et al. 1998; Giveon et al. 2005). We ﬁnd maser or H II region association for only seven cores. 4.1. Mass limit for massive dense cores However four cores have a methanol maser with velocity peaking at −51, −41 or −16 km s−1 respectively suggesting they do not belong to NGC 6357 (the systemic LSR velocity of the region is −4 km s−1, Caswell & Haynes 1987) and two cores have compact radio counterpart, but their masses are low (M ≤10 M⊙) suggesting either that their association is erroneous or they are H II regions at different distances along the NGC 6357 line of sight. Only one core exhibits a CH3OH maser and a strong and compact MSX 21 µm emission (it is in a saturated area on MIPSGAL 24 µm and WISE 22 µm images). In addition this core is classiﬁed as an “extended green object” (an object within an enhanced Spitzer 4.5 µm emission usualy attributed to shock exited H2 features tracing outﬂow, Cyganowski et al. 2008) by Chambers et al. (2014). Assuming the evolutionnary tracks from Molinari et al. (2008), the evolved nature of this core, with a present mass of 48 M⊙, could suggest an earlier mass around 140 M⊙consistent with the adopted mass limit. no IR-bright protostellars MDC. As in Tigé et al. (2017), an MDC is qualiﬁed as an IR-quiet protostellar if it is associated with a 70 µm compact emission even if it is detected or not at 21/22/24 µm (but lower than 10/12/15 Jy). allowed us to make an homogeneous comparison between the two regions. by looking for signposts of high-mass star forma- tion. Nevertheless, to validate this choice we look for signs of high-mass star formation by cross-correlating (cone search 10′′) the 155 robust sources with Class II CH3OH masers (Caswell et al. 2010; Urquhart et al. 2013) and radio centimeter UCH II regions (White et al. 2005; Condon et al. 1998; Giveon et al. 2005). We ﬁnd maser or H II region association for only seven cores. However four cores have a methanol maser with velocity peaking at −51, −41 or −16 km s−1 respectively suggesting they do not belong to NGC 6357 (the systemic LSR velocity of the region is −4 km s−1, Caswell & Haynes 1987) and two cores have compact radio counterpart, but their masses are low (M ≤10 M⊙) suggesting either that their association is erroneous or they are H II regions at different distances along the NGC 6357 line of sight. g y p 16 see http://herschel.asdc.asi.it/index.php?page=cutex. html 4.2. Nature and evolutionary status of MDCs To estimate the evolutionary status of MDCs we followed a similar approach to that in Motte et al. (2007), Csengeri et al. (2014), König et al. (2017), Giannetti et al. (2017), and Tigé et al. (2017). We classiﬁed the MDCs into IR-bright protostellar MDC, IR-quiet protostellar MDC, starless MDC or undeﬁned cloud. An MDC is qualiﬁed as an “IR-bright protostellar” if it has a 21, 22, or 24 µm ﬂux larger than 10, 12, 15 Jy, respectively (for d = 1.75 kpc, Motte et al. 2010; Russeil et al. 2010), a maser and/or H II region (radio continuum) association and a clear centrally-located Spitzer-8 µm point source. But, because of the large area saturared at 22 and 24 µm and due to the poor coverage and quality of the radio and maser surveys it is difﬁcult to ﬁnd sources fullﬁlling all these criteria and we can miss IR-bright MDCs identiﬁcation. Despite relaxing the IR-bright protostellar status to sources fullﬁlling at least one of these criteria we found A134, page 6 of 42 A134, page 6 of 42 Table 4. Physical parameters of the MDCs found in NGC 6357. MDC MDC (a) FWHMDec ⟨Tdust⟩ Mass Lbol ⟨nH2⟩ Lsub−mm/Lbol Comments (b) Id. nb. (pc) (K) (M⊙) (L⊙) (× 106 cm−3) (%) IR-quiet protostellar MDC HOBYS_J172631.1-340236 6 0.12 13.1 ± 0.3 155.0 ± 20.3 76 2.64 ± 0.34 10.7 70 µm compact source Starless MDC candidate HOBYS_J172516.5-342446 1 0.21 17.0 ± 0.6 385.8 ± 50.7 905 1.19 ± 0.15 4.3 HOBYS_J172535.8-342051 5 0.25 23.2 ± 1.3 160.5 ± 29.2 2420 0.28 ± 0.05 1.3 HOBYS_J172601.1-342955 7 0.26 20.3 ± 2.3 137.2 ± 37.2 919 0.22 ± 0.05 2.2 HOBYS_J172412.3-341307 10 0.13 15.3 ± 0.7 111.6 ± 22.0 139 1.45 ± 0.28 6.3 HOBYS_J172446.2-341048 11 0.08 23.3 ± 1.5 103.2 ± 21.1 1568 6.19 ± 1.27 1.2 HOBYS_J172538.8-343058 12 0.10 14.9 ± 1.0 102.4 ± 28.0 107 3.05 ± 0.83 6.9 HOBYS_J172357.2-340545 14 0.25 15.6 ± 0.5 97.5 ± 14.7 168 0.17 ± 0.02 4.4 No Hi-GAL cov. HOBYS_J172451.0-341018 17 0.08 25.3 ± 2.2 83.1 ± 19.5 2065 4.98 ± 1.17 0.9 HOBYS_J172350.5-340813 18 0.12 15.9 ± 0.6 82.2 ± 13.8 125 1.29 ± 0.21 5.6 No Hi-GAL cov. 4.2. Nature and evolutionary status of MDCs HOBYS_J172623.1-343223 21 0.15 16.7 ± 2.1 80.6 ± 28.7 167 0.60 ± 0.21 4.6 HOBYS_J172548.0-342852 22 0.08 16.2 ± 2.4 79.0 ± 40.3 138 3.77 ± 1.92 5.1 HOBYS_J172459.2-341427 23 0.13 18.7 ± 1.0 75.4 ± 14.5 431 (c) 0.85 ± 0.16 2.2 Undeﬁned cloud structure HOBYS_J172519.7-342427 2 0.13 21.2 ± 2.4 237.1 ± 62.5 2076 2.84 ± 0.75 1.8 HOBYS_J172422.4-341218 3 0.29 15.7 ± 1.7 182.1 ± 58.0 265 0.20 ± 0.06 1.0 Over two Hi-GAL HOBYS_J172410.3-340216 4 0.20 12.9 ± 1.3 163.1 ± 56.9 72 0.58 ± 0.20 11.4 HOBYS_J172439.6-340924 8 0.12 20.1 ± 1.2 134.2 ± 27.5 847 2.14 ± 0.44 2.3 Over two Hi-GAL HOBYS_J172646.5-343102 9 0.30 16.3 ± 1.5 111.9 ± 29.2 201 0.11 ± 0.03 2.2 HOBYS_J172350.7-340726 13 0.25 15.7 ± 1.7 98.9 ± 33.3 145 0.16 ± 0.05 5.6 No Hi-GAL cov. HOBYS_J172444.3-341031 15 0.08 24.9 ± 1.7 93.0 ± 18.7 2122 4.84 ± 0.97 0.6 HOBYS_J172539.3-342839 16 0.09 18.2 ± 0.9 89.8 ± 15.5 308 3.46 ± 0.59 3.4 HOBYS_J172635.6-340230 19 0.12 12.1 ± 0.6 81.1 ± 17 24 1.32 ± 0.28 13.9 HOBYS_J172515.1-340940 20 0.20 21.5 ± 3.1 80.8 ± 27.6 769 0.26 ± 0.08 1.7 Notes. (a)The MDC’s numbering is done by decreasing mass. (b)All MDCs are associated with a Hi-GAL source (Molinari et al. 2016) except when it is indicated as “No Hi-GAL cov”. (not covered by the Hi-GAL survey) or “Over two Hi-GAL” (meaning that the MDC is straddling two Hi-GAL sources). (c)For MDC #23, the lack of mid-IR and 70 µm ﬂuxes make a large and unreliable over-estimation of Lbol. Then the quoted Lbol is assigned to be LData but calculated using the expected 70 µm ﬂux evaluated from the ﬁtted curve. Table 4. Physical parameters of the MDCs found in NGC 6357. e 4. Physical parameters of the MDCs found in NGC 6357. Notes. (a)The MDC’s numbering is done by decreasing mass. (b)All MDCs are associated with a Hi-GAL source (Molinari et al. 2016) except when it is indicated as “No Hi-GAL cov”. (not covered by the Hi-GAL survey) or “Over two Hi-GAL” (meaning that the MDC is straddling two Hi-GAL sources). (c)For MDC #23, the lack of mid-IR and 70 µm ﬂuxes make a large and unreliable over-estimation of Lbol. Then the quoted Lbol is assigned to be LData but calculated using the expected 70 µm ﬂux evaluated from the ﬁtted curve. 17 To increase the signal-to-noise ratio the spectra were extracted from 1.26′ to 1′ area for MALT90 and Mopra data, respectively. 4.2. Nature and evolutionary status of MDCs semi-continuous elliptical-like feature of 6.7′ × 8.3′ size, centred at l,b = 353.08◦,+0.83◦(α, δ = 17h 24m 45.7s, −34◦18′ 21.9′′), while it is found towards Hα extinction areas and superimposed on the −4 km s−1 emission around l,b = 353.15◦,+0.67◦. There- fore, this position appears to be special due to the different veloc- ity components mixing and extended proﬁles. This can be due to a combination of component superposition and self-absorption effects. However, this place corresponds to the contact zone between the cavity and the regions H II 353.09+0.63 and H II 353.24+0.60 (Russeil et al. 2016) where dynamical inter- action of the ionised gas with the molecular cloud can occur. There are also young stars and OB stars (Getman et al. 2014; Russeil et al. 2017) which can participate to the local turbulence by their feedback. Assuming that the −9 and the +2 km s−1 are the extreme velocities and −4 km s−1 the systemic veloc- ity, we can estimate an expansion velocity of ∼5 km s−1 for the NGC 6357 central cavity. Finally, the patchy +6 km s−1 emission is usually attributed to a foreground layer (Russeil et al. 2017). are ﬁtted using the hyperﬁne ﬁtting routine of the spectroscopic analysis toolkit “pyspeckit v0.1.20” (Ginsburg & Mirocha 2011) assuming a single temperature. However, for lot of N2H+ spectra the modelled proﬁle over- or under-estimate some of the hyper- ﬁne components suggesting that it is not consistent with a single temperature assumption. The HCO+ spectra are ﬁtted by simple Gaussian(s). semi-continuous elliptical-like feature of 6.7′ × 8.3′ size, centred at l,b = 353.08◦,+0.83◦(α, δ = 17h 24m 45.7s, −34◦18′ 21.9′′), while it is found towards Hα extinction areas and superimposed on the −4 km s−1 emission around l,b = 353.15◦,+0.67◦. There- fore, this position appears to be special due to the different veloc- ity components mixing and extended proﬁles. This can be due to a combination of component superposition and self-absorption effects. However, this place corresponds to the contact zone between the cavity and the regions H II 353.09+0.63 and H II 353.24+0.60 (Russeil et al. 2016) where dynamical inter- action of the ionised gas with the molecular cloud can occur. There are also young stars and OB stars (Getman et al. 2014; Russeil et al. 2017) which can participate to the local turbulence by their feedback. 4.2. Nature and evolutionary status of MDCs cloud Starless MDC IR-quiet MDC 10 12 22 17 17 11 Galactic latitude Galactic longitude Fig. 4. NGC 6357 column density (NH2) map overlain with the evolutionary colour-coded sub-samples of MDCs (MD ndicated). The cyan and magenta diamonds indicate the position of the OB clusters Pismis 24 and AH03J1725-34.4, r Fig. 4. NGC 6357 column density (NH2) map overlain with the evolutionary colour-coded sub-samples of MDCs (MDCs 10, 11, 12, 17, and 22 are indicated). The cyan and magenta diamonds indicate the position of the OB clusters Pismis 24 and AH03J1725-34.4, respectively. four have \| δv \| > 0.25 while for undeﬁned clouds none of them have a signiﬁcant asymmetry. Interestingly, the starless MDC #23 has the most negative δv (suggesting collapse), and prob- ably is in a slightly more evolved stage than the other starless MDCs. that 36 clumps have single associated Herschel sources, 11 have two or three associated sources, and 27 have none. Of the 36 clumps associated with a single Herschel core, ten are asso- ciated with a M > 75 M⊙cores18. Among the 11 clumps with two Herschel sources, one encompasses two massive cores, four encompass one massive core and six encompass low mass cores only. The 1.2 mm clumps with no associated Herschel source always have M < 75 M⊙or a size larger than 0.3 pc. Inversely, 16 of the 23 MDCs are found in the direction of a 1.2 mm clump, 13 being located within a M ≥100 M⊙clump. To estimate the stability of the MDCs, virial parameter (deﬁned as αvir = Mvir/MMDC by for example, Bertoldi & McKee 1992) is calculated. For this, from the velocity width of the N2H+ we derive the turbulent component as σ2 turb = σ2 N2H+ −σ2 th where σth is calculated from the dust temperature. The virial mass and the virial parameter are calculated using σturb. These quantities are reported in Table 5, for MDCs with no N2H+, σturb and αvir are calculated from the HCO+ line. The virial parameter can be used for estimating whether a structure is in a state of being grav- itationally bound. 18 A massive dense core is deﬁned as a core with M > 75 M⊙; see Sect. 4.1. 4.2. Nature and evolutionary status of MDCs In this way, we are able to probe the virial equilibrium for most of the MDCs ﬁnding that 14 of them have αvir ≤1, suggesting they are gravity dominated (and may be undergoing collapse), while four have 1 < αvir < 2 suggesting they are at least gravitationally bound and 1 MDCs has αvir > 2 suggesting it is unbound and may expand (in agreement with the fact that it is also classiﬁed as undeﬁned cloud). On aver- age undeﬁned clouds structures have larger αvir but most of them have αvir < 2, suggesting they are not transient features. Giannetti et al. (2012) studied the molecular cloud associated with the region G353.2+0.9 facing Pismis 24. They distin- guished, from molecular lines, 14 clumps (with size between 0.27 and 0.56 pc and a total mass of ∼2000 M⊙). From our sam- ple, ten reliable cores are detected in the same area among which four are MDCs and appear located in the Giannetti et al. (2012) clumps of mass between 180 and 360 M⊙. We also note that 18 of our 23 MDCs fall in an ATLASGAL compact source footprint (Csengeri et al. 2014; Urquhart et al. 2018) and one (MDC #18) partly overlaps an ATLASGAL source. All this highlights how this study allows us to see the frag- mentation process within clumps. This is also in agreement with the results of Csengeri et al. (2017a) who ﬁnd a limited frag- mentation (at the typical scale of ∼0.06 pc) from a sample of 35 massive infrared quiet ATLASGAL clumps. This underlines the importance of the Herschel resolution and multi-wavelength information for the MDCs census and characterisation in com- parison to previous studies. 4.2. Nature and evolutionary status of MDCs Assuming that the −9 and the +2 km s−1 are the extreme velocities and −4 km s−1 the systemic veloc- ity, we can estimate an expansion velocity of ∼5 km s−1 for the NGC 6357 central cavity. Finally, the patchy +6 km s−1 emission is usually attributed to a foreground layer (Russeil et al. 2017). The optically thick HCO+ line is usualy used to probe col- lapse or outﬂows (e.g. Fuller et al. 2005) as a double peaked proﬁle with an excess of emission on the blue (red) side is gen- erally interpreted as an indication of collapse or infall (outﬂow). In parallel, combining HCO+ and N2H+, considered as optically thick and thin lines respectively, we can calculate the asymme- try parameter δv, as deﬁned by Mardones et al. (1997), which indicates a clear asymmetry if \| δv \| > 0.25. y y The results are listed in Table 5. The MDCs show a mean velocity of −3.74 km s−1, while the MDC #23 has a velocity approximately −10 km s−1 (in agreement with its location in the pillar like feature previously noted at l,b = 353.16◦, +0.82◦). For the IR-quiet protostellar MDC the spectral analysis is in agreement with its proto-stellar classiﬁcation as it shows a double-peaked asymetric HCO+ proﬁle. However its HCO+ blue and red peaks relative intensity suggest an outﬂow while the asymmetry factor suggests an infall motion. For starless MDCs, y g y We have extracted the spectra17 at the position of 20 among 23 MDCs in order to measure the core’s local environment velocity and to identify particular proﬁle. The N2H+ spectra A134, page 7 of 42 A134, page 7 of 42 A&A 625, A134 (2019) 20.48 20.97 21.35 21.68 22.00 22.30 22.60 22.91 23.21 23.51 354.0 353.6 353.2 352.8 352.4 1.2 0.8 0.4 Galactic longitude Galactic latitude log (Col. Dens.) (cm ) -2 Undef. cloud Starless MDC IR-quiet MDC 10 12 22 17 17 11 Fig. 4. NGC 6357 column density (NH2) map overlain with the evolutionary colour-coded sub-samples of MDCs (MDCs 10, 11, 12, 17, and 22 are indicated). The cyan and magenta diamonds indicate the position of the OB clusters Pismis 24 and AH03J1725-34.4, respectively. 20.48 20.97 21.35 21.68 22.00 22.30 22.60 22.91 23.21 23.51 354.0 353.6 353.2 352.8 352.4 1.2 0.8 0.4 Galactic longitude Galactic latitude log (Col. Dens.) (cm ) -2 Undef. 5.2. Quest for the best birthplaces for high-mass stars different slope and intercept values for their ﬁtted power-laws than Baldeschi et al. (2017). In addition, as noted by Baldeschi et al. (2017) the massive star formation (MSF) thresholds cer- tainly breaks at mass lower than 20 M⊙. This is because the adopted values of the core-to-star conversion factor (between 0.5 and 0.33 from Alves et al. 2007) suggest it is not reason- able for a core (if there is no ambient accretion) that will form a high-mass star to have a mass lower than 20 M⊙. Besides, some theoretical MSF thresholds suggest for radii smaller than 0.05 pc a minimum core mass between 50 and 90 M⊙(e.g. see Fig. 1 in Kauffmann & Pillai 2010). In this context we need to precise the mass, density and size criteria to reﬁne the number of starless MDCs able to form a high-mass star. Indeed, densities as low as nH2 ∼104 – 106 cm−3 is character- istic of low-mass pre-stellar cores (Könyves et al. 2015; Motte et al. 2007; Ward-Thompson et al. 1999) while small (<0.1 pc), high mass (M > 100 M⊙) and high density (nH2 > 5 × 106 cm−3) are good criteria to select starless MDCs which could produce high-mass stars (e.g. Tigé et al. 2017; Urquhart et al. 2018). More generally, high resolution observations show that the dense (nH2 > 105 cm−3, compact (≃0.1 pc) and massive clumps tend to In NGC 6334, Tigé et al. (2017) identiﬁed 46 MDCs among which 16 are starless candidates and thus possible sites for the pre-stellar stage of high-mass star formation. However among these 16 starless candidates only one was held as the best candidate, while nine were excluded due to poor SED ﬁt- ting, and the others due to their low density (1 −5 × 105 cm−3) or because they were not centrally concentrated. Similarly in NGC 6357 we can evaluate the reliability of detected starless MDCs to be truly precursors of high-mass stars. In NGC 6357, except MDC #1, the SED ﬁtting of the starless MDCs is good (χ2 < 5). In addition, their mass is above the lower limit for high-mass star formation (given e.g. by the mass-radius relation M(r) ≥1282 × (r/pc)1.42 M⊙from Baldeschi et al. 2017) and their surface density, Σ, is larger than 0.05 g cm−2 (He et al. 5. Discussion 5.1. Comparison with previous massive dense core studies in NGC 6357 5.1. Comparison with previous massive dense core studies in NGC 6357 We have compared our Herschel cores with the 74 clumps (∼0.4 pc) extracted at 1.2 mm by Russeil et al. (2010). We ﬁnd A134, page 8 of 42 A134, page 8 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 353.50 353.25 353.00 +1.0 +0.8 +0.6 -12 km/s -9 km/s 353.50 353.25 353.00 +1.00 +0.75 +0.50 -12 km/s -9 km/s 353.50 353.25 353.00 +1.0 +0.8 +0.6 -4 km/s 353.50 353.25 353.00 +1.00 +0.75 +0.50 -4 km/s 353.50 353.25 353.00 +1.0 +0.8 +0.6 +2 km/s +6 km/s 353.50 353.25 353.00 +1.00 +0.75 +0.50 +2 km/s +6 km/s AO-UKST Hα image (left) and column density map (right) overlaid by JCMT-HARPS 12CO(3–2) emission iso-contours at −12, −9, −4, +2 and +6 km s−1). 353.50 353.25 353.00 +1.0 +0.8 +0.6 -12 km/s -9 km/s 353.50 353.25 353.00 +1.00 +0.75 +0.50 -12 km/s -9 km/s 353.50 353.25 353.00 +1.00 +0.75 +0.50 +2 km/s +6 km/s Fig. 5. AAO-UKST Hα image (left) and column density map (right) overlaid by JCMT-HARPS 12CO(3–2) emission iso-contours at different velocity (−12, −9, −4, +2 and +6 km s−1). A134, page 9 of 42 A&A 625, A134 (2019) Table 5. Velocity information for the MDCs associated with NGC 6357. MDC VLSR VLSR Line HCO+ N2H+ VN2H+ σN2H+ Asymmetry σturb αvir VLSR (primary) (secondary) litterature Id km s−1 km s−1 note note km s−1 km s−1 factor km s−1 km s−1 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) IR-quiet protostellar MDC 6 −2.12 −5.24 HCO+ B«R Reg −1.74 0.62 −0.26 0.57 0.15 −3.1 Starless MDC candidate 1 −4.15 HCO+ G Reg −4.17 0.88 +0.01 0.84 0.23 −3.5 5 −3.53 HCO+ Flat Faint −4.44 1.09 +0.36 1.05 1.03 −4.4 7 −3.40 HCO+ BS Reg −3.04 0.70 −0.22 0.64 0.47 −3.3 10 −4.24 HCO+ G Reg −4.45 0.80 +0.11 0.76 0.40 −4.6 11 −5.78 HCO+ G No det. 5. Discussion 1.05 0.49 −5.1 12 −2.98 HCO+ BS Irr −1.39 1.49 −0.45 1.48 1.25 −1 14 17 −2.76 HCO+ BW Irr −2.41 0.63 −0.24 0.55 0.17 −2 18 −4 21 2.49 HCO+ G Reg 1.95 0.83 +0.28 0.79 0.72 +1.7 22 −4.12 HCO+ RS Reg −4.01 0.75 −0.06 0.70 0.31 23 −10.31 HCO+ RW 2P −10.64 0.66 +0.21 0.60 0.39 −10.9 Undeﬁned cloud structure 2 −4.26 HCO+ G Reg −4.18 0.91 −0.03 0.87 0.25 −3.5 3 −4.23 HCO+ RW? No det. 1.08 1.11 +1.4 4 −4.84 12CO(3−2) (a) −4.2 8 −4.36 HCO+ BWRW Reg −4.24 0.52 −0.10 0.44 0.10 −4.5 9 −1.02 HCO+ G Irr −0.92 0.5 −0.09 0.43 0.30 13 15 −5.20 HCO+ RW No det. 1.47 1.15 −5.1 16 −3.42 HCO+ G Reg −3.26 0.61 −0.11 0.55 0.18 −3.3 19 −1.78 −5.66 HCO+ B»R Irr −5.59 0.68 −0.04 0.65 0.37 20 −4.90 HCO+ G no det. 1.38 2.91 −5.4 Notes. Column (4): line used for the velocity measurements given in Cols. 2 and 3. (a)Indicates a poor quality spectrum. Column (5): HCO+ line morphology. G: gaussian shape. BW (RW): wing on the blue (red) side. BS (RS): shoulder on the blue (red) side. Irr: irregular shape. B > R, B < R o B = R: the blue peak higher (smaller or equal) than the red one. Flat: means that it is a ﬂat-top proﬁle. Column (6): “Reg” means a regular hyperﬁne line structure while “Irr” suggests possible multiple components or low S/N. 2P: two different velocities along the line of sight. Column (12) velocity of the ATLASGAL clump from Urquhart et al. (2018). Notes. Column (4): line used for the velocity measurements given in Cols. 2 and 3. (a)Indicates a poor quality spectrum. Column (5): HCO+ line morphology. G: gaussian shape. BW (RW): wing on the blue (red) side. BS (RS): shoulder on the blue (red) side. Irr: irregular shape. B > R, B < R or B = R: the blue peak higher (smaller or equal) than the red one. Flat: means that it is a ﬂat-top proﬁle. Column (6): “Reg” means a regular hyperﬁne line structure while “Irr” suggests possible multiple components or low S/N. 2P: two different velocities along the line of sight. Column (12): velocity of the ATLASGAL clump from Urquhart et al. (2018). 5.2. Quest for the best birthplaces for high-mass stars 2015) suggesting that all the starless MDCs are on ﬁrst approxima- tion possible sites of high-mass star formation. However, from mass-radius plots the massive-star formation limit is empirical, and based on ﬁts to different clump or core samples. For exam- ple, Kauffmann & Pillai (2010) and Urquhart et al. (2014) ﬁnd A134, page 10 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 Fig. 6. Mass versus size diagram. Starless and protostellar MDC can- didates in NGC 6357 are displayed as blue and red dots respectively. For comparison, following Nony et al. (2018), we also display massive starless and protostellar MDCs (Motte et al. 2007; Tigé et al. 2017; Tan et al. 2013; Louvet et al. 2014) as red crosses and circles respectively, and pre-stellar and protostellar sub-fragments and cores (Motte et al. 2018b; Tan et al. 2013; Bontemps et al. 2010; Louvet et al. 2019) as blue crosses and circles respectively. The red and blue lines represent mass radial power-laws of Mass(<r) ∝r and Mass(<r) ∝r2. pre-stellar cores and protostars (Louvet et al., priv. comm.). This appears as a strong observationnal argument in agreement with the empirical model by Motte et al. (2018a) in which few low- and intermediate mass pre-stellar cores form ﬁrst in MDCs and then grow in mass from the surrounding gas but some may not form high-mass stars but a low-mass cluster. Similar results for NGC 6357 starless MDCs can be suspected, but we have to understand why both regions show a similar number of starless MDCs, while they have a very different number of protostellar MDCs (∼18 and ∼1 per square degree for NGC 6334 and NGC 6357, respectively). This can be linked to the ﬁlamentary structure of the regions. Indeed, Tigé et al. (2017) underline that the massive star formation in NGC 6334 is strongly related to ridge and hub features where mass can be accreted during the formation of the massive star while in NGC 6357 such features are not observed. Similarly Rayner et al. (2017) observe that in Mon R2 the massive star formation is at the centre of a ﬁlament hub. Fig. 6. Mass versus size diagram. Starless and protostellar MDC can- didates in NGC 6357 are displayed as blue and red dots respectively. For comparison, following Nony et al. 19 The Vialactea Filamentary Structures Extraction Package is available at http://vialactea.iaps.inaf.it/vialactea/eng/ tools.php 20 APEX-ArtMiS, http://www.apex-telescope.org/ instruments/pi/artemis/, proposals E-094.C-0743 PI. P. André and O-094.F-9320 PI. T. Hill. 21 Mline,crit is deﬁned as Mline,crit = 2 c2 s G−1 ∼23 to 42 M⊙pc−1 for a dust temperature of T = 14 and 25 K respectively (e.g. André et al. 2010). 5.2. Quest for the best birthplaces for high-mass stars (2018), we also display massive starless and protostellar MDCs (Motte et al. 2007; Tigé et al. 2017; Tan et al. 2013; Louvet et al. 2014) as red crosses and circles respectively, and pre-stellar and protostellar sub-fragments and cores (Motte et al. 2018b; Tan et al. 2013; Bontemps et al. 2010; Louvet et al. 2019) as blue crosses and circles respectively. The red and blue lines represent mass radial power-laws of Mass(<r) ∝r and Mass(<r) ∝r2. To investigate and compare ﬁlamentary structures in NGC 6357 and NGC 6334 we ran the Vialactea Filamentary Structures Extraction Package19 on the column density maps. This package allows us to identify spatially coherent structures and determine their morphological (like lengths and geometrical shape) and physical (e.g. column density, mass) parameters. Filament spine and branches are displayed on Fig. 7. Selecting structures with length-to-width ratio larger than two (see Appendix B) we plot their properties in Fig. 8. be those that are fragmented and that contain the most massive cores (Bontemps et al. 2010; Csengeri et al. 2017b; Figueira et al. 2018; Lu et al. 2018). It is the case for example of CygX-N63 MDC (which has nH2 = 1.64 106 cm−3) and shows, at higher res- olution, one massive fragment (Bontemps et al. 2010) as well the few massive protostellar cores of Lu et al. (2018) which have size ≤0.15 pc and nH2 > 1.1 106 cm−3. These characteristics are in agreement with the IR-quiet protostellar MDCs identiﬁed both in NGC 6357 and NGC 6334. For this reason, in this study, we have assumed that the compact (size ≤0.15 pc) and dens- est (nH2 > 1 106 cm−3) starless MDCs are the best candidates to form a high-mass star. At this step, the starless MDCs #10, #11, #12, #17, #18, and #22 are the most favourable MDCs to form high-mass stars. To explore further the probability of MDCs containing high-mass starless cores or high-mass protostars we look at their mass concentration by plotting starless MDCs in the mass versus size diagram (Fig. 6). Motte et al. (2018a) and Tigé et al. (2017) recall that a mass concentration as Mass(<r) ∝r is expected for ρ ∝r−2 radial density structure (like it is observed for protostellar envelopes and the outskirt of pre-stellar cores) while Mass(<r) ∝r2 characterise much less concentrated clouds. Tigé et al. 5.2. Quest for the best birthplaces for high-mass stars (2017) led such analysis for NGC 6334 starless MDCs (for which 40′′ and 10′′ resolution data exist) allowing them to ﬁnally select only one best starless MDC candidate. Following the same approach, in NGC 6357 (from Fig. 6), because they stray the most from the Mass(<r) ∝r2 law, MDCs #10, #11, #12, #17 and #22 appear more concentrated and then more favourable to form a high-mass star. We can note that these MDCs are mainly located in features facing the clusters Pismis 24 (MDCs #11 and #17) and AH03J1726-34.4 (MDCs #12 and #22). The histograms (Fig. 8) show that the ﬁlament properties in NGC 6334 and NGC 6357 are statistically similar, but in NGC 6334 they have parameters reaching higher values. In Fig. 8 the histogram of the deconvolved widths is presented. However, due to the distance of the regions, the ﬁlament width is not resolved at Herschel/PACS 250 µm (used to produce our high- resolution column density map) and are thus very uncertain. We do not use the ﬁlament width further in our analysis, their analy- sis is the purpose of the paper of Könyves et al. (in prep.) based on the 350 µm and 8′′ resolution map20. The ﬁlamentary difference between the two regions becomes obvious on Fig. 7. NGC 6334 is dominated by a ridge corre- sponding to three aligned ﬁlaments with a total mass ∼31807 M⊙ ﬁlament with a mass per unit length (Mline) between 380 and 1893 M⊙pc−1, with a total length of 21.5 pc and a mean width of 0.08 pc. In NGC 6357 no ridge or hub is noted, the only distinct feature is the ﬁlament (composed of three segments) around l,b = 353.5◦, +0.66◦(hosting the IR-quiet MDC # 6) with a mass of 585.6 M⊙ (Mline between 15 and 107 M⊙pc−1), a total length of 8 pc and a mean width of 0.16 pc. Such difference in ﬁlament and MDCs concentration is already observed at different locations of other regions as, for example, Cygnus (Motte et al. 2007), in Lupus (Rygl et al. 2013) and Perseus (Sadavoy et al. 2014). There is increasing observational evidence that ﬁlaments with Mline larger (thermally supercritical) than the critical value Mline,crit21 show evidence of pre-stellar cores and YSOs whereas thermally sub-critical ﬁlaments (Mline < Mline,crit) appear 5.3. Relation between MDCs and ﬁlaments Recently Louvet et al. (2019), targeting with ALMA 13 of the 16 starless MDCs of NGC 6334, observed that four of them contain low-mass protostars while amid the others only one appears sub-structured into two low-mass pre-stellar cores. Even the best starless candidate in NGC 6334 (MDC-5) shows only low mass A134, page 11 of 42 A134, page 11 of 42 A134, page 11 of 42 A134, page 11 of 42 A&A 625, A134 (2019) 353.8 353.6 353.4 353.2 353.0 352.8 352.6 1.2 1.0 0.8 0.6 0.4 352.2 352.0 351.8 351.6 351.4 351.2 351.0 350.8 350.6 1.0 0.8 0.6 ig. 7. NGC 6357 (upper panel) and NGC 6334 (lower panel) column density maps overlaid with class 0/I YSOs from Povich et al. (2017) (mage ots), ﬁlaments spines (black lines) and branches (red lines) and young stellar clusters from Kuhn et al. (2015) (cyan ellipses). The MDCs col oding is the same as in Fig. 4. enerally devoid of Herschel pre stellar cores and protostars 2013; Hill et al 2011; Motte et al 2018a) As highlighted by A&A 625, A134 (2019) 353.8 353.6 353.4 353.2 353.0 352.8 352.6 1.2 1.0 0.8 0.6 0.4 353.8 353.6 353.4 353.2 353.0 352.8 352.6 1.2 1.0 0.8 0.6 0.4 352.2 352.0 351.8 351.6 351.4 351.2 351.0 350.8 350.6 1.0 0.8 0.6 Fig. 7. NGC 6357 (upper panel) and NGC 6334 (lower panel) column density maps overlaid with class 0/I YSOs from Povich et al. (2017) (mage dots), ﬁlaments spines (black lines) and branches (red lines) and young stellar clusters from Kuhn et al. (2015) (cyan ellipses). The MDCs col coding is the same as in Fig. 4. 352.2 352.0 351.8 351.6 351.4 351.2 351.0 350.8 350.6 1.0 0.8 0.6 Fig. 7. NGC 6357 (upper panel) and NGC 6334 (lower panel) column density maps overlaid with class 0/I YSOs from Povich et al. (2017) (magen dots), ﬁlaments spines (black lines) and branches (red lines) and young stellar clusters from Kuhn et al. (2015) (cyan ellipses). The MDCs colo coding is the same as in Fig. 4. Fig. 7. NGC 6357 (upper panel) and NGC 6334 (lower panel) column density maps overlaid with class 0/I YSOs from Povich et al. (2017) (magenta dots), ﬁlaments spines (black lines) and branches (red lines) and young stellar clusters from Kuhn et al. (2015) (cyan ellipses). The MDCs colour coding is the same as in Fig. 4. 2013; Hill et al. 5.3. Relation between MDCs and ﬁlaments Physical properties of the ﬁlaments associated to the best starless candidates. 5e19 1e20 2e20 5e20 1e21 2e21 5e21 1e22 2e22 5e22 1e23 2e23 Mean column density [cm¡2] 0 2 4 6 8 10 12 14 16 18 20 Number 5e21 1e22 2 density [cm¡2] 1 10 100 1000 1e4 Mass [M¯] 0 2 4 6 8 10 12 14 16 18 20 22 Number to ∼2000 M⊙pc−1 over nearly 10 pc and a width of 0.15 pc. A gap is noted around the middle of the NGC 6334 ﬁlament probably created by the H II region seen in Hα at this position and illus- trating how recent massive star formation disrupts ﬁlaments. In addition, Zernickel et al. (2013) observe gas ﬂow along the ridge which can be interpreted as another hint for the model by Motte et al. (2018a), model in which MDCs grow in mass from the surrounding gas. g g We clearly note that ﬁlaments more massive than 1000 M⊙ belong to NGC 6334 while the most massive ﬁlament in NGC 6357 reaches only 766 M⊙. In both regions, MDCs are located on the ﬁlament, spine, or branch and they belong to the most massive and highest Mline ﬁlaments (Fig. 8) indicating that ﬁlaments are important structures for the formation of massive stars. Among the ﬁve best candidates the MDCs #11 and #17 are located on a spine; the others are either on a branch or at the end of a small spine. Therefore, if we assumed that mass feed- ing along ﬁlaments is an effective process, MDC #17 could be the best to grow in mass as it is located at a branch to spine junction. 1 10 100 1000 Mass ¡ line [M¯pc¡1] 0 2 4 6 8 10 12 14 16 18 Number Mass ¡ line [M¯pc 1] 0 0:05 0:10 0:15 0:20 0:25 0:30 0:35 0:40 0:45 0:50 0:55 Width [pc] 0 5 10 15 20 25 Number We can use the mass and the Mline to estimate which of the starless MDCs in NGC 6357 has enough mass reservoir to form a massive star. The IR-quiet MDC (MDC # 6) belongs to a 5 pc long, 537 M⊙, Mline = 107 M⊙pc−1 segment (while in NGC 6334 IR-quiet MDCs belong to ﬁlaments with Mline between 224 M⊙pc−1 and 1152 M⊙pc−1). 5.3. Relation between MDCs and ﬁlaments In this way, the star- less MDCs #10, #11, #12, #17 and #22 are all in ﬁlaments that have Mline > 107 M⊙pc−1 (see Table 6). MDC #12 is quite pecu- liar because it is located at the end of a short (0.61 pc) ﬁlament (clump-like ﬁlament). Its velocity analysis (Sect. 4.3) indicates infall but the ﬁlament mass is similar to its mass suggesting that either the mass reservoir is not enough to form a massive star or that it is in a slightly more evolved stage than the other starless MDCs. Width [pc] 1 2 3 4 5 6 7 8 9 10 11 12 13 Length [pc] 0 10 20 30 40 50 Number We also use class 0/I YSOs (from Povich et al. 2017) to probe the star formation activity of ﬁlaments where the starless MDCs are. Indeed, Rivilla et al. (2013, 2014) show that YSOs and low mass pre-main sequence stars tend to be clustered at the massive star-forming sites. They interpret that as evidence of the “com- petitive theory” for massive star-formation (e.g. Bonnell & Bate 2006), where a low-mass stellar cluster creates a potential well which funnels gas and dust towards its centre where the most massive stars will form. In this frame, we can speculate that MDCs not surrounded by YSOs could be less evolved or have less probability to form a massive star. Fig. 8. Filament parameters. Light blue and red histograms correspond to NGC 6334 and NGC 6357 respectively. Filled histograms are ﬁl- aments hosting MDCs. In the third panel the ﬁlament width is the deconvolved width. Several YSOs are identiﬁed around the MDCs #10, #12, and #22. Especially, around MDC#22 their spatial distribution seems to delineate an elongated structure (from l,b = 353.04◦, +0.57◦to l,b = 353.07◦, +0.40◦) which encompasses three ﬁl- aments (Fig. A.1). In this picture the parental ﬁlament would be 7.4 pc long, with Mass = 1188 M⊙and Mline = 161 M⊙pc−1. Towards it we note four WISE detected compact H II regions clusters (e.g. Nguyen Luong et al. 2011b, 2013; Hill et al. 2011; Schneider et al. 2012; Kainulainen et al. 2013; Contreras et al. 2016; Motte et al. 2018a). Tigé et al. (2017) and André et al. 5.3. Relation between MDCs and ﬁlaments 2011; Motte et al. 2018a). As highlighted by Li et al. (2016), the morphology of ﬁlaments varies from marginally resolved elongated structures to very complex networks of ﬁla- ments and that they can be isolated or at the periphery of H II regions (as they are found by Inutsuka et al. 2015 to be preferen- tial sites of ﬁlament formation). In addition, ﬁlaments that have high linear mass density (Mline ≫100 M⊙pc−1) contain large enough mass reservoirs to give birth to high-mass stars and star generally devoid of Herschel pre-stellar cores and protostars (André et al. 2010). This led to a proposed paradigm for solar- type star formation in which low-mass stars form primarily by gravitational fragmentation of supercritical ﬁlaments (André et al. 2014). Hill et al. (2011) and Schisano et al. (2014) also suggest that the ﬁlamentary regions are more favourable to form massive stars and that MDCs are either in ridges or in hub within an AV > 100 mag environment (e.g. Nguyen Luong et al. 2011b, A134, page 12 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 y 5e19 1e20 2e20 5e20 1e21 2e21 5e21 1e22 2e22 5e22 1e23 2e23 Mean column density [cm¡2] 0 2 4 6 8 10 12 14 16 18 20 Number 1 10 100 1000 1e4 Mass [M¯] 0 2 4 6 8 10 12 14 16 18 20 22 Number 1 10 100 1000 Mass ¡ line [M¯pc¡1] 0 2 4 6 8 10 12 14 16 18 Number 0 0:05 0:10 0:15 0:20 0:25 0:30 0:35 0:40 0:45 0:50 0:55 Width [pc] 0 5 10 15 20 25 Number 1 2 3 4 5 6 7 8 9 10 11 12 13 Length [pc] 0 10 20 30 40 50 Number Fig. 8. Filament parameters. Light blue and red histograms correspond to NGC 6334 and NGC 6357 respectively. Filled histograms are ﬁl- aments hosting MDCs. In the third panel the ﬁlament width is the deconvolved width. clusters (e.g. Nguyen Luong et al. 2011b, 2013; Hill et al. 2011; Table 6. Physical properties of the ﬁlaments associated to th starless candidates. MDC Fil. Mass Fil. length Fil. Mline M⊙ pc M⊙pc−1 10 424 2.3 179 11 & 17 507 4.7 108 12 129 0.6 211 22 407 1.8 220 Table 6. 5.3. Relation between MDCs and ﬁlaments (a)Free-fall time (tﬀ) measured from the median values of the density averaged over the full MDC volume, which is approx- imately a sphere with a FWHM radius: ⟨nH2⟩full = ⟨nH2⟩/8 and tfree−fall = q 3 π 32 G µ mH ⟨nH2 ⟩full . (b)Due to the large uncertainty on Lbol and any possible NIR source fortuitous association this value can reach a lower limit of 319 L⊙(considering only that Lbol = LFit). , p y The young stellar population also appears very different in the two regions. While the young stellar clusters, identiﬁed by Kuhn et al. (2015), are mainly located along the NGC 6334’s ridge and hub, in NGC 6357 they are either associated to the clusters Pismis 24 or AH03J1725-34.4. In parallel, Getman et al. (2014) underline an age gradient (between 2.3 and 0.7 Myr and from the south-west to the north-east) of the young stellar clus- ter along the NGC 6334’s ﬁlaments while this is not the case in NGC 6357 (clusters and the uniformly distributed stellar popula- tion have similar ages between 1.0 and 1.5 Myr), suggesting that the recent star formation proceeded nearly simultaneously across NGC 6357. However, because the Wolf–Rayet phase is expected to occur at a stellar age of 3 Myr (e.g. Sokal et al. 2016) and because OB stars with ages around 4.6 Myr were also reported towards NGC 6357 (Russeil et al. 2017), we can suspect that star- formation in NGC 6357 has been active for at least 5 Myr. The large (radius 15 pc) Hα ring (as deﬁned by Massi et al. 2015) could even have been shaped by a previous event as Russeil et al. (2017) note shock-heated gas towards the ﬁlaments on its north- east side. Adopting an expansion velocity of 5 km s−1 would give and age of 3.6 Myr for this ring, which could make it a relic of a previous massive star formation episode. (Anderson et al. 2014) suggesting that massive star-formation is already processing at this place, disrupting the ﬁlament. In this context, we can suspect that MDC#22 would not have enough mass to accrete to form a high-mass star. MDC#10 is in a branch (Fig. A.1) of a thick ﬁlament which has started at least low mass star-formation at its other spine-to-branch junction. 5.4. NGC 6357 and NGC 6334 history So we can speculate that previous feedback and the present feedback from O-type stars in Pismis 24 (e.g. Massey et al. 2001 list in Pismis-24 at least two O3 and one WR stars), have stopped the star formation by dispersing its molecular cloud. This is in agreement with the results of Walch et al. (2012) who show that a single O7 star is able to photo-ionise and disperse a 104 M⊙molecular cloud in 1–2 Myr. NGC 6334 harbouring fewer and less-massive stars (e.g. Persi & Tapia 2008) the gas removal timescale would then be longer. In addition, NGC 6357 follows qualitatively the evolutionnary picture of the star for- mation (at kpc scale) with EUV and SN feedback as simulated by Butler et al. (2017). They show that feedback tends to dis- perse the clustering of the star-formation and to reduce the star formation rate (especially when the mechanical feedback from radiation and supernovae is combined). Comparing MDCs for both regions (see Table 7) we can note that they have similar properties except that Lbol is about seven times larger for starless MDCs in NGC 6357. Following Ward-Thompson et al. (2002) this higher Lbol reﬂects that the external heating of the starless cores by the local radiation ﬁeld is higher in NGC 6357 than in NGC 6334. This is expected as NGC 6357 is powered by a the rich OB star cluster Pismis 24 while no such strong radiation source is noted in NGC 6334. g In addition, from his sample of protostellar MDCs, Tigé et al. (2017) estimate a statistical lifetime of 3.5 × 105 yr. Such statistical lifetime is estimated from the relative number of a given MDC phase to the OB stars. For NGC 6357 we estimate a total number of 60 O-B3 stars from Russeil et al. (2012, 2017) and Povich et al. (2017). We assume a median age of the O-B3 stars to be 1 × 106 yr according to Fang et al. (2012) and Getman et al. (2014). To evaluate the typical lifetime in NGC 6357 we have to estimate the number of massive stars our MDCs can form. Then, following Tigé et al. (2017), and in agreement with Csengeri et al. (2017a), we adopt the same fragmentation level found in Cygnus X protostellar MDCs by Bontemps et al. (2010) to assume that IR-quiet MDCs should host on average two high-mass stars. 5.3. Relation between MDCs and ﬁlaments It is difﬁcult to predict if MDC#10 will be able to grow in mass because it is in a quite isolated area. Towards the ﬁlament where MDC#17 and MDC#11 sit, no YSOs are noted. MDC#17, however, being located at a branch to spine junction, appears then as the most favourable starless MDC to form a high-mass star. In addition, on the 350 µm and 8′′ resolution map (Könyves et al., in prep.), only MDC#11 and MDC#17 are centrally peaked sources, addi- tionnally suggesting they are the most favourable MDCs to form high-mass star. 5.3. Relation between MDCs and ﬁlaments (2016) characterise the NGC 6334 ridge showing it is a very dense and massive (∼15 000 M⊙) ﬁlament with a Mline ranging from ∼500 M⊙pc−1 A134, page 13 of 42 A&A 625, A134 (2019) Table 7. Median properties for starless candidates MDCs in NGC 6334 and NGC 6357. NGC 6334 NGC 6357 Tigé et al. (2017) Number 16 12 Size (pc) 0.14 0.13 ⟨Tdust⟩(K) 15 17 Lbol (L⊙) 130 431(b) Mass (M⊙) 103 102 ⟨nH2⟩(cm−3) 1.4 × 106 1.3 × 106 Free-fall time (a) (yr) 3 × 104 3.2 × 104 Notes. (a)Free-fall time (tﬀ) measured from the median values of the density averaged over the full MDC volume, which is approx- imately a sphere with a FWHM radius: ⟨nH2⟩full = ⟨nH2⟩/8 and tfree−fall = q 3 π 32 G µ mH ⟨nH2 ⟩full . (b)Due to the large uncertainty on Lbol and any possible NIR source fortuitous association this value can reach a lower limit of 319 L⊙(considering only that Lbol = LFit). Table 7. Median properties for starless candidates MDCs in NGC 6334 and NGC 6357. Table 7. Median properties for starless candidates MDCs in NGC 6334 and NGC 6357. that the massive star formation has stopped for at least the last Myr and that NGC 6357 will not form any more massive star. From the Miville-Deschênes et al. (2017) molecular cloud catalogue, based on 12CO emission, we estimate a total gas mass of 1.6 × 105 M⊙(this can be compared with the mass esti- mation of 2.3 × 105 M⊙from Willis et al. 2013 and Schneider et al. 2015) and 2.4 × 105 M⊙(which can be compared with the mass estimation of 4 × 105 from Cappa et al. 2011) for NGC 6334 and NGC 6357 respectively. The total ﬁlament masses are ∼5.4 × 104 M⊙and 1.02 × 104 M⊙giving that about 25 and 4% of the mass is in the form of ﬁlaments in NGC 6334 and NGC 6357, respectively. Considering the total MDC mass, we esti- mate that about 9 and 14% of the ﬁlament mass is in the form of MDCs, for both regions respectively. Comparatively, the esti- mated total massive core formation efﬁcienty (TCFE) is about 3 and 0.7% for NGC 6334 and NGC 6357, respectively. Notes. 6. Conclusions 2015, ApJ, 802, 60 Li, G.-X., Urquhart, J. S., Leurini, S., et al. 2016, A&A, 591, A5 Liu, H.-L., Stutz, A., & Yuan, J.-H. 2018, MNRAS, 478, 2119 Louvet, F., Motte, F., Hennebelle, P., et al. 2014, A&A, 570, A15 Louvet, F., Neupane, S., Garay, G., et al. 2019, A&A, 622, A99 Lu, X., Zhang, Q., Liu, H. B., et al. 2018, ApJ, 855, 9 Mardones, D., Myers, P. C., Tafalla, M., et al. 1997, ApJ, 489, 719 Massey, P., DeGioia-Eastwood, K., & Waterhouse, E. 2001, AJ, 121, 1050 Massi, F., Brand, J., & Felli, M. 1997, A&A, 320, 972 Massi, F., Giannetti, A., Di Carlo, E., et al. 2015, A&A, 573, A95 [record ascl:1109.001] Giveon, U., Becker, R. H., Helfand, D. J., & White, R. L. 2005, AJ, 129, 348 Grifﬁn, M. J., Abergel, A., Abreu, A., et al. 2010, A&A, 518, L3 He, Y.-X., Zhou, J.-J., Esimbek, J., et al. 2015, MNRAS, 450, 1926 Hildebrand, R. H. 1983, QJRAS, 24, 267 Hildebrand, R. H. 1983, QJRAS, 24, 267 Acknowledgement. SPIRE has been developed by a consortium of institutes led by Cardiff University (UK) and including University of Lethbridge (Canada); NAOC (China); CEA, LAM (France); IFSI, University of Padua (Italy); IAC (Spain); Stockholm Observatory (Sweden); Imperial College London, RAL, UCL-MSSL, UKATC, University of Sussex (UK); and Caltech, JPL, NHSC, University of Colorado (USA). This development has been supported by national funding agencies: CSA (Canada); NAOC (China); CEA, CNES, CNRS (France); ASI (Italy); MCINN (Spain); SNSB (Sweden); STFC, UKSA (UK); and NASA (USA). PACS has been developed by a consortium of institutes led by MPE (Germany) and including UVIE (Austria); KU Leuven, CSL, IMEC (Belgium); CEA, LAM (France); MPIA (Germany); INAF-IFSI/OAA/OAP/OAT, LENS, SISSA (Italy); IAC (Spain). This development has been supported by the funding agencies BMVIT (Austria), ESA-PRODEX (Belgium), CEA/CNES (France), DLR (Germany), ASI/INAF (Italy), and CICYT/MCYT (Spain). This research has made use of the SIMBAD database, operated at CDS, Strasbourg, France. K.L.J.R. acknowledges ﬁnancial support by the Italian Ministero dell’Istruzione Università e Ricerca through the grant Progetti Premiali 2012-iALMA (CUP C52I13000140001). N.S. and S.B. acknowledge support by the french ANR and the german DFG through the project “GENESIS” (ANR-16-CE92-0035- 01/DFG1591/2-1). GLW gratefully acknowledges support from The Leverhulme Trust. We acknowledge ﬁnancial support from “Programme National de Physique Stellaire” (PNPS) and programme “Physique et Chime du Milieu Interstel- laire” (PCMI) of CNRS/INSU, France. A.Z. 5.4. NGC 6357 and NGC 6334 history For high-mass protostellar cores this gives us a statistical lifetime of 3.3 × 104 yr corresponding to the free-fall time. This unrealistically small value suggests On a larger scale, a 100-pc scale feature can be underlined by connecting the young stellar clusters located in NGC 6334 and NGC 6357 (Fang et al. 2012; Kuhn et al. 2014, 2015; Massi et al. 2015), the ridge in NGC 6334, the ﬁlament (at l,b = 353.5◦, +0.66◦) in NGC 6357, the ﬁlament (seen in on extinction map) connecting NGC 6334 and NGC 6357 (Russeil et al. 2010) and the G350.54+0.69 ﬁlament (Liu et al. 2018). They trace a 100-pc long feature aligned with the Galactic plane at b ∼0.67◦(20 pc above the Galactic plane at a distance of 1.75 kpc) which could trace the parental ﬁlament of both A134, page 14 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 star-forming regions. In this scheme, following Fukui et al. 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Conclusions Csengeri, T., Bontemps, S., Schneider, N., Motte, F., & Dib, S. 2011, A&A, 527, A135 In the framework of the Herschel/HOBYS key programme and in the same way as is done for NGC 6334 by Tigé et al. (2017), we performed a study of the massive dense cores in NGC 6357 to better understand how high-mass stars form. We combined the Herschel/HOBYS images to mid-infrared and (sub-)millimeter ground-based data to obtain a complete census of 23 MDCs, among which ﬁve are expected to be the most probable pro- genitors of high-mass stars at 0.1 pc scale. These starless MDCs belong mainly to the edge of the Pismis-24 cavity and the region H II 353.09+0.63 (excited by AH03J1726-34.4). We con- ﬁrm that, contrarily to NGC 6334, no ridge and hub which can feed the MDCs are observed in NGC 6357. Filaments in NGC 6334 reach higher mass and higher Mline than in NGC 6357. 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Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 Appendix A: Additional ﬁgure 353.25 353.20 353.15 353.10 353.05 1.00 0.95 0.90 0.85 0.80 MDC-11 MDC-17 MDC-10 0.55 0.50 5 MDC-22 MDC-12 Appendix A: Additional ﬁgure Appendix A: Additional ﬁgure dd t o a gu e 353.25 353.20 353.15 353.10 353.05 1.00 0.95 0.90 0.85 0.80 MDC-11 MDC-17 MDC-10 353.15 353.10 353.05 353.00 352.95 0.55 0.50 0.45 0.40 MDC-22 MDC-12 ws (with Galactic l and b coordinates) of the MDCs #10, #11, #12, #17, and #22 (symbols are the s column density (red) and Spitzer-8 µm (green). For the upper panel the WISE-22 µm emission is not d k dashed circles are the WISE detected compact H II regions from Anderson et al. (2014). Magenta sym al. (2017). A 353.25 353.20 353.15 353.10 353.05 1.00 0.95 0.90 0.85 0.80 MDC-11 MDC-17 MDC-10 353.25 353.20 353.15 353.10 353.05 0.80 353.15 353.10 353.05 353.00 352.95 0.55 0.50 0.45 0.40 MDC-22 MDC-12 Fig. A.1. Colour views (with Galactic l and b coordinates) of the MDCs #10, #11, #12, #17, and #22 (symbols are the same as in Fig. 4): WISE-22 µm (blue), column density (red) and Spitzer-8 µm (green). For the upper panel the WISE-22 µm emission is not displayed because it is saturated. The black dashed circles are the WISE detected compact H II regions from Anderson et al. (2014). Magenta symbols are class 0/I YSOs from Povich et al. (2017). 353.15 353.10 353.05 353.00 352.95 0.55 0.50 0.45 0.40 MDC-22 MDC-12 Fig. A.1. Colour views (with Galactic l and b coordinates) of the MDCs #10, #11, #12, #17, and #22 (symbols are the same as in Fig. 4): WISE-22 µm (blue), column density (red) and Spitzer-8 µm (green). For the upper panel the WISE-22 µm emission is not displayed because it is saturated. The black dashed circles are the WISE detected compact H II regions from Anderson et al. (2014). Magenta symbols are class 0/I YSOs from Povich et al. (2017). A134, page 17 of 42 A&A 625, A134 (2019) , ( ) Appendix B: NGC 6357 ﬁlament validation 353.8 353.6 353.4 353.2 353.0 352.8 352.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 Fig. B.1. Column density map (symbols are the same as in Fig. 4, coordinates are Galactic l,b) overlaid with the spines (black lines) and branches (red lines) deﬁned by the Vialactea Filamentary Structures Extraction Package (with a threshold of 4) and with the ﬁlament isocontours (in green) from getfilaments (up to a spatial scale of 72′′). Appendix B: NGC 6357 ﬁlament validation Fig. B.1. Column density map (symbols are the same as in Fig. Appendix C: Catalogues Appendix C: Catalogues Table C.1. Physical properties of the 155 robust sources. Table C.1. Physical properties of the 155 robust sources. Source FWHMdec ⟨Tdust⟩ Mass LData LFit ⟨nH2⟩ RA2000, Dec2000 MDC nb. (pc) (K ) (M⊙) (L⊙) (L⊙) (×106 cm−3) (h m s, ◦′ ′′) nb. 1 0.05 27.7 ± 0.4 32.4 ± 2.4 2275 1388 7.32 ± 0.54 17:26:51.60, −34:08:25.14 2 0.05 32.0 ± 0.7 2.1 ± 0.2 352 216 0.47 ± 0.06 17:25:26.52, −34:38:12.28 3 0.05 31.2 ± 2.3 48.7 ± 8.6 6900 4309 11.01± 1.95 17:26:01.61, −34:15:14.72 4 0.05 34.7 ± 0.9 1.1 ± 0.1 279 177 0.24 ± 0.03 17:28:03.26, −34:18:28.77 5 0.05 30.8 ± 0.6 4.6 ± 0.5 675 372 1.03 ± 0.1 17:27:10.27, −34:16:39.86 6 0.05 19.1 ± 0.9 27.3 ± 5.1 186 127 6.16 ± 1.16 17:26:46.96, −33:59:24.13 7 0.05 18.7 ± 1.5 5.5 ± 1.8 131 23 1.24 ± 0.41 17:24:41.98, −34:40:37.63 8 0.05 18.6 ± 1.2 16.2 ± 4.5 146 64 3.67 ± 1.02 17:26:45.90, −33:59:27.97 9 0.07 28.0 ± 2.5 2.8 ± 0.7 251 127 0.22 ± 0.06 17:27:46.85, −34:16:55.39 10 0.05 15.9 ± 0.8 17.4 ± 4.3 47 27 3.27 ± 0.8 17:26:21.50, −34:47:02.20 Notes. The full table is available at the CDS. The 155 selected robust sources are presented in Table C.1 (only the ten ﬁrst sources are displayed, the full table is available at the CDS). In addition, the NGC 6357 HOBYS catalogue tables for the 23 MDCs are given here (ordered by mass). Table C.2 gives the source position and the Herschel/PACS getsources output. The outputs presented here are: the peak and integrated ﬂuxes, the major and minor FWHM (noted A and B respec- tively) of the elliptic footprint and its position angle (PA). Tables C.3 and C.4 give the Herschel-SPIRE and non-Herschel getsources outputs respectively. We note that the maps for all wavelengths used in the getsources extraction are also available at CDS. Appendix A: Additional ﬁgure 4, coordinates are Galactic l,b) overlaid with the spines (black lines) and branches (red lines) deﬁned by the Vialactea Filamentary Structures Extraction Package (with a threshold of 4) and with the ﬁlament isocontours (in green) from getfilaments (up to a spatial scale of 72′′). (investigating the eigenvalues of the Hessian matrix of the intensity ﬁeld) directly related to the contrast. In addition to extracting ﬁlaments with the Vialactea Filamen- tary Structures Extraction Package (hereafter VFSEP, Schisano et al. 2014 and in prep.) we also performed the extrac- tion using getfilaments a multi-scale, multi-wavelength ﬁla- ment extraction method (Men’shchikov 2013). getfilaments analyses decompositions of original image (here the col- umn density map) across a wide range of spatial scales, the latter being separated by a small amount (a factor of ∼1.05) while the Vialactea Filamentary Structures Extrac- tion Package deﬁnes a ﬁlament as a two-dimensional elon- gated region with a relatively higher brightness contrast with respect to its surroundings, and uses a differential method We note that the two methods are in overall agreement and that VFSEP resolved small marginally elongated structures. All of the MDCs are within a VFSEP ﬁlament while about half of them do not belong a ﬁlament when using getfilaments. In the paper we have used the VFSEP ﬁlaments but Schisano et al. (2014) indicate that relatively roundish structures like large and elongated compact clumps, or clusters of compact objects lying on a strong intensity ﬁeld, might also be detected. To discard such “roundish” structures, we then consider only ﬁlaments with length to width ratio larger than 2. A134, page 18 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 Appendix D: Multi-wavelength images and spectral energy distribution We present in this appendix the multi-wavelength images and spectral energy distributions (SEDs) for the 23 MDCs of NGC 6357, which are discussed in the main body of the text. A134, page 19 of 42 A134, page 19 of 42 A&A 625, A134 (2019) 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172516.5-342446 MDC 1 anel: ﬂux density versus wavelegnth (SED). The curve is the best ﬁt model and the associated ﬁtted values 21/22/24, 70, 160, 250, 350, 500, and 870 µm, 1.2 mm, and high-resolution NH2 column density maps. E ces footprints. The MDCs is identiﬁed by a cross on the 70 µm image.“Lum. Fit” is the ﬂux integration u main text) while “Lum. Data.” corresponds to the integral, using the trapezoid rule, over the ﬁnite number of Data in the main text). HOBYS_J172516.5-342446 MDC 1 HOBYS_J172516.5-342446 MDC 1 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. Upper panel: ﬂux density versus wavelegnth (SED). The curve is the best ﬁt model and the associated ﬁtted values are indicated. Lower maps: 3.6, 5.8, 8, 21/22/24, 70, 160, 250, 350, 500, and 870 µm, 1.2 mm, and high-resolution NH2 column density maps. Ellipses represent the 160 µm getsources footprints. The MDCs is identiﬁed by a cross on the 70 µm image.“Lum. Fit” is the ﬂux integration under the ﬁtted curve (noted LFit in the main text) while “Lum. Data.” corresponds to the integral, using the trapezoid rule, over the ﬁnite number of data-points sampling the SED (noted LData in the main text). 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. ﬂux density versus wavelegnth (SED). The curve is the best ﬁt model and the associated ﬁtted value 2/24, 70, 160, 250, 350, 500, and 870 µm, 1.2 mm, and high-resolution NH2 column density maps. footprints. The MDCs is identiﬁed by a cross on the 70 µm image.“Lum. Fit” is the ﬂux integration text) while “Lum. Data.” corresponds to the integral, using the trapezoid rule, over the ﬁnite number o n the main text). Fig. D.1. Upper panel: ﬂux density versus wavelegnth (SED). Fig. D.2. Continued Fig. D.1. continued. Fig. D.1. continued. Appendix D: Multi-wavelength images and spectral energy distribution The curve is the best ﬁt model and the associated ﬁtted values are indicated. Lower maps: 3.6, 5.8, 8, 21/22/24, 70, 160, 250, 350, 500, and 870 µm, 1.2 mm, and high-resolution NH2 column density maps. Ellipses represent the 160 µm getsources footprints. The MDCs is identiﬁed by a cross on the 70 µm image.“Lum. Fit” is the ﬂux integration under the ﬁtted curve (noted LFit in the main text) while “Lum. Data.” corresponds to the integral, using the trapezoid rule, over the ﬁnite number of data-points sampling the SED (noted LData in the main text). A134, page 20 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172519.7-342427 MDC 2 HOBYS_J172519.7-342427 MDC 2 HOBYS_J172519.7-342427 MDC 2 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172519.7-342427 MDC 2 A1 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.2. Continued Fig. D.1. continued. Fig. D.2. Continued Fig. D.1. continued. A134, page 21 of 42 A&A 625, A134 (2019) 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172422.4-341218 MDC 3 HOBYS_J172422.4-341218 MDC 3 HOBYS_J172422.4-341218 MDC 3 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 22 of 42 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. Fig. D.1. continued. A134, page 22 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 0.5 pc 3.6 µm 5.8 µm 8 µm 22 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. Fig. D.1. continued. Fig. D.1. continued. Fig. D.1. continued. Fig. D.1. continued. Appendix D: Multi-wavelength images and spectral energy distribution HOBYS_J172410.3-340216 MDC 4 HOBYS_J172410.3-340216 MDC 4 HOBYS_J172410.3-340216 MDC 4 0.5 pc 3.6 µm 5.8 µm 8 µm 22 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. A Fig. D.1. continued. A134, page 23 of 42 A&A 625, A134 (2019) 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172535.8-342051 MDC 5 ued. HOBYS_J172535.8-342051 MDC 5 HOBYS_J172535.8-342051 MDC 5 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 24 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172631.1-340236 MDC 6 ued. A HOBYS_J172631.1-340236 MDC 6 HOBYS_J172631.1-340236 MDC 6 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. Fig. D.1. continued. A134, page 25 of 42 A&A 625, A134 (2019) 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172601.1-342955 MDC 7 d. HOBYS_J172601.1-342955 MDC 7 HOBYS_J172601.1-342955 MDC 7 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 26 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172439.6-340924 MDC 8 ued. HOBYS_J172439.6-340924 MDC 8 HOBYS_J172439.6-340924 MDC 8 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. MDC 8 . Fig. D.1. continued. Fig. D.1. continued. A134, page 27 of 42 A&A 625, A134 (2019) 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. Fig. D.1. continued. Fig. D.1. continued. Fig. D.1. continued. Fig. D.1. continued. Appendix D: Multi-wavelength images and spectral energy distribution HOBYS_J172646.5-343102 MDC 9 HOBYS_J172646.5-343102 MDC 9 HOBYS_J172646.5-343102 MDC 9 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. Fig. D.1. continued. A134, page 28 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172412.3-341307 MDC 10 HOBYS_J172412.3-341307 MDC 10 HOBYS_J172412.3-341307 MDC 10 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 29 of 42 A&A 625, A134 (2019) ( ) 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172446.2-341048 MDC 11 ued. HOBYS_J172446.2-341048 MDC 11 HOBYS_J172446.2-341048 MDC 11 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 30 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172538.8-343058 MDC 12 ued. A HOBYS_J172538.8-343058 MDC 12 HOBYS_J172538.8-343058 MDC 12 Fig. D.1. continued. A134, page 31 of 42 A&A 625, A134 (2019) 0.5 pc 3.6 µm 5.8 µm 8 µm 22 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172350.7-340726 MDC 13 d. 0.5 pc 3.6 µm 5.8 µm 8 µm 22 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172350.7-340726 MDC 13 HOBYS_J172350.7-340726 MDC 13 HOBYS_J172350.7-340726 MDC 13 0.5 pc 3.6 µm 5.8 µm 8 µm 22 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 32 of 42 D. Fig. D.1. continued. Fig. D.1. continued. Fig. D.1. continued. Fig. D.1. continued. Appendix D: Multi-wavelength images and spectral energy distribution Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 0.5 pc 3.6 µm 5.8 µm 8 µm 22 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172357.2-340545 MDC 14 HOBYS_J172357.2-340545 MDC 14 HOBYS_J172357.2-340545 MDC 14 0.5 pc 3.6 µm 5.8 µm 8 µm 22 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. Fig. D.1. continued. A134, page 33 of 42 A&A 625, A134 (2019) 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172444.3-341031 MDC 15 d. HOBYS_J172444.3-341031 MDC 15 HOBYS_J172444.3-341031 MDC 15 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 34 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172539.3-342839 MDC 16 HOBYS_J172539.3-342839 MDC 16 HOBYS_J172539.3-342839 MDC 16 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. MDC 16 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 35 of 42 A&A 625, A134 (2019) A&A 625, A134 (2019) 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172451.0-341018 MDC 17 HOBYS_J172451.0-341018 MDC 17 HOBYS_J172451.0-341018 MDC 17 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 36 of 42 0.5 pc 3.6 µm 5.8 µm 8 µm 21 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 36 of 42 D. Fig. D.1. continued. Fig. D.1. continued. Fig. D.1. continued. Fig. D.1. continued. Appendix D: Multi-wavelength images and spectral energy distribution Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 0.5 pc 3.6 µm 5.8 µm 8 µm 22 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172350.5-340813 MDC 18 nued. A HOBYS_J172350.5-340813 MDC 18 HOBYS_J172350.5-340813 MDC 18 Fig. D.1. continued. Fig. D.1. continued. A134, page 37 of 42 A&A 625, A134 (2019) 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172635.6-340230 MDC 19 d. HOBYS_J172635.6-340230 MDC 19 HOBYS_J172635.6-340230 MDC 19 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 38 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172515.1-340940 MDC 20 HOBYS_J172515.1-340940 MDC 20 HOBYS_J172515.1-340940 MDC 20 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. d. 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 39 of 42 A&A 625, A134 (2019) 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172623.1-343223 MDC 21 d. HOBYS_J172623.1-343223 MDC 21 HOBYS_J172623.1-343223 MDC 21 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 40 of 42 D. Russeil et al.: Herschel-HOBYS study of the earliest phases of high-mass star formation in NGC 6357 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172548.0-342852 MDC 22 ued. HOBYS_J172548.0-342852 MDC 22 Fig. D.1. continued. Fig. D.1. continued. Appendix D: Multi-wavelength images and spectral energy distribution A134, page 41 of 42 A&A 625, A134 (2019) A&A 625, A134 (2019) 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. HOBYS_J172459.2-341427 MDC 23 ued. HOBYS_J172459.2-341427 MDC 23 HOBYS_J172459.2-341427 MDC 23 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. A134, page 42 of 42 0.5 pc 3.6 µm 5.8 µm 8 µm 24 µm 70 µm 160 µm 250 µm 350 µm 500 µm 870 µm 1.2 mm Col.dens. Fig. D.1. continued. Fig. D.1. continued. A134, page 42 of 42
https://openalex.org/W2022069640	https://europepmc.org/articles/pmc3335069?pdf=render	English	null	Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding	PloS one	2,012	cc-by	6,353	Introduction situ studies [2], consistent with evidence that its concentration of macromolecules is similar to that of the cytoplasm in interphase [14] which has been measured to be 130–200 mg/ml of diffusible macromolecules [15–17]. In these highly crowded conditions within and outside chromosomes the close proximity of macro- molecules results in strong attractive forces, termed entropic or depletion forces, between them [18–20], and it has been amply demonstrated that linear polyelectrolyte polymers [21,22] includ- ing DNA [23] and polynucleosomes [24] adopt collapsed, compact conformations in similar conditions. The chromosome of Esche- richia coli is maintained in its compact conformation in vivo due to crowding by cytoplasmic macromolecules, and its compaction is conserved in vitro if an inert volume-occupying macromolecule is included in the medium to reproduce this crowding [20]. It is notable that in these conditions, the divalent cations and/or polyamines which were used earlier to stabilise these chromosomes are no longer required [20]. Here, in experiments aimed to examine if the packing of chromatin in metaphase chromosomes could be influenced by the crowding effects of cytoplasmic macromolecules, chromosomes were found to conserve their characteristic structure when they were isolated in media containing an inert, volume-occupying macromolecule (polyethyl- ene glycol, dextran, or Ficoll) without significant concentrations of exogenous ions and with no polyamines. These findings suggest that crowding effects due to cytoplasmic macromolecules may play Metaphase chromosomes are formed by two giant polynucleo- some chains, one in each chromatid and 1.7–8.5 cm long in human cells, compacted to a measured average density of several hundred mg/ml [1,2] consistent with values calculated from their DNA content and volume [3,4]. The conformation of the polynucleosome chains and the mechanism(s) by which this dense packing is achieved are not understood. The primary contribution is generally believed to be from electrostatic effects mediated by interactions of monovalent and/or divalent cations, principally K+, Na+, and/or Mg2+, because in vitro these cations cause polynucleosomes to fold to a compact helical conformation termed the 30-nm fibre [5–7], and media containing these cations at millimolar concentrations, often with the polycations spermine and/or spermidine, are usually used to isolate chromosomes [8– 12]. Chromatin fibres of ,30 nm diameter cannot be detected in chromosomes in situ [13], however, suggesting that other factors may contribute to the dense packing of chromatin in chromosomes in vivo. Abstract Funding: This author has no support or funding to report. Competing Interests: The author has declared that no competing interests exist. Competing Interests: The author has declared that no competing interests exist. * E-mail: ronald.hancock@crhdq.ulaval.ca Ronald Hancock* Laval University Cancer Research Centre, Hoˆtel-Dieu Hospital, Que´bec, Que´bec, Canada PLoS ONE \| www.plosone.org Abstract In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ,30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100–200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 mM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIa and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ,30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes. Citation: Hancock R (2012) Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding. PLoS ONE 7(4): e36045. doi:10.1371/ journal.pone.0036045 012) Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding. PLoS ONE 7(4): e36045. doi:10.1371/ Editor: Katrin Karbstein, The Scripps Research Institute, United States of America Received January 25, 2012; Accepted March 29, 2012; Published April 23, 2012 Copyright: 2012 Ronald Hancock. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ald Hancock. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Funding: This author has no support or funding to report. Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding Ronald Hancock* Isolation of chromosomes in medium containing a crowding macromolecule Chromosomes were released from mitotic chinese hamster ovary (CHO) fibroblasts by disrupting them in a solution containing a volume-occupying macromolecule of the type which is widely employed to study crowding effects in vitro [25–28]. The macromolecules used were polyethylene glycol (PEG) (Mr 8 kDa), dextran (Mr 10.5 kDa), or Ficoll (Mr 70 kDa) at a concentration expressed as (w/v), with 100 mM K-Hepes buffer, pH 7.4, as the only supplement. To disrupt mitotic cells, disperse membranes and cytoplasmic material, and release chromosomes these solutions were supplemented with Triton X-100 (0.5% v/v), and the chromosomes were cytocentrifuged onto slides in conditions which reduced the contamination by smaller cellular components to a minimum. Chromosomes released in a solution containing 12% PEG, 12% dextran, or 40% Ficoll conserved the characteristic structure of those isolated by conventional procedures (Figure 1A–E). Their size and compaction showed some variation in solutions containing different concentrations of a crowding macromolecule, an effect which is discussed below. For comparison, Figure 1F shows chromosomes released in a conventional polyamine-containing buffer [29] from a sample of the mitotic cells used in Figure 1A. buffer [29] from a sample of the mitotic cells used in Figure 1A. This conservation of the characteristic structure of chromosomes in solutions containing 100 mM K-Hepes buffer as the only ionic component contrasted with the large increase in volume of chromosomes isolated by conventional procedures [30–32] and of chromosomes in situ [33,34] in media of low ionic strength. To confirm that their structure was not influenced by contaminating cations in the solutions of crowding macromolecules, these were assayed by atomic emission spectrometry. In a 12% solution of PEG the concentrations were ,4 mM Mg2+, 1.1 mM Ca2+, 18 mM Na+, and 710 mM K+; most of this K+ (,650 mM) originated from KOH required to neutralise unidentified components in commercial PEG and was not present in solutions of the other crowding macromol- ecules. In solutions containing cations at these concentrations chromatin fibres and polynucleosomes have an extended conforma- tion, and they become progressively more compact only when the concentration reaches ,60 mM for Na+ or ,0.3 mM for Mg2+ [7]. Figure 1. (A–E) Metaphase chromosomes released from mitotic CHO cells in a solution containing a crowding macromolecule in 100 mM K-Hepes buffer. Representative fields of chromosomes cytocentrifuged and fixed in the same medium as that used for cell lysis. Structure of chromosomes by electron microscopy Images of chromosomes sectioned for electron microscopy after release in 12% PEG are shown in Figure 2. In general, these images resemble those of chromosomes prepared by other methods [8–12]. The diameter of chromosomes measured on longitudinal sections was 1370685 nm (mean 6SEM, n = 14), larger than that of chromosomes isolated in cation- or polyamine- containing buffers (700–800 nm) [10]. The diameter of individual chromatids from transverse sections (Figure 2B) was 590640 nm. The dense packing of chromatin fibres precluded reliable measurements of their diameter and tracing their paths, but in less densely-packed regions at the periphery of chromosomes their width was variable and between 10 and 40 nm (Figure 2C). Isolation of chromosomes in medium containing a crowding macromolecule (A, B, F) phase-contrast images; (C–E) DNA labeled with YOYO-1. Chromosomes were released in (A) 12% PEG (Mr 8 kD); (B) 25% PEG; (C) 20% PEG; (D) 40% Ficoll (Mr 70 kD); (E) 12% dextran (Mr 10.5 kD). (F) Chromosomes isolated by a conventional method [29] from a sample of the mitotic cells used in panel A. Magnification is the same in all panels; scale bar in A, 5 mm. doi:10.1371/journal.pone.0036045.g001 CHO cell karyotype [35], which could be identified unambiguously when the density of chromosomes on slides was low (Figure 3A). Measurements of chromosome width after incubation in different concentrations of PEG, which was relatively constant for chromo- somes of all sizes, together with the length of the longest chromosome showed that these dimensions varied approximately isotropically (Figure 3B). Transverse linescans of the fluorescence intensity of YOYO-1-stained chromosomes showed the radial distribution of DNA (Figure 3C), but the limited resolution of optical microscopy was insufficient to detect if a region of lower density existed in the central region of chromatids (,3% of their width) as predicted by a recent polymer model of chromosomes [36]. Incubation of chromosomes in the absence of a crowding macromolecule resulted in marked expansion, but they did not disperse completely during the incubation time of 1 h (Figure 3D). Together, these observations show that the concentration of crowding macromolecule in the solution was the crucial factor which determined the compaction of isolated chromosomes. CHO cell karyotype [35], which could be identified unambiguously when the density of chromosomes on slides was low (Figure 3A). Measurements of chromosome width after incubation in different concentrations of PEG, which was relatively constant for chromo- somes of all sizes, together with the length of the longest chromosome showed that these dimensions varied approximately isotropically (Figure 3B). Transverse linescans of the fluorescence intensity of YOYO-1-stained chromosomes showed the radial distribution of DNA (Figure 3C), but the limited resolution of optical microscopy was insufficient to detect if a region of lower density existed in the central region of chromatids (,3% of their width) as predicted by a recent polymer model of chromosomes [36]. Incubation of chromosomes in the absence of a crowding macromolecule resulted in marked expansion, but they did not disperse completely during the incubation time of 1 h (Figure 3D). Introduction A further parameter which has not been considered is predicted to influence strongly the structure of chromosomes in vivo, namely the high concentration of macromolecules in the cytoplasm surrounding them after the nuclear envelope is disassembled in prophase. The cytoplasm of mitotic cells contains proteins at ,105 mg/ml together with RNA at ,42 mg/ml according to in April 2012 \| Volume 7 \| Issue 4 \| e36045 April 2012 \| Volume 7 \| Issue 4 \| e36045 1 Macromolecular Crowding and Chromosome Structure Figure 1. (A–E) Metaphase chromosomes released from mitotic CHO cells in a solution containing a crowding macromolecule in 100 mM K-Hepes buffer. Representative fields of chromosomes cytocentrifuged and fixed in the same medium as that used for cell lysis. (A, B, F) phase-contrast images; (C–E) DNA labeled with YOYO-1. Chromosomes were released in (A) 12% PEG (Mr 8 kD); (B) 25% PEG; (C) 20% PEG; (D) 40% Ficoll (Mr 70 kD); (E) 12% dextran (Mr 10.5 kD). (F) Chromosomes isolated by a conventional method [29] from a sample of the mitotic cells used in panel A. Magnification is the same in all panels; scale bar in A, 5 mm. doi:10.1371/journal.pone.0036045.g001 a significant role in determining the compact structure of the genome in metaphase chromosomes. Isolation of chromosomes in medium containing a crowding macromolecule Together, these observations show that the concentration of crowding macromolecule in the solution was the crucial factor which determined the compaction of isolated chromosomes. Discussion DNA fragments whose monomer length was initially ,180 bp (Figure 4A), a value essentially identical to that (177 bp) in chromosomes of CHO cells isolated by a conventional method [37]. As well as a canonical pattern of histones, some larger acid- soluble polypeptides were detectable (Figure 4B); these probably originate from ribosomes and RNP particles since the chromo- somes were not purified further after centrifugation from the cell lysate. Topoisomerase IIa and the SMC2 subunit of condensin, which are predominant non-histone proteins in chromosomes isolated by conventional methods [38–42], were identified by immunofluorescence (Figure 4C, D). The patterns of labelling of these proteins along the chromatid axes were irregular, like those observed in other studies [38,41], for reasons which are not clear. Topoisomerase IIa more intense signal in the centromeric region, as observed in other cell types particularly in the prometaphase or metaphase stage [42]. DNA fragments whose monomer length was initially ,180 bp (Figure 4A), a value essentially identical to that (177 bp) in chromosomes of CHO cells isolated by a conventional method [37]. As well as a canonical pattern of histones, some larger acid- soluble polypeptides were detectable (Figure 4B); these probably originate from ribosomes and RNP particles since the chromo- somes were not purified further after centrifugation from the cell lysate. Topoisomerase IIa and the SMC2 subunit of condensin, which are predominant non-histone proteins in chromosomes isolated by conventional methods [38–42], were identified by immunofluorescence (Figure 4C, D). The patterns of labelling of these proteins along the chromatid axes were irregular, like those observed in other studies [38,41], for reasons which are not clear. Topoisomerase IIa more intense signal in the centromeric region, as observed in other cell types particularly in the prometaphase or metaphase stage [42]. The essential conclusion of these experiments is that the characteristic structure and compaction of metaphase chromo- somes are conserved when they are isolated in media which contain a volume-occupying crowding macromolecule, with concentrations of K+, Na+, Ca2+, and Mg2+ ions in the low micromolar range. Theory predicts that assemblies of macromol- ecules are stabilised in crowded conditions [19,28], and this has been confirmed experimentally in numerous cases including filaments of actin [43] and of tubulin [44], ribosomes [25], oligomers of the chaperonin GroEL [26], HIV capsids [27], bacterial chromosomes [20], and intranuclear structures [45]. Nucleosomal structure, topoisomerase IIa, and SMC2 in chromosomes The images in Figure 1 show that chromosome dimensions varied with the concentration of crowding macromolecules in the surrounding medium. This effect could be visualised more clearly by reconstructing the 3-D volume of the largest chromosome in the Chromosomes isolated in 12% PEG and incubated with micrococcal nuclease showed a pattern of nucleosome-protected PLoS ONE \| www.plosone.org April 2012 \| Volume 7 \| Issue 4 \| e36045 2 Macromolecular Crowding and Chromosome Structure Macromolecular Crowding and Chromosome Structure Figure 2. Images by transmission electron microscopy of chromosomes released in 12% PEG. Sections are approximately longitudinal or transversal in (A) and (B), respectively. (C) Chromatin fibres in regions of lower density at the periphery of chromosomes; white arrows illustrate regions where fibres of ,30 nm diameter are seen. Scale bars (A, B), 1 mm; (C), 30 nm. doi:10.1371/journal.pone.0036045.g002 Figure 2. Images by transmission electron microscopy of chromosomes released in 12% PEG. Sections are approximately longitudinal or transversal in (A) and (B), respectively. (C) Chromatin fibres in regions of lower density at the periphery of chromosomes; white arrows illustrate regions where fibres of ,30 nm diameter are seen. Scale bars (A, B), 1 mm; (C), 30 nm. doi:10.1371/journal.pone.0036045.g002 Discussion Nucleosomal structure and nonhistone proteins of chromosomes released in 12% PEG. (A) DNA fragments from chromosomes incubated with micrococcal nuclease, separated on a 2% agarose gel; M, length markers. (B) Proteins extracted from chromosomes in 0.2 N H2SO4 and separated in a 4–20% denaturing SDS-PAGE gel; markers (M) were purified histones from calf thymus. (C) Topoisomerase IIa and (D) SMC2 visualised by immunofluorescence (red); DNA was labeled with YOYO-1 (green). Scale bars, 1 mm. doi:10.1371/journal.pone.0036045.g004 deduced from the diameter of chromatids measured by electron microscopy (Figure 2B). In 12% PEG the diameter of chromatids was 590640 nm (Figure 2B), within the range of values measured for single chromatids in living CHO cells (400–600 nm) [41] and for entire chromosomes in living CHO and NRK cells (,1 mm) [46,47]. The osmotic pressure in this solution, which is an alternative manner of viewing macromolecular crowding forces [20,48], is ,200 kPa [49] or approximately equivalent to that of a solution containing BSA at ,200 mg/ml [50]. interspersed repeated DNA sequences, isochores, and nucleosomes with different histone variants and post-translational modifications, like a multiblock polymer [62]. Polymers of appropriate stiffness can adopt compact cylindrical conformations not unlike a metaphase chromatid [63], and recent simulations show dramat- ically how conformations of this type could be formed by entropically-favoured looping of a chromatin fibre [36]. The concept that entropic forces make crucial contributions to the conformation of chromatin in vivo is not novel, and indeed is central to current models of interphase chromosomes where they contribute to forming chromatin loops [64–67] and discrete chromosome territories [68]. These models do not, however, exclude a contribution of electrostatic effects; ions which were strongly bound in chromosomes would not be extracted in the conditions used here, and a subtle interplay is seen between the effects of crowding and electrostatic forces when a polyelectrolyte polymer bearing counterions, a model for a polynucleosome chain, collapses in crowded conditions [21,69]. The conservation of chromosome structure in crowded media in which the concentrations of K+, Na+, Ca2+, and Mg2+ ions were 100–1000-fold lower than those usually employed for their isolation, often together with polyamines [8–12], is consistent with the elimination of a requirement for ions for stabilisation of other macromolecular assemblies in crowded conditions [25– 27,44]. Discussion The concentration of a crowding macromolecule required to reproduce the compaction of chromosomes in vivo cannot be estimated precisely from the present data, but an approximate value could be Figure 3. Influence of the concentration of crowding agent on chromosome dimensions. Chromosomes released in 12% PEG were deposited on slides and incubated for 1 h with PEG at the concentration shown in 100 mm K-Hepes buffer, fixed in the same solution, and DNA was labeled with YOYO-1. (A) 3-D volume of the largest chromosome of CHO cells reconstructed from serial confocal sections; scale bar, 1 mm. (B) Length of the largest chromosome, diameter of randomly selected chromosomes, and these values expressed as the % of those in 12% PEG; error bars show SEM from measurements of $15 chromosomes. (C) Transverse linescans of fluorescence intensity across representative chomosomes labeled with YOYO-1. (D) Representative images of chromosomes incubated in 100 mm K-Hepes buffer with no PEG for 1 h and labeled with YOYO-1. Scale bar, 1 mm. doi:10.1371/journal.pone.0036045.g003 Figure 3. Influence of the concentration of crowding agent on chromosome dimensions. Chromosomes released in 12% PEG were deposited on slides and incubated for 1 h with PEG at the concentration shown in 100 mm K-Hepes buffer, fixed in the same solution, and DNA was labeled with YOYO-1. (A) 3-D volume of the largest chromosome of CHO cells reconstructed from serial confocal sections; scale bar, 1 mm. (B) Length of the largest chromosome, diameter of randomly selected chromosomes, and these values expressed as the % of those in 12% PEG; error bars show SEM from measurements of $15 chromosomes. (C) Transverse linescans of fluorescence intensity across representative chomosomes labeled with YOYO-1. (D) Representative images of chromosomes incubated in 100 mm K-Hepes buffer with no PEG for 1 h and labeled with YOYO-1. Scale bar, 1 mm. doi:10.1371/journal.pone.0036045.g003 April 2012 \| Volume 7 \| Issue 4 \| e36045 3 Macromolecular Crowding and Chromosome Structure Figure 4. Nucleosomal structure and nonhistone proteins of chromosomes released in 12% PEG. (A) DNA fragments from chromosomes incubated with micrococcal nuclease, separated on a 2% agarose gel; M, length markers. (B) Proteins extracted from chromosomes in 0.2 N H2SO4 and separated in a 4–20% denaturing SDS-PAGE gel; markers (M) were purified histones from calf thymus. (C) Topoisomerase IIa and (D) SMC2 visualised by immunofluorescence (red); DNA was labeled with YOYO-1 (green). Scale bars, 1 mm. doi:10.1371/journal.pone.0036045.g004 Figure 4. Discussion The extent to which ionic conditions in the cell are reproduced by media commonly used to isolate chromosomes is difficult to evaluate; concentrations of diffusible (osmotically active) ions in vivo cannot be derived from measurements of their total quantities because significant fractions of K+ and Na+ appear to be bound to macromolecules [51–55] and of Mg2+ to ATP, mitochondria, and the sarcoplasmic reticulum [56], and it has been argued that the cytoplasm contains essentially no free ions [57]. Polyamines at micromolar concentrations cause compaction of chromatin fibres and have significant effects on other properties of chromatin [58,59], and their effects on the structure of chromosomes merit consideration as noted in [12]. The results described here, together with the evidence that macromolecular crowding is a crucial factor in structuring the interphase genome [64], bacterial chromosomes [20,70], and possibly polytene chromosomes [71] and the liquid crystalline chromosomes of dinoflagellates [72], are consistent with the hypothesis that a crowded environment is an essential character- istic of all genomes. This model has particularly interesting implications for meiotic chromosomes, because pairing of homologous DNAs [73,74] and recA-promoted exchange of DNA strands [75] are stimulated in crowded conditions. As already emphasised [13], observations on the conformation of chromatin fibres at low concentrations in vitro must be extrapolated with caution to conditions in vivo where the concentration of nucleosomes in chromosomes is vastly higher, resulting in strong entropic inter-fibre attractive forces which create compact conformations resembling a polymer melt [13,60]. The compaction of linear polymers like polynucleosome chains or DNA by these forces is well established by both simulation and experiments [21–24]. A significant contribution to the compaction of polynucleosome chains is likely to be provided by nucleosome- nucleosome interactions, which are sufficiently strong to form liquid crystals in crowded conditions [61], and theory predicts that the fibres formed will be irregular with different degrees of local compaction because polynucleosome chains are mosaics with PLoS ONE \| www.plosone.org References 1. Kellenberger E, Arnold-Schulz-Gahmen B (1992) Chromatins of low-protein content: Special features of their compaction and condensation. FEMS Microbiol Lett 100: 361–370. 13. Eltsov M, Maclellan KM, Maeshima K, Frangakis AS, Dubochet J (2008) Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibres in mitotic chromosomes in situ. Proc Natl Acad Sci USA 105: 19732–19737. 2. Pliss A, Kuzmin AN, Kachynski AV, Prasad PN (2010) Nonlinear Optical Imaging and Raman Microspectrometry of the Cell Nucleus throughout the Cell Cycle. Biophys J 99: 3483–3491. 14. Weiss M, Elsner M, Kartberg F, Nilsson T (2004) Anomalous subdiffusion is a measure for cytoplasmic crowding in living cells. Biophys J 87: 3518–3524. 3. Bennett MD, Heslop-Harrison JS, Smith JB, Ward JP (1983) DNA density in mitotic and meiotic metaphase chromosomes of plants and animals. J Cell Sci 63: 173–179. 15. Arrio-Dupont M, Cribier S, Foucault G, Devaux PF, d’Albis A (1996) Diffusion of Fluorescently Labeled Macromolecules in Cultured Muscle Cells. Biophys J 70: 2327–2332. 4. Daban JR (2000) Physical constraints in the condensation of eukaryotic chromosomes. Local concentration of DNA versus linear packing ratio in higher order chromatin structures. Biochemistry 39: 3861–3866. 16. Luby-Phelps K (2000) Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int Rev Cytol 92: 189–221. g y 5. Finch JT, Klug A (1976) Solenoid model for superstructure in chromatin. Proc Natl Acad Sci USA 73: 1897–1901. ˚ 17. Pielak GJ (2005) A model of intracellular organization. Proc Natl Acad Sci USA 102: 5901–5902. J g ( ) Natl Acad Sci USA 73: 1897–1901. 6. Widom J (1986) Physicochemical studies of the folding of the 100 A˚ nucleosome filament into the 300 A˚ filament: cation dependence. J Mol Biol 190: 411–424. 18. Asakura S, Oosawa F (1954) On interaction between two bodies immersed in a solution of macromolecules. J Chem Phys 22: 1255–1256. 7. Hansen JC (2002) Conformational dynamics of the chromatin fibre in solution: Determinants, Mechanisms and Functions. Annu Rev Biophys Biomol Struct 31: 361–392. 19. Zimmerman SB (1993) Macromolecular crowding effects on macromolecular interactions: some implications for genome structure and function. Biochim Biophys Acta 1216: 175–185. 8. Chorazy M, Bendich A, Borenfreund E, Hutchison DJ (1963) Studies on the isolation of metaphase chromosomes. J Cell Biol 19: 59–69. 20. Cunha S, Woldringh CL, Odijk T (2001) Polymer-mediated compaction and internal dynamics of isolated Escherichia coli nucleoids. Acknowledgments I thank John Marko, Mikhail Eltsov, and anonymous reviewers for constructive comments, Anne Loranger, Normand Marceau, Carl St- Pierre, Alain Goulet, and Richard Janvier for support of imaging, and Peter Smith (University of Guelph) for measuring cation concentrations. Author Contributions Conceived and designed the experiments: RH. Performed the experiments: RH. Analyzed the data: RH . Wrote the paper: RH. Conceived and designed the experiments: RH. Performed the experiments: RH. Analyzed the data: RH . Wrote the paper: RH. Nucleosomal structure Chromosomes released in 12% PEG solution were centrifuged (500 g, 10 min), incubated with micrococcal nuclease at 37uC as described in [37]. and DNA fragments were phenol-extracted and separated on a 2% agarose gel. Histones were extracted from chromosomes in 0.2 N H2SO4 (30 min, 4uC), precipitated with 80% ethanol, and separated by denaturing SDS-PAGE in a 4– 20% gradient gel. Macromolecular Crowding and Chromosome Structure experiment the pH of these solutions was verified and adjusted to pH 7.4 if neccessary. Cation concentrations in polymer solutions were measured by atomic emission spectrometry (Varian Vista- Pro). Cells were centrifuged (300 g, 10 min in 12% PEG or 12% dextran; 500 g, 20 min in 40% Ficoll) and resuspended at ,56106 cells/ml in the same solution containing 0.5% (v/v) Triton X-100 (Sigma-Aldrich). After 5 min chromosomes were released by ,50 hand strokes in a 2 ml Teflon-glass homogeniser (Wheaton) and one volume of the same solution without Triton was added with gentle mixing. Chromosomes were also prepared by a conven- tional procedure for comparison; mitotic cells were homogenised in 7.5 mM Tris-HCI (pH 7.4), 0.1 mM spermine, 0.25 mM spermidine, 1 mM EDTA (pH 7.4) and 40 mM KCI [29], and cytocentrifugation as described below. linescans were made with ImageJ (http://rsb.info.nih.gov/ij; developed by Wayne Rasband, NIH). Grayscale images were pseudocoloured and merged using Photoshop 7.0 (Adobe). Optical imaging and immunofluorescence p g g Chromosomes were cytocentrifuged onto polylysine-coated slides (300 g, 20 min in PEG and dextran; 500 g, 40 min in Ficoll). When indicated, they were overlayed with 500 ml of solution of a crowding macromolecule and incubated in a humidified container for 1 h. Fixation was for 10 min in the same solution as the previous step supplemented with 2% formaldehyde by adding 16% aqueous formaldehyde solution, pH 7.4 (Ted Pella); this fixation was used to immunolabel topoisomerase II and methanol (220uC, 15 min) for SMC2. Antibodies were rabbit anti-human topoisomerase IIa (Topogen) (1/20, 4 h) or rabbit anti-human SMC2 (Abcam antibody 10399) (1/500, 1 h) followed by Alexa 594-secondary antibody (Invitro- gen) (1/500, 1 h). DNA was labeled with YOYO-1 (1 mM, 10 min). Phase-contrast images were acquired with a CoolSNAP camera (Roper Scientific) on a Nikon E800 microscope with a 1006NA 1.3 oil-immersion objective. Confocal images of 0.2 mm sections acquired on an MRC1024 microscope (BioRad) with a 606 NA 1.4 oil-immersion objective were deconvoluted (nearest neighbour) and are shown as maximum intensity projections made with Metamorph 7.65 (Universal Imaging). 3-D volumes were constructed with Volocity 5.4 (PerkinElmer) and dimensions and Transmission electron microscopy Chromosomes released in 12% PEG solution were centrifuged (700 g, 10 min), resuspended in the same solution, and fixed by adding 16% formaldehyde to a concentration of 2% (see above) and glutaraldehyde (Sigma-Aldrich) to 0.1%. After 1 h on ice they were cytocentrifuged onto a 2 mm film of Aclar (EMS) fixed to a slide and the entire sample was detached, dehydrated, and embedded in Poly/Bed 812 (Polysciences). Sections (90–100 mm) cut parallel or perpendicular to the Aclar film were stained with uranyl acetate and lead citrate by standard methods. Digital images were acquired on a Jeol 1200 microscope at 20,000–40,000 magnification. Isolation of chromosomes Mitotic cells were detached from semi-confluent monolayers of CHO cells (CHO-K1, ATCC) growing in McCoy’s 5a medium with 10% FCS by shaking horizontally for 2 min after incubation for 2 h with nocodazole (60 ng/ml; Sigma-Aldrich). Cells were centrifuged and resuspended at room temperature in a solution of PEG (average Mr 8 kDa, Fluka BioUltra), dextran (10.5 kDa, Sigma-Aldrich), or Ficoll (70 kDa, Fluka) in bidistilled H2O, deionised by shaking with AG 501-X8 resin (Bio-Rad) for 6–8 h, supplemented with 100 mM K-Hepes buffer, pH 7.4. Before each April 2012 \| Volume 7 \| Issue 4 \| e36045 PLoS ONE \| www.plosone.org 4 Macromolecular Crowding and Chromosome Structure Macromolecular Crowding and Chromosome Structure St-Jean P, Vaillant C, Audit B, Arneodo A (2008) Spontaneous emergence of sequence-dependent rosettelike folding of chromatin fiber. Phys Rev E 77: 061923. 39. 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Poirier MG, Monhait T, Marko JF (2002) Reversible hypercondensation and decondensation of mitotic chromosomes studied using combined chemical- micromechanical techniques. J Cell Biochem 85: 422–434. 59. Vergani L, Mascetti G, Nicolini C (1998) Effects of polyamines on higher-order folding of in situ chromatin. Mol Biol Rep 25: 237–244. q J 33. Hungerford DA, Diberardino M (1958) Cytological Effects of Prefixation Treatment. J Biophys Biochem Cytol 4: 391–400. 60. Sikorav J-L, Jannink G (1994) Kinetics of chromosome condensation in the presence of topoisomerases: a phantom chain model. Biophys J 66: 827–837. ence of topoisomerases: a phantom chain model. Biophys J 66: 827–8 34. Howell WM, Hsu TC (1979) Chromosome core structure revealed by silver staining. Chromosoma 73: 61–66. 61. Livolant F, Mangenot S, Leforestier A, Bertin A, Frutos M, et al. (2006) Are liquid crystalline properties of nucleosomes involved in chromosome structure and dynamics? Philos Transact A Math Phys Eng Sci 364: 2615–2633. 35. Deaven LL, Petersen DF (1973) The chromosomes of CHO, an aneuploid Chinese hamster cell line: G-band C-band and autoradiographic analyses. Chromosoma 41: 129–144. 62. Cooke IR, Williams DRM (2004) Collapse of flexible-semiflexible copolymers in selective solvents: single chain rods, cages, and networks. Macromolecules 37: 5778–5783. 36. Zhang Y, Heermann DW (2011) Loops Determine the Mechanical Properties of Mitotic Chromosomes. PLoS ONE 6: e29225. 63. Vasilevskaya VV, Markov VA, Khalatur PG, Khokhlov AR (2006) Semiflexible amphiphilic polymers: Cylindrical-shaped, collagenlike, and toroidal structures. J Chem Phys 124: 144914. 37. Compton JL, Hancock R, Oudet P, Chambon P (1976) Biochemical and Electron-Microscopic Evidence that the Subunit Structure of Chinese-Hamster- Ovary Interphase Chromatin Is Conserved in Mitotic Chromosomes. Eur J Biochem 7: 555–568. 64. Hancock R (2011) The crowded environment of the genome. In: Rippe K, ed. Genome Organization And Function In The Cell Nucleus, Wiley-VCH, Weinheim. pp 169–184. 38. Rattner JB, Hendzel MJ, Furbee CS, Muller MT, Bazett-Jones DP (1996) Topoisomerase IIa Is Associated with the Mammalian Centromere in a Cell Cycle- and Species-specific Manner and Is Required for Proper Centromere/ Kinetochore Structure. J Cell Biol 134: 1097–1107. 65. Macromolecular Crowding and Chromosome Structure Macromolecular Crowding and Chromosome Structure 24. Hancock R (2007) Packing of the polynucleosome chain in interphase chromosomes: evidence for a contribution of crowding and entropic forces. Semin Cell Dev Biol 18: 668–675. 50. Maughan DW, Godt RE (2001) Protein osmotic pressure and the state of water in frog myoplasm. Biophys J 80: 435–442. 51. Horowitz SB, Paine PL (1979) Reference phase analysis of free and bound intracellular solutes. II. Isothermal and isotopic studies of cytoplasmic sodium potassium and water. Biophys J 25: 45–62. 25. Zimmerman SB, Trach SO (1988) Effects of macromolecular crowding on the association of E, coli ribosomal particles. Nucleic Acids Res 16: 6309–6326. 26. Gala´n A, Sot B, Llorca O, Carrascosa JL, Valpuesta JM, et al. (2001) Excluded Volume Effects on the Refolding and Assembly of an Oligomeric Protein. GroEL, a Case Study. J Biol Chem 276: 957–964. ´ 52. Edelmann L (1989) The physical state of potassium in frog skeletal muscle studied by ion-sensitive microelectrodes and by electron microscopy. Scanning Microsc 3: 1219–1230. y J 27. del A´ lamo M, Rivas G, Mateu MG (2005) Effect of Macromolecular Crowding Macromolecules on Human Immunodeficiency Virus Type 1 Capsid Protein Assembly In vitro. J Virol 79: 14271–14281. 53. Kellermayer M, Ludany A, Jobst K, Szucs G, Trombitas K, et al. (1986) Cocompartmentation of proteins and K+ within the living cell. Proc Natl Acad Sci USA 83: 1011–1015. 28. Zhou HX, Rivas G, Minton AP (2008) Macromolecular crowding and confinement: biochemical biophysical and potential physiological consequences. Annu Rev Biophys 37: 375–397. 54. Ling GN (1990) The physical state of potassium ion in the living cell. Scanning Microsc 4: 737–750. 55. Negendank M, Shaller C (2005) Multiple fractions of sodium exchange in human lymphocytes. J Cell Physiol 104: 443–459. p y 29. Lewis CD, Laemmli UK (1982) Higher order metaphase chromosome structure: Evidence for metalloprotein interactions. Cell 29: 171–181. y p y J y 56. Gu¨nther T (2006) Concentration, compartmentation and metabolic function of intracellular free Mg2+. Magnes Res 19: 225–236. 56. Gu¨nther T (2006) Concentration, compartmentation intracellular free Mg2+. Magnes Res 19: 225–236. 30. Cole A (1967) Chromosome Structure. In: Cole A, ed. Theoretical and Experimental Biophysics: A Series Of Advances, Vol. 1, Marcel Dekker, New York. pp 305–375. intracellular free Mg2+. Magnes Res 19: 225–236 57. Spitzer JJ, Poolman B (2005) Electrochemical structure of the crowded cytoplasm. Trends Biochem Sci 30: 536–541. pp 31. References J Struct Biol 136: 53–66. 9. Blumenthal AB, Dieden JD, Kapp LN, Sedat JW (1979) Rapid isolation of metaphase chromosomes containing high molecular weight DNA. J Cell Biol 81: 255–259. 21. Micka U, Holm C, Kremer K (1999) Strongly Charged Flexible Polyelectrolytes in Poor Solvents: Molecular Dynamics Simulations. Langmuir 15: 4033–4044. 10. Adolph KW (1980) Isolation and structural organization of human mitotic chromosomes. Chromosoma 76: 23–33. 22. Chang R, Yethiraj A (2006) Dilute Solutions of Strongly Charged Flexible Polyelectrolytes in Poor Solvents: Molecular Dynamics Simulations with Explicit Solvent. Macromolecules 39: 821–828. 11. Gooderham K, Jeppesen P (1983) Chinese hamster metaphase chromosomes isolated under physiological conditions. A partial characterization of associated non-histone proteins and protein cores. Exp Cell Res 144: 1–14. 23. Pastre´ D, Hamon L, Mechulam A, Sorel I, Baconnais S, et al. (2007) Atomic force microscopy imaging of DNA under macromolecular crowding conditions. Biomacromolecules 8: 3712–3717. 12. Belmont AS, Braunfeld MB, Sedat JW, Agard DA (1989) Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro. Chromosoma 98: 129–143. PLoS ONE \| www.plosone.org April 2012 \| Volume 7 \| Issue 4 \| e36045 April 2012 \| Volume 7 \| Issue 4 \| e36045 5 Macromolecular Crowding and Chromosome Structure Danilowicz C, Lee CH, Kim K, Hatch K, Coljee VW, et al. (2009) Single molecule detection of direct, homologous, DNA/DNA pairing. Proc Natl Acad Sci USA 106: 19824–19829. metaphase chromosomes. J Cell Biol 162: 23–35. 47. Mora-Bermu´dez F, Gerlich D, Ellenberg J (2007) Maximal chromosome compaction occurs by axial shortening in anaphase and depends on Aurora kinase. Nat Cell Biol 9: 822–831. 74. Lavery PE, Kowalczykowski SC (1992) Enhancement of recA protein-promoted DNA strand exchange activity by volume-occupying agents. J Biol Chem 267: 9307–9314. 48. Parsegian VA, Rand RP, Rau DC (1995) Macromolecules and water: probing with osmotic stress. Meth Enzymol 259: 43–94. 75. Feng B, Frykholm K, Norde´n B, Westerlund F (2010) DNA strand exchange catalyzed by molecular crowding in PEG solutions. Chem Commun 46: 8231–8233. 49. Stanley CB, Strey HH (2003) Measuring Osmotic Pressure of Poly(ethylene glycol) Solutions by Sedimentation Equilibrium Ultracentrifugation. Macromol- ecules 36: 6888–6893. PLoS ONE \| www.plosone.org April 2012 \| Volume 7 \| Issue 4 \| e36045 6
https://openalex.org/W4382581818	https://www.scielo.br/j/pci/a/CdcLhTccskBfrVrCdcb6m3q/?lang=pt&format=pdf	Portuguese	null	UMA ONTOLOGIA DE DOMÍNIO PARA A PRESTAÇÃO DE CONTAS DOS GESTORES PÚBLICOS FEDERAIS: ONTOACCOUNT	Perspectivas em Ciência da Informação	2,023	cc-by	8,323	Seção: artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: on DOI http://dx.doi.org/10.1590/1981 Seção: artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: on DOI http://dx.doi.org/10.1590/1981 RESUMO RESUMO A difusão de inovação em gestão pública e o uso de tecnologias em prol do acesso à informação, da transparência, e da participação social são marcas dos esforços governamentais para o desenvolvimento e expansão do paradigma do Governo Aberto no Brasil. O presente artigo pretende divulgar os resultados de uma pesquisa de doutorado que investigou o paradoxo entre a vasta quantidade de dados governamentais disponibilizados na tomada e prestação de contas dos administradores públicos federais e a baixa percepção e apropriação do conhecimento gerado pelo cidadão. Seu propósito central consistiu em realizar uma modelagem do domínio de conhecimento da tomada e da prestação de contas anual dos gestores públicos federais a fim de facilitar e acelerar o desenvolvimento de agentes de software cívicos. Do ponto de vista metodológico, desenvolveu-se uma pesquisa exploratória qualitativa com procedimentos técnicos de pesquisa bibliográfica e documental. Utilizou-se o framework Protégé 5.5.0 e a metodologia OntoForInfoScience, proposta por Mendonça (2015), para desenvolver o protótipo de ontologia. Como resultado, foi criada a ontologia de domínio chamada OntoAccount para representar os conceitos e os relacionamentos do domínio, além de possibilitar responder a questionamentos sobre as instâncias do domínio da tomada e prestação de contas anual dos gestores públicos federais. Palavras-chave: Ontologia de domínio. Representação do conhecimento. Prestação de contas. UMA ONTOLOGIA DE DOMÍNIO PARA A PRESTAÇÃO DE CONTAS DOS GESTORES PÚBLICOS FEDERAIS: ONTOACCOUNT Reuber da Silva Fonseca 79486 – https://orcid.org/0000-0002-3316-9684 reuberf@gmail.com Universidade Federal de Minas Gerais (UFMG) Belo Horizonte, MG, Brasil Reuber da Silva Fonseca http://lattes.cnpq.br/6490326251279486 – https://orcid.org/0000-0002-3316-9684 reuberf@gmail.com Universidade Federal de Minas Gerais (UFMG) Belo Horizonte, MG, Brasil Gercina Angela Lima http://lattes.cnpq.br/3183050056105009 – https://orcid.org/0000-0003-0735-3856 limagercina@gmail.com Universidade Federal de Minas Gerais (UFMG) Belo Horizonte, MG, Brasil Recebido em: 29 setembro 2022. Aceito em: 23 fevereiro 2023. Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 ABSTRACT The diffusion of innovation in public management and the use of technologies in favor of access to information, transparency, and social participation are hallmarks of government efforts to develop and expand the Open Government paradigm in Brazil. The present article intends to disclose the results of a doctoral research that investigated the paradox between the vast amount of governmental data available in the taking and rendering of accounts of federal public administrators and the low perception and appropriation of the knowledge generated by the citizen. Its main purpose was to carry out a modeling of the domain of knowledge of the taking and annual accountability of federal public managers in order to facilitate and accelerate the development of civic software agents. From the methodological point of view, an exploratory qualitative research was developed with technical procedures of bibliographic and documental research. The Protégé 5.5.0 framework and the OntoForInfoScience methodology, proposed by Mendonça (2015), were used to develop the ontology prototype. As a result, a domain ontology called OntoAccount was created to represent the concepts and relationships of the domain, in addition to making it possible to answer questions about the instances of the domain of taking and rendering annual accounts of federal public managers. Keywords: Domain ontology; Knowledge representation; Accountability DOI http://dx.doi.org/10.1590/1981-5344/41347 DOI http://dx.doi.org/10.1590/1981-5344/41347 1 de 21 1 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 Reuber da Silva Fonseca; Gercina Angela Lima INTRODUÇÃO A evolução tecnológica contínua e ininterrupta dos recursos computacionais contemporâneos ampliou as capacidades de captura, de processamento, de disponibilização e de acesso à informação pública. Do outro lado, a adoção de novos referenciais político-administrativos, pela Administração Pública, baseados no conceito de Governo Aberto, compatibilizou agendas governamentais no intuito de transformar governos em entidades mais responsáveis, responsivas e eficientes, e cidadãos em agentes mais conscientes e participativos. A difusão de inovação em gestão pública e o uso de tecnologias em prol do acesso à informação, da transparência, e da participação social são marcas dos esforços governamentais para o desenvolvimento e expansão do paradigma do Governo Aberto no Brasil. Nesta seara, é relevante problematizar o cenário de produção, de disponibilização e de consumo de dados abertos governamentais provenientes da tomada e prestação de contas do administrador público federal e discutir alternativas para representar este domínio de conhecimento de modo a aumentar a percepção e a apropriação pelo cidadão. Dentre os propósitos da prestação de contas anual, se sobrepõe a finalidade de informar aos cidadãos (principais provedores de recursos e destinatários de serviços públicos) o alcance dos objetivos de interesse coletivo estabelecidos. Esta pesquisa, contudo, questiona a efetividade dos dados que compõem a prestação de contas em subsidiar a geração de conhecimento para o controle social sobre o Poder Público. Questiona-se, sobretudo, a ausência de política que apoie a disponibilização dos dados em formato estruturado e semântico a fim de favorecer o reuso das informações por máquinas. Ao longo dos anos, uma extensa quantidade de dados sobre resultados da gestão pública federal, provenientes dos processos de tomada e de prestação de contas, foi disponibilizada ao público em formatos não estruturados. (ex: Portable Document Format - PDF). Contudo, entende-se que a maneira isolada como estes processos são relatados implica barreiras para o reuso e agregação deste conhecimento. Uma ideia, ainda que incompleta, do valor destes dados para os indivíduos e organizações, pode ser alcançada 2 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contínuo, 2023: e-41347 rmação, Belo Horizonte, v. 23, Fluxo Contínuo, 2023: e-41347 Seção: artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: ontoaccount DOI http://dx.doi.org/10.1590/1981-5344/41347 analisando o seu potencial de reuso e de construção de novos contextos de análise. Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 INTRODUÇÃO Logo, a ausência ou incipiência de uma política de disponibilização destes dados em formato estruturado e semântico, tendo como fim o seu reuso, é um problema a ser enfrentado por esta pesquisa. Neste cenário político e tecnológico, emerge a necessidade de “libertar dados” dos relatórios que compõem a tomada e prestação de contas e de se desenvolver estratégias, abordagens e instrumentos para a construção de modelos conceituais representativos desta realidade e consistentes com as necessidades e interesses dos cidadãos. Dentre as propostas, as ontologias são instrumentos uteis de organização e representação do conhecimento para representar domínios para uso tanto de humanos como de agentes de software. Este estudo, portanto, é baseado na premissa de que a ausência de modelagem semântica formal e explícita do domínio de conhecimento da tomada e da prestação de contas anual dos gestores públicos federais prejudica o eventual consumo dos dados públicos por agentes que queiram assimilar o valor público gerado na gestão. No tocante à publicação dos dados públicos provenientes da prestação de contas, cumpre-se alertar que se esta for inadequadamente instituída, constituir-se-á em barreira ao avanço da participação e do controle social. Em última análise, isto pode levar à alienação política do cidadão. Deste modo, esta pesquisa teve como objetivo desenvolver uma modelagem de conhecimento baseada em um protótipo de ontologia instanciada com dados estruturados provenientes do domínio de conhecimento da tomada e da prestação de contas anual dos gestores públicos federais a fim de oferecer suporte ao desenvolvimento de aplicações cívicas. Durante os trabalhos, buscou-se conhecer e revelar significados e relações semânticas presentes no domínio e contribuir para a publicação de dados abertos estruturados e semânticos deste domínio. As relações semânticas foram exploradas por meio de uma solução ontológica com vista a atribuir sentido e significado ao processo e ao conteúdo dos relatórios, oportunizando o reuso de recursos e a troca de informação e conhecimento entre pessoas e aplicações. 3 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 Reuber da Silva Fonseca; Gercina Angela Lima Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contínuo, 2023: e-41347 DADOS ABERTOS, DADOS ABERTOS CONECTADOS E WEB SEMÂNTICA Segundo a Open Definition (2014), o significado do termo “aberto”, no contexto do movimento de acesso livre e aberto ao conhecimento, é aquele que qualquer pessoa pode acessar, utilizar, modificar, e compartilhar com exigência máxima de preservação da proveniência e da abertura. A Open Knowledge Foundation (OKF), define Dados Abertos como os dados “que podem ser livremente usados, reutilizados e redistribuídos por qualquer pessoa - sujeitos, no máximo, à exigência de atribuição da fonte e compartilhamento pelas mesmas regras” (OKF, 2020). Isotani e Bittencourt (2015) esclarecem que a abertura de dados se concretiza por meio da retirada de controles legais, técnicos e econômicos sobre os dados. Este movimento, contudo, implica em custos e benefícios para produtores e consumidores de dados abertos. Uma proposta de classificação da maturidade das publicações de dados abertos foi apresentada por Berners-Lee (2006). O Esquema 5-Estrelas dos Dados Abertos tem como propósito encorajar pessoas e instituições (em especial as governamentais) a desenvolver ações de publicação e consumo de dados estruturado na Web de forma aberta. O sistema de classificação alvitrado atribui maior número de estrelas para os dados mais abertos, isto é, aqueles com maior facilidade de conexão a outros dados. Em síntese, o esquema expressa as seguintes conclusões segundo o número de estrelas: • 1 Estrela: O recurso está disponível na Internet em qualquer formato e com licença aberta. (exigência mínima para que seja considerado dado aberto); • 2 Estrelas: Seguindo todas as regras acima, o recurso está disponível de forma estruturada; • 3 Estrelas: Seguindo todas as regras acima, o recurso está disponível em formato não proprietário; • 4 Estrelas: Seguindo todas as regras acima, o recurso está disponível nos padrões RDF e SPARQL; • 5 Estrelas: Seguindo todas as regras acima, o recurso conecta seus dados a outros dados a fim de fornecer um contexto. Kim e Hausenblas (2012), refletindo sobre o esquema de avaliação, apresentado acima, identificou um conjunto de custos e benefícios para 4 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contínuo, 2023: e-41347 artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: ontoaccount DOI http://dx.doi.org/10.1590/1981-5344/41347 produtores e consumidores de dados abertos em cada nível de classificação. Os resultados foram estruturados no Quadro 1. DADOS ABERTOS, DADOS ABERTOS CONECTADOS E WEB SEMÂNTICA Quadro 1 - custos e benefícios de dados abertos na web Nível Categoria Consumidores Produtores 1 estrela Benefícios Podem visualizar, imprimir, armazenar, usar, modificar e compartilhar os dados. É fácil publicar os dados e comunicar as regras de licenciamento. Custos Requer desenvolvimento de código para extrair os dados presos ao documento. 2 estrelas Benefícios Podem tudo anteriormente e adicionalmente podem processar os dados diretamente com um software proprietário e exportá-los para outros formatos suportados. Continua fácil publicar os dados. Custos Para manipular os dados é necessária uma licença de software proprietário. 3 estrelas Benefícios Podem tudo anteriormente e adicionalmente podem usar, modificar e compartilhar os dados de qualquer forma (sem as restrições do software proprietário). Continua fácil publicar os dados. Custos Os dados continuam no documento e não são diretamente acessíveis na Web. Pode ser necessário conversores ou plugins para exportar os dados. 4 estrelas Benefícios Podem tudo anteriormente e adicionalmente podem acessá-los diretamente para reusar parte ou todos os dados. Podem conectá-los a outros dados. Tem controle granular detalhado sobre os itens de dados e pode tê-los referenciados por outros publicadores. Custos Entender a estrutura de dados RDF Requer mais tempo para organizar e representar os dados (definir URIs e padrões para reuso) 5 estrelas Benefícios Podem tudo anteriormente e adicionalmente podem descobrir e conectar mais dados relacionados aprendendo sobre a sua organização. Os dados são encontráveis na Web, aumentando o seu valor. Com o efeito de rede, passam a obter todos os benefícios de consumidores de dados. Custos Devem controlar “erros 404” e links quebrados de dados nas páginas Web e monitorar o conteúdo de outras páginas Web reutilizadas. Requer investimento de recursos para manter os dados na Web. Requer reparo de links quebrados ou erros 404. Fonte: Elaborado pelo autor baseado no trabalho de Kim e Hausenblas (2012). Percebe-se, com a leitura do Quadro 1, que a partir da classificação de 4 estrelas do modelo, os itens de dados podem ser referenciados por outros publicadores, possibilitando o efeito de rede. Também é verdade que este compromisso com a abertura de dados requer mais tempo e investimento para organizar e representar os dados a serem consumidos. Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 5 de 21 Reuber da Silva Fonseca; Gercina Angela Lima O desenvolvimento da Web Semântica1, cunhou o surgimento do conceito de Dados Conectados (do Inglês, Linked Data). 1 A Web Semântica pode ser definida como uma extensão da Web atual na qual os dados recebem um significado mais bem definido (BERNERS-LEE; HENDLER; LASSILA, 2001). DADOS ABERTOS, DADOS ABERTOS CONECTADOS E WEB SEMÂNTICA Segundo Isotani e Bittencourt (2015), “este pode ser definido como um conjunto de boas práticas para publicar e conectar conjuntos de dados estruturados na Web”. Estas boas práticas são recomendações, reveladas em padrões de desenvolvimento, que promovem a interoperabilidade de dados na Web. Os padrões de publicação dos dados conectados na Web permitem a identificação de recursos (objetos reais ou conceitos) habilitando a descoberta e o processamento automatizado de dados por agentes de software. Apesar de existirem ações de publicação de dados conectados de forma fechada na Web, esta pesquisa busca refletir sobre as ações de publicação e consumo de Dados Abertos Conectados (do inglês, Linked Open Data - LOD). De acordo com Berners-Lee (2006, tradução nossa), LOD pode ser definido como “um dado conectado que é lançado sob uma licença aberta, que não impede sua reutilização gratuitamente“. Isto é, para ser considerado LOD, o recurso deve existir na Web sem restrições legais que impeçam sua reutilização (princípio de Dados Abertos) e deve observar padrões de desenvolvimento que promovam a interoperabilidade. As iniciativas de LOD, portanto, posicionam-se na interseção dos dois conceitos anteriormente discutidos e refletem decisões de abertura de dados no mais alto nível do Esquema 5-Estrelas dos Dados Abertos proposto por Berners-Lee (2006). Segundo Berners-Lee, Hendler e Lassila (2001, p. 3, tradução nossa) “para que a Web Semântica funcione, os computadores devem ter acesso a coleções estruturadas de informações e conjuntos de regras de inferência que podem usar para conduzir o raciocínio automatizado.” Para que os sistemas interoperem, eles devem ter a capacidade de descobrir os significados comuns (a semântica) dos dados que encontrar. Logo, esta visão da Web requer compromisso ontológico para se concretizar. 1 A Web Semântica pode ser definida como uma extensão da Web atual na qual os dado recebem um significado mais bem definido (BERNERS-LEE; HENDLER; LASSILA, 2001). 6 de 21 6 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contínuo, 2023: e-41347 Seção: artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: ontoaccount DOI http://dx.doi.org/10.1590/1981-5344/41347 eção: artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: ontoa DOI http://dx.doi.org/10.1590/1981-53 relacionar as informações em uma página às estruturas de conhecimento e regras de inferência associadas. (BERNERS-LEE; HENDLER; LASSILA, 2001, p. 7, tradução nossa). As ontologias, portanto, são fundamentais para transformar os dados em recursos compreensíveis por agentes de software. DADOS ABERTOS, DADOS ABERTOS CONECTADOS E WEB SEMÂNTICA Deste modo elas permitem romper a barreira semântica existente e criam oportunidades para a interação automatizada por meio do reuso da infraestrutura de dados. Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 ONTOLOGIAS E APLICAÇÕES CÍVICAS As ontologias assumem importância crescente no tratamento temático da informação na Web, em especial na Web Semântica. (BRÄSCHER; CARLAN, 2010). Os estudos crescentes sobre ontologia carregam contribuições de várias disciplinas e ciências. Cada uma delas, partindo de sua própria visão de mundo e amparadas em fundamentos teóricos e metodológicos mais ou menos distintos, ajuda a moldar o pensar e o fazer ontológico. Uschold e Gruninger (1996) afirmam que uma ontologia se compromete com uma visão compartilhada de mundo sobre um determinado domínio. Já Guarino (1998), propõe uma definição para ontologia quando diz que: Uma ontologia é uma teoria lógica que explica o significado pretendido de um vocabulário formal, ou seja, seu compromisso ontológico com uma conceituação particular do mundo. Os modelos pretendidos de uma linguagem lógica usando tal vocabulário são restritos pelo seu compromisso ontológico. Uma ontologia reflete indiretamente esse compromisso (e a conceituação subjacente) aproximando esses modelos pretendidos. (GUARINO, 1998, p. 7, tradução nossa). Como se observa, o autor propõe a ontologia como uma teoria que reflete o significado dos objetos no mundo. Almeida (2014) identifica dois principais sentidos para o termo neste campo de pesquisa, são eles: a) a ontologia como teoria representativa que descreve fatos e regras que governam parte da realidade; e b) a ontologia como declarações expressas em uma linguagem formal de representação. No primeiro sentido, a função da ontologia é formalizar o conhecimento de um grupo de especialistas com fins computacionais. No segundo sentido, a função da ontologia na modelagem “é tornar explícitos axiomas que restringem modelos, de forma a igualar, tanto quanto possível, os modelos que contém o significado pretendido” (ALMEIDA, 2014, p. 249), permitindo inferências automatizadas. 7 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 Reuber da Silva Fonseca; Gercina Angela Lima As autoras Bräscher e Carlan (2010) postulam que as ontologias “definem conceitos e relações de alguma área do conhecimento, de forma compartilhada e consensual” (BRÄSCHER; CARLAN, 2010, p. 160). Já Campos (2004), refletindo sobre os princípios subjacentes ao processo de modelização de domínios do conhecimento, compreende a ontologia formal, como um formalismo que sistematiza conhecimento pretendendo a formalização de definições axiomáticas. Como tratado, a definição de ontologia varia em decorrência da tradição disciplinar em que é operacionalizado. Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contínuo, 2023: e-41347 ONTOLOGIAS E APLICAÇÕES CÍVICAS Nesta pesquisa, utilizou-se a definição postulada por Gruber (1993), neste sentido, a ontologia é a especificação formal e explícita de uma conceitualização compartilhada em determinado domínio do conhecimento (GRUBER, 1993). Enquanto ferramenta apta a representar domínios de conhecimento computacionalmente, as ontologias adquirem importância crescente na Web semântica. Para que os dados possam ser consumidos independentemente da estrutura original, as ontologias precisam atuar. Considerando o papel das ontologias na Web semântica, estas também poderão sustentar o desenvolvimento de aplicativos cívicos baseados em conhecimento. A Internet e a Web têm inspirado pesquisadores de várias matizes de pensamento a discorrer sobre o papel que estas tecnologias assumem no processo democrático. Há uma expectativa delas poderem contribuir para o aperfeiçoamento das instituições e das práticas democráticas. Nesta pesquisa importa discutir o significado que se atribui ao conceito de aplicações cívicas e revelar sua ligação com outros conceitos supracitados. Segundo Jäske e Ertiö (2019, p. 21) aplicações cívicas podem ser definidas como “tecnologias ascendentes que usam dados abertos para resolver desafios de governança e políticas”. A adoção de padrões de publicação e consumo de dados abertos na Web permitiu ao homem, à máquina ou a uma combinação entre os dois explorar dados que antes estavam ocultos em documentos. Segundo O’Reilly (2011), “a magia dos dados abertos é que a mesma abertura que permite transparência também permite inovação” (O’REILLY, 2011, p. 26, tradução nossa). Na visão do autor, este movimento se dá a partir da reutilização 8 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contínuo, 2023: e-41347 Seção: artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: ontoaccount DOI http://dx.doi.org/10.1590/1981-5344/41347 criativa dos dados abertos governamentais (DAG) no desenvolvimento de aplicativos. Assim sendo, os panoramas possíveis para a utilização de dados abertos governamentais são ilimitados, amparando ações empreendedoras, governamentais e cívicas. Estes podem viabilizar a criação de novos modelos de negócios baseados em plataformas e aplicativos; o aperfeiçoamento e desenvolvimento de serviços públicos; e o incremento de ações de participação, deliberação e controle sobre o Estado e outras organizações. Os DAG habilitam formas de participação direta do cidadão (crowdsourcing) na formulação e execução de serviços públicos potencialmente mais efetivos, além de fornecer bases para economia de custos no governo. ONTOLOGIAS E APLICAÇÕES CÍVICAS Por meio de APIs de DAG, desenvolvedores de software podem usar dados governamentais para escrever códigos que criam interfaces novas e profícuas com o governo e/ou com o mercado. Algumas destas aplicações podem auxiliar ou mesmo substituir funções de governo. Um aplicativo, por exemplo, que possibilita aos indivíduos de uma cidade relatar buracos na via pública, iluminações e sinalizações deficientes, entre outros problemas, pode auxiliar um órgão público (eventualmente sobrecarregado) a oferecer melhores serviços públicos (O’REILLY, 2011). É crescente a adoção pelo poder público dos padrões W3C para a Web semântica, permitindo o reuso e integração de DAG (LEE; ALMIRALL; WAREHAM, 2015). Este movimento promoverá a padronização de publicação de DAG entre os poderes públicos constituídos e alavancará os efeitos de rede que permitirão, entre outros feitos, o desenvolvimento de plataformas comuns entre cidades. Estas mudanças, caso se concretizem, podem conduzir a gestão pública a uma visão de “government as a platform2” (O’REILLY, 2011). 2 Uma abordagem para a transformação digital do setor público apresentado por O’Reilly, (2011). b) Etapa 1: especificação b) Etapa 1: especificação Nesta etapa foram destacadas algumas dimensões básicas que demarcam o propósito da construção da ontologia de domínio. Seguindo orientações da metodologia OntoForInfoScience, foram especificados por meio do template de especificação os seguintes pontos: a) o domínio e escopo da ontologia; b) os prováveis cenários de aplicação da ontologia; c) o público-alvo da ontologia; e o d) o grau de formalidade da ontologia. Além destes elementos que compõe a metodologia, foram acrescentados outros metadados sobre a arquitetura da ontologia e da sua interface Web. Entre eles: a) o namespace e a URI da ontologia; b) a página da Internet do projeto; e c) o idioma padrão. ONTOACCOUNT: METODOLOGIA E ESPECIFICAÇÃO Para a construção de uma ontologia existem diversas metodologias de referência. A metodologia de construção adotada nesta pesquisa é a OntoForInfoScience, proposta por Mendonça (2015). Ela compreende uma pré-etapa e oito etapas de desenvolvimento de ontologias. São elas: 1) especificação; 2) aquisição e extração de conhecimento; 3) 2 Uma abordagem para a transformação digital do setor público apresentado por O’Reilly, (2011). 2 Uma abordagem para a transformação digital do setor público apresentado por O’Reil (2011). Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 9 de 21 Reuber da Silva Fonseca; Gercina Angela Lima conceitualização; 4) fundamentação ontológica; 5) formalização; 6) avaliação; 7) documentação e 8) disponibilização. 6) A escolha desta metodologia se deu em função de quatro qualidades atribuídas a ela: 1) possui um melhor detalhamento das etapas de construção de ontologias; 2) foi concebida para apoiar especialistas em organização e representação do conhecimento; e 3) tem como origem etapas de metodologias já conhecidas (Methontology, NeOn e o 101 Method); e 4) foi desenvolvida no âmbito de um Programa de Pós-Graduação, o que confere uma garantia razoável de que a sua proposição obedeceu ao rigor científico. A OntoAccount foi construída mediante a utilização do framework Protégé 5.5.0, um editor de ontologia de código aberto e gratuito, que suporta as especificações OWL 2 e RDF do W3C. A seguir, descrevem-se os procedimentos sucessivos adotados em cada uma das etapas da metodologia e, quando disponível, uma prévia dos resultados. a) Pré-etapa: avaliação da necessidade a) Pré-etapa: avaliação da necessidade Nesta fase foi realizada uma descrição da real necessidade de construção da ontologia ao contrário de outros instrumentos de organização do conhecimento (tesauros, taxonomia etc.). 3 Foram selecionados de forma intencional os relatórios de gestão de 2018 das seguintes instituições: Universidade de Brasília; Universidade Federal do Rio de Janeiro e Universidade Federal de Minas Gerais. 4 Sketch Engine é uma ferramenta online que analisa textos e identifica o que é típico, raro ou de uso emergente na linguagem. Disponível em: https://www.sketchengine.eu/. Acesso em: 07 nov 2021. 5 Além da legislação específica, foi consultada a ferramenta Vocabulário de Controle Externo (VCE), um tesauro que padroniza a terminologia utilizada na atividade de Controle Externo e auxilia o tratamento e a recuperação das informações no TCU. Disponível em: https://contas.tcu.gov.br/ords/f?p=701663:1:3919793213294. Acesso em: 08 Nov 2021. c) Etapa 2: aquisição e extração de conhecimento c) Etapa 2: aquisição e extração de conhecimento Os documentos de referência utilizados no desenvolvimento da OntoAccount compreendem normas jurídicas e documentos públicos, em especial relatórios. Resoluções, instruções normativas, publicações e acórdãos emitidos pelos colegiados do Tribunal de Contas da União (TCU) em 10 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contínuo, 2023: e-41347 decorrência da tomada de contas ordinária, assim como relatórios de prestação de contas anual elaborados por universidades federais foram coletadas e analisadas. decorrência da tomada de contas ordinária, assim como relatórios de prestação de contas anual elaborados por universidades federais foram coletadas e analisadas. Os materiais de referência foram analisados informal e formalmente com o objetivo de identificar as estruturas textuais do domínio e as definições primárias. Uma amostra3 de relatórios de gestão foi submetida ao processo de extração terminológica automática dos termos mais frequentes por meio da funcionalidade “Wordlist” da ferramenta Sketch Engine4. As listas de frequência dos termos foram analisadas em conjunto com a lista de termos provenientes da extração manual dos demais documentos de referência. Os termos resultantes foram organizados em três conjuntos (glossário de conceitos, glossário de verbos e glossário de relações) que serviram de base para a formação de classes, propriedades e relações na etapa de conceitualização. Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 d) Etapa 3: conceitualização Nesta etapa foram realizadas as atividades de identificação e de análise dos conceitos do domínio a fim de subsidiar a construção de modelos conceituais. Tendo em vista a vivência profissional do autor e a existência de fontes de autoridade legal e formal,5 que dão suporte a tarefa de definição textual de conceitos, optou-se por não envolver outros especialistas do domínio nesta etapa. Os produtos deste estágio compreendem: a) a tabela de conceitos e propriedades; b) o dicionário de verbos; e c) os modelos conceituais. A tabela de conceitos e propriedades foi elaborada a partir da análise semântica do glossário de conceitos. A cada fase do processo de conceitualização, foram acrescidos novos elementos à estrutura original do glossário, gerando os seguintes subprodutos: a) o dicionário de conceitos; e b) a tabela de conceito e valores. 11 de 21 11 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 Reuber da Silva Fonseca; Gercina Angela Lima O dicionário de conceitos foi criado a partir da associação e da inclusão da definição textual dos conceitos selecionados a partir do glossário. Nesta fase, também foram identificados os sinônimos. Para fins de definição textual dos conceitos e de associação de sinônimos aos termos preferenciais, foram considerados os conceitos constantes do Anexo I da Instrução Normativa-TCU 84, de 22 de abril de 2020 e, como fonte secundária, a terminologia do tesauro do TCU, operado por meio da ferramenta Vocabulário de Controle Externo (VCE). No caso de conceitos ausentes nos documentos referenciais, procurou-se a definição na legislação aplicável à Administração Pública. Já a tabela de conceito e valores foi elaborada a partir do relacionamento entre os conceitos do dicionário e seus respectivos “valores possíveis”. (MENDONÇA, 2015, p. 201). Por fim, foram identificadas algumas possíveis propriedades dos conceitos ou classes. A tabela de conceitos e propriedades corresponde à tabela de conceitos e valores acrescida das propriedades identificadas nesta fase. O dicionário de verbos também foi elaborado a partir de um produto da etapa anterior, o glossário de verbos. Esse foi elaborado acrescentando, a cada termo do glossário, uma definição do significado do verbo no domínio. Os verbos selecionados foram representados uma única vez no dicionário de verbos e associados com seus sinônimos. Por fim, foram acrescentados exemplos de uso do verbo no domínio. 6 É uma plataforma online colaborativa que auxilia na construção de mapas mentais, conceituais, diagramas e outras representações gráficas. 7 Utilizou-se a versão traduzida da BFO 2.0, denominada BFO-PT, que foi elaborada por Simone Torres de Souza durante a pesquisa de doutorado que resultou na ontologia denominada 12 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Ontolegis, aplicada à informação legislativa para o Direito Médico. Disponível em: http://mba.eci.ufmg.br/downloads/BFO-PT/BFO-PT.owl. Acesso em: 28 nov 2021. 8 Um servidor de dados conectados projetado para promover o reuso de ontologias. Disponível em: http://www.ontobee.org/. Acesso em: 18 Nov. 2021. d) Etapa 3: conceitualização O dicionário de verbos, assim como ocorre com a tabela de conceitos e propriedades, contém um conjunto de termos candidatos às relações conceituais. Os modelos conceituais correspondem ao terceiro produto da etapa de conceitualização e foram gerados a partir da ferramenta Miro6. Esta ferramenta apoiou a construção de mapas conceituais, taxonomias e outras estruturas gráfica que representam o domínio. e) Etapa 4: fundamentação ontológica ) p ç g Adotou-se os princípios de formalização e as entidades e relações do Basic Formal Ontology (BFO), versão 2, em português7, como ontologia de alto Adotou-se os princípios de formalização e as entidades e relações do Basic Formal Ontology (BFO), versão 2, em português7, como ontologia de alto Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contínuo, 2023: e-41347 12 de 21 Seção: artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: ontoaccount DOI http://dx.doi.org/10.1590/1981-5344/41347 Seção: artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: ontoaccount DOI http://dx.doi.org/10.1590/1981-5344/41347 nível para fundamentar o modelo da OntoAccount. Justifica-se a opção pela BFO e pelo paradigma do realismo científico pelas características deste domínio, que para sua descrição verdadeira necessita representar fenômenos e objetos reais. Sendo assim, a OntoAccount surge como uma ontologia que busca descrições verdadeiras (ou aproximadamente verdadeiras) das entidades do domínio da tomada e prestação de contas. A ontologia de fundamentação foi importada para o Protégé 5.5.0 para início da formalização. Como referência para a implementação do BFO, utilizou-se o Basic Formal Ontology 2.0: Specification and User’s Guide (SMITH et al., 2015). f) Etapa 5: formalização f) Etapa 5: formalização Nesta fase as estruturas conceituais do domínio, tratadas anteriormente no nível textual, passaram por uma série de restrições ontológicas. A metodologia OntoForInfoScience considera três passos no processo de construção da taxonomia da ontologia em desenvolvimento, são eles: • Passo 1: Classificar cada conceito do conjunto de modelos conceituais produzidos na etapa de conceitualização. No desenvolvimento da OntoAccount, os conceitos contidos nos artefatos gráficos e textuais foram organizados a fim de contrastá-los com as classes equivalentes existentes em repositórios de ontologia. • Passo 2: Promover o reuso de classe de outras ontologias que represente o significado do conceito no domínio. Quando nenhuma classe é encontrada, deve-se criar uma classe na ontologia em desenvolvimento. Na construção da OntoAccount, o autor realizou buscas de recursos ontológicos por meio da ferramenta Web OntoBee8. Além dos termos da ontologia de fundamentação (BFO), foram importadas classes e axiomas associados de outras ontologias de nível médio. Na construção da OntoAccount, utilizou-se classes originalmente definidas na Information Artifact Ontology (IAO), na NCBI organismal classification (NCBITaxon), na Obstetric and Neonatal Ontology Ontolegis, aplicada à informação legislativa para o Direito Médico. Disponível em: http://mba.eci.ufmg.br/downloads/BFO-PT/BFO-PT.owl. Acesso em: 28 nov 2021. 8 Um servidor de dados conectados projetado para promover o reuso de ontologias. Disponível em: http://www.ontobee.org/. Acesso em: 18 Nov. 2021. 13 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 Reuber da Silva Fonseca; Gercina Angela Lima (ONTONEO), na Ontology for Biomedical Investigations (OBI) e na Ontology of Medically Related Social Entities (OMRSE). • Passo 3 – Uma vez criada ou identificada a classe equivalente, a metodologia OntoForInfoScience determina a inclusão desta classe na taxonomia da ontologia em construção. Na construção da OntoAccount, cada classe específica do domínio foi classificada como um tipo de classe BFO e inserida como uma subclasse da classe superior. Na atividade de definição das propriedades descritivas (textuais) das classes, a metodologia OntoForInfoScience motiva o uso da Tabela de Conceitos e Propriedades como base desta tarefa, contudo alerta para a necessidade de se adequar as propriedades ao nível ontológico, tendo em vista se tratar de definição livre e textual (MENDONÇA, 2015). Nesse passo de construção da OntoAccount, foram inseridas as propriedades textuais por meio da funcionalidade “Annotations” do Protégé 5.5.0. A Figura 1 exemplifica as propriedades descritivas para a classe “organização pública”. Figura 1 - propriedades descritivas da classe “organização pública” no protégé 5.5.0. 9 A OntoForInfoScience orienta a utilizar no nome de classe (label) apenas letras minúsculas e sem acentos gráficos. Contudo, não se verificou incompatibilidade na manutenção de acentos gráficos em nomes de classes originados da língua portuguesa. 10 Os dados inseridos na ontologia, para efeito de testes e avaliação, são dados não sensíveis, de origem pública e não requerem autorização prévia para exibição conforme artigo 7º, parágrafos 3º, 4º e 7º da Lei nº 13.709/2018 (Lei Geral de Proteção de Dados Pessoais - LGPD), resguardados os direitos do titular e os princípios previstos nesta Lei. Como forma de proteção, os dados do Cadastro de Pessoas Físicas (CPF) foram inseridos num formato anonimizado. Por exemplo, o CPF 123.456.789-00, se tornou *.456.789-. Já o nome próprio dos gestores públicos foi inserido no mesmo formato tornado público pelo próprio titular, em documentos de prestação de contas, ou pela Administração Pública, tendo em vista os princípios da publicidade e da transparência, além do direito de informação dos cidadãos. f) Etapa 5: formalização Figura 1 - propriedades descritivas da classe “organização pública” no protégé 5.5.0. Fonte: Elaborado pelo autor (2022). Fonte: Elaborado pelo autor (2022). Fonte: Elaborado pelo autor (2022). A classe “organização pública” recebeu atributos ontológicos associados aos seguintes atributos do Protégé: nome de classe (label), definição (definition), comentários (comments) e termo alternativo (alternative 14 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contínuo, 2023: e-41347 Seção: artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: ontoaccount DOI http://dx.doi.org/10.1590/1981-5344/41347 Seção: artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: ontoaccount DOI http://dx.doi.org/10.1590/1981-5344/41347 term). O preenchimento dos atributos foi realizado no formato string e manteve-se os acentos gráficos, inclusive no nome de classe9. Na atividade de definição formal das classes da ontologia a OntoForInfoScience prescreve uma série de restrições ontológicas que objetivam viabilizar processos de inferência à base de conhecimento da ontologia. Estes procedimentos foram adotados nas classes da OntoAccount e um dos resultados podem ser observados no Quadro 2, a seguir. Quadro 2 – exemplo da definição formal da classe “organização” da Ontoaccount Termo (Organização Pública) Definição textual Uma organização que é portadora do papel de gestão pública e tem membro algum agente público. Anotação Uma organização [OBI/ONTOACCOUNT: organização] que é portadora do papel de gestão pública [RO/ONTOACCOUNT: tem papel], e tem membro algum agente público. [BFO/ONTOACCOUNT: tem membro]. Definição formal (OWL-DL) Organização pública é um organização E (tem papel SOME papel de gestão pública) E (tem membro SOME agente público) Fonte: Elaborado pelo autor (2022). Quadro 2 – exemplo da definição formal da classe “organização” da Ontoaccount Termo (Organização Pública) No que tange à definição de propriedades de dados das classes, algumas restrições foram aplicadas. Uma amostra do resultado é apresentada a seguir (Quadro 3). Quadro 3 – parte das propriedades de dados da ontoaccount ID Propriedades de dados Valor 147 'tem ano de atuação' Value = integer 134 'tem cargo' Value = string 164 'tem data sessão' Value = dateTime 403 'tem dirigente UCI' Value = string 118 'tem entidade' Value = string 131 'tem início de exercício' Value = dateTime 115 'tem nome próprio' Value = string 149 'tem relator' Value = string 396 'tem tipo' Value = string Fonte: Elaborado pelo autor (2022). Perspectivas em Ciência da Informação, Belo Horizonte, v. f) Etapa 5: formalização 28, Fluxo Contínuo, 2023: e-41347 Reuber da Silva Fonseca; Gercina Angela Lima A criação de instâncias das classes é mais um procedimento previsto na etapa de formalização da ontologia, a OntoAccount foi instanciada com dados abertos estruturados10, que representam objetos específicos do domínio da tomada e prestação de contas. Entre eles, destacam-se, como exemplo, os particulares da classe ‘órgão de controle interno’ visualizados na Figura 2. Figura 2 – exemplos de instâncias da classe ‘órgão de controle interno’ da ontoaccoun Fonte: Elaborado pelo autor (2022). Fonte: Elaborado pelo autor (2022). Fonte: Elaborado pelo autor (2022). No escopo da especificação de relações ontológicas, foram incorporadas na OntoAccount algumas relações ontológicas específicas do domínio da tomada e prestação de contas, conforme o Quadro 4, a seguir. Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contínuo, 2023: e-41347 16 de 21 Quadro 4 – parte das relações específicas do domínio na Ontoaccount Relação Origem Definição informal C julga C1 OntoAccount Relação ontológica identificada entre dois continuantes, C e C1, sendo que C é um órgão de controle externo que julga o processo de contas que corresponde a uma organização pública C1. C é julgado por C1 OntoAccount Relação ontológica (inversa) identificada entre dois continuantes, C e C1, sendo que C é uma organização pública que tem as contas julgadas por um órgão de controle externo C1. C é apresentadora de contas C1 OntoAccount Relação ontológica identificada entre dois continuantes, C e C1, sendo que C é uma unidade apresentadora de contas que apresenta a prestação de contas correspondente a uma unidade prestadora de contas C1. C tem apresentadora de contas C1 OntoAccount Relação ontológica (inversa) identificada entre dois continuantes, C e C1, sendo que C é uma unidade prestadora de contas que tem sua prestação de contas corresponde apresentada por uma unidade apresentadora de contas C1. C tem responsável C1 OntoAccount Relação ontológica identificada entre dois continuantes, C e C1, sendo que C é uma unidade prestadora de contas que tem como responsável um gestor público C1. C é responsável por C1 OntoAccount Relação ontológica (inversa) identificada entre dois continuantes, C e C1, sendo que C é um gestor público responsável pela prestação de contas corresponde a uma unidade prestadora de contas C1. Fonte: Elaborado pelo autor (2022). Fonte: Elaborado pelo autor (2022). Por fim, as propriedades das relações ontológicas foram estabelecidas no Protégé 5.5.0. Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 f) Etapa 5: formalização O conjunto completo de relações e propriedades da ontologia compõe a documentação da ontologia. g) Etapa 6: avaliação g) Etapa 6: avaliação g) Etapa 6: avaliação O sexto estágio envolve a avaliação da ontologia com os objetivos de validação (adequação ao domínio) como de verificação ontológica (adequação interna). Na metodologia proposta, um conjunto de critérios avaliativos, consistente e suficiente, para avaliar o conteúdo da ontologia, tanto do ponto de vista da validação quanto da verificação ontológicas foi sugerido. As bases destes critérios remetem aos princípios ontológicos e às regras de avaliação de outras metodologias tradicionais de construção de ontologias (por exemplo, NeOn, Methontology e o 101). O resultado da avaliação da ontologia OntoAccount, utilizando-se tais parâmetros e critérios, também compõe a documentação formal da ontologia. 17 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 Reuber da Silva Fonseca; Gercina Angela Lima h) Etapa 7: documentação Nesta etapa foi concatenada a documentação formal da ontologia produzida ao longo de todo o processo. O documento formal inclui todos os produtos das etapas anteriores e é oferecido em linguagem natural. Ficaram de fora da documentação formal os artefatos produzidos com representações preliminares, isto é, aqueles que sofreram transformações ao longo do processo, tais como: a) o pré-glossário de termos; b) os conjuntos de candidatos à ontologia; e c) a tabela de conceitos e valores. i) Etapa 8: disponibilização i) Etapa 8: disponibilização A última versão da OntoAccount foi disponibilizada na Web em linguagem de lógica descritiva, no formato OWL/XML, para que o seu conteúdo ontológico possa ser interpretado por máquinas de inferências e para que seus recursos possam ser consumidos na Web. A escolha por este formato se deu pela familiaridade do autor com o editor Protégé 5.5.0, sendo este o formato padrão do arquivo criado nesta ferramenta. Essa escolha também foi motivada pela aparente popularidade deste formato em ontologias de domínio. Por meio do Protégé 5.5.0, foi registrado um identificador de recurso à ontologia, um Internationalized Resource Identifier (IRI)11, por meio do qual os usuários podem fazer referências ao conteúdo da ontologia. A OntoAccount também recebeu um endereço Web12 e uma homepage com interface de busca ao conteúdo ontológico, que permite conhecer seus elementos (classes, propriedades e indivíduos) a fim de permitir a reutilização de seus termos em outros projetos. O documento de especificação, inserido no Quadro 5, a seguir, reúne as dimensões básicas da arquitetura e do propósito da OntoAccount. Quadro 5 – especificação da ontologia Ontoaccount 1 Namespace https://purl.archive.org/ontoaccount/ 2 IRI https://purl.archive.org/ontoaccount/ontoaccount.owl 3 Identificador local/ Local Identifier https://purl.archive.org/ontoaccount/<PREFIX>_<9999999> 4 PREFIX or IDSPACE ONTOACCOUNT 5 Website https://ontoaccount.org/ 6 Idioma padrão/ Default language Português 18 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contín Quadro 5 – especificação da ontologia Ontoaccount 1 Namespace https://purl.archive.org/ontoaccount/ 2 IRI https://purl.archive.org/ontoaccount/ontoaccount.owl 3 Identificador local/ Local Identifier https://purl.archive.org/ontoaccount/<PREFIX>_<9999999> 4 PREFIX or IDSPACE ONTOACCOUNT 5 Website https://ontoaccount.org/ 6 Idioma padrão/ Default language Português 11 Disponível em: https://purl.archive.org/purl/ontoaccount/ontoaccount.owl. Acesso em: 12 dez 2021. 12 Disponível em: https://ontoaccount.org/. Acesso em: 12 dez 2021. 18 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 23, Fluxo Contínu Quadro 5 – especificação da ontologia Ontoaccount 1 Namespace https://purl.archive.org/ontoaccount/ 2 IRI https://purl.archive.org/ontoaccount/ontoaccount.owl 3 Identificador local/ Local Identifier https://purl.archive.org/ontoaccount/<PREFIX>_<9999999> 4 PREFIX or IDSPACE ONTOACCOUNT 5 Website https://ontoaccount.org/ 6 Idioma padrão/ Default language Português 11 Disponível em: https://purl.archive.org/purl/ontoaccount/ontoaccount.owl. Acesso em: 12 dez 2021. 12 Disponível em: https://ontoaccount.org/. Acesso em: 12 dez 2021. Perspectivas em Ciência da Informação, Belo Horizonte, v. i) Etapa 8: disponibilização 23, Fluxo Contínuo, 2023: e-41347 artigos – Uma ontologia de domínio para a prestação de contas dos gestores públicos federais: ontoaccount DOI http://dx.doi.org/10.1590/1981-5344/41347 7 Idioma alternativo/ Alternative language English 8 Licença / License Creative Commons CC-BY license version 4.0 9 Ontologia de alto nível base / Top- level ontology Basic Formal Ontology version 2.0 10 Domínio e Escopo geral/ Domain and General Scope A Ontology of Accountability (OntoAccount) é uma ontologia de domínio que representa o conhecimento relativo à tomada e à prestação de contas anual dos gestores públicos federais. Seu escopo geral abrange os elementos constituintes do processo tomada e de prestação de contas anual dos gestores públicos federais. 11 Propósito Geral/ General Purpose A OntoAccount tem como propósito oferecer suporte ao desenvolvimento de aplicações cívicas sobre o domínio da tomada e da prestação de contas anual dos gestores públicos federais. Esta ação poderá auxiliar no aumento da transparência e na maior participação do cidadão na atividade de controle social. 12 Classes de usuários/ User classes A OntoAccount destina-se a desenvolvedores de aplicações cívicas e profissionais que trabalham em processos de abertura de dados, transparência ativa e controle da gestão pública. 13 Uso pretendido/ Intended use O uso geral pretendido com a OntoAccount é no suporte à modelagem de conhecimento sobre o domínio da tomada e da prestação de contas anual dos gestores públicos federais. 14 Tipo da ontologia/ Ontology type A OntoAccount é classificada como ontologia de domínio 15 Grau de formalidade/ Degree of formality A OntoAccount está representada na linguagem OWL-DL, considerada de médio rigor formal. Fonte: elaborado pelo autor (2022). Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 CONSIDERAÇÕES FINAIS Este estudo se baseia na premissa de que a ausência de modelagem semântica formal e explícita do domínio da tomada e da prestação de contas anual dos gestores públicos federais prejudica o consumo dos dados públicos por agentes que queiram assimilar o valor público gerado. Esta ausência gera barreiras ao avanço da participação e do controle social. Deste modo, esta pesquisa buscou contribuir para a publicação de dados abertos estruturados e semânticos deste domínio por meio de uma solução ontológica, nomeada OntoAccount, que atribuiu sentido e significado ao processo e ao conteúdo dos relatórios da amostra. Esta iniciativa oportuniza o reuso de recursos e a troca de informação e conhecimento entre pessoas e aplicações. Como se verificou na metodologia apresentada, a ontologia foi desenvolvida em etapas estruturadas que se somam e se apoiam para garantir a recuperação e a reutilização de conhecimento do domínio. Recomenda-se para trabalhos futuros a exploração de soluções de extração 19 de 21 Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 Perspectivas em Ciência da Informação, Belo Horizonte, v. 28, Fluxo Contínuo, 2023: e-41347 Reuber da Silva Fonseca; Gercina Angela Lima automática de dados de documentos do domínio para contribuir para a publicação de dados abertos estruturados que podem instanciar a estrutura básica da ontologia de modo a permitir seu uso como base de conhecimento. automática de dados de documentos do domínio para contribuir para a publicação de dados abertos estruturados que podem instanciar a estrutura básica da ontologia de modo a permitir seu uso como base de conhecimento. 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https://openalex.org/W4308545195	https://link.springer.com/content/pdf/10.1007/s00466-022-02236-0.pdf	English	null	Correction: An isogeometric finite element formulation for frictionless contact of Cosserat rods with unconstrained directors	Computational mechanics	2,022	cc-by	511	Computational Mechanics (2023) 71:349 https://doi.org/10.1007/s00466-022-02236-0 Computational Mechanics (2023) 71:349 https://doi.org/10.1007/s00466-022-02236-0 CORRECTION CORRECTION Correction: An isogeometric ﬁnite element formulation for frictionless contact of Cosserat rods with unconstrained directors Myung-Jin Choi1 · Sven Klinkel1 · Roger A. Sauer2,3,4 Published online: 8 November 2022 © The Author(s) 2022 Published online: 8 November 2022 © The Author(s) 2022 Published online: 8 November 2022 © The Author(s) 2022 Correction to: Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- tation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indi- cate 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 permitteduse,youwillneedtoobtainpermissiondirectlyfromthecopy- right holder. To view a copy of this licence, visit http://creativecomm ons.org/licenses/by/4.0/. Computational Mechanics In the original publication of the article typesetters have induced errors as given below: Algorith 3 Prime symbol was induced In Appendix C, there is no space between the caption of Fig. 36 and this section title (C.1.2 Case 2) C i f Fi 30 d Fi 31 i l d Algorith 3 Prime symbol was induced In Appendix C, there is no space between the caption of Fig. 36 and this section title (C.1.2 Case 2) Caption of Fig. 30 and Fig. 31 is overlapped. These corrections have been corrected now. Algorith 3 Prime symbol was induced In Appendix C, there is no space between the caption of Fig. 36 and this section title (C.1.2 Case 2) Caption of Fig. 30 and Fig. 31 is overlapped. These corrections have been corrected now. These corrections have been corrected now. Publisher’s Note Springer Nature remains neutral with regard to juris- dictional claims in published maps and institutional afﬁliations. The original article can be found online at https://doi.org/10.1007/ s00466-022-02223-5. B Myung-Jin Choi choi@lbb.rwth-aachen.de Sven Klinkel klinkel@lbb.rwth-aachen.de Roger A. Sauer sauer@aices.rwth-aachen.de 1 Chair of Structural Analysis and Dynamics, RWTH Aachen University, Mies-van-der-Rohe Str. 1, 52074 Aachen, Germany 2 Aachen Institute for Advanced Study in Computational Engineering Science (AICES), RWTH Aachen University, Templergraben 55, 52062 Aachen, Germany 3 Faculty of Civil and Environmental Engineering, Gda´nsk University of Technology, ul. Narutowicza 11/12, 80-233 Gda´nsk, Poland 4 Department of Mechanical Engineering, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India The original article can be found online at https://doi.org/10.1007/ s00466-022-02223-5. 1 Chair of Structural Analysis and Dynamics, RWTH Aachen University, Mies-van-der-Rohe Str. 1, 52074 Aachen, Germany 2 Aachen Institute for Advanced Study in Computational Engineering Science (AICES), RWTH Aachen University, Templergraben 55, 52062 Aachen, Germany 3 Faculty of Civil and Environmental Engineering, Gda´nsk University of Technology, ul. Narutowicza 11/12, 80-233 Gda´nsk, Poland 4 Department of Mechanical Engineering, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India 123 123
https://openalex.org/W4200262990	https://www.frontiersin.org/articles/10.3389/fped.2021.796143/pdf	English	null	Vascular Endothelial Growth Factor Signaling in Models of Oxygen-Induced Retinopathy: Insights Into Mechanisms of Pathology in Retinopathy of Prematurity	Frontiers in pediatrics	2,021	cc-by	7,035	MINI REVIEW published: 09 December 2021 doi: 10.3389/fped.2021.796143 MINI REVIEW MINI REVIEW published: 09 December 2021 doi: 10.3389/fped.2021.796143 published: 09 December 2021 doi: 10.3389/fped.2021.796143 Edited by: Alison Chu, University of California, Los Angeles, United States y g United States Reviewed by: Paola Bagnoli, University of Pisa, Italy Reviewed by: Paola Bagnoli, University of Pisa, Italy Reviewed by: Paola Bagnoli, University of Pisa, Italy Correspondence: M. Elizabeth Hartnett me.hartnett@hsc.utah.edu Keywords: ROP, OIR, VEGF, VEGFRs, VEGF receptors, neuropilins Specialty section: This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics Specialty section: This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics Received: 16 October 2021 Accepted: 10 November 2021 Published: 09 December 2021 Vascular Endothelial Growth Factor Signaling in Models of Oxygen-Induced Retinopathy: Insights Into Mechanisms of Pathology in Retinopathy of Prematurity Aniket Ramshekar and M. Elizabeth Hartnett Department of Ophthalmology and Visual Sciences, John A. Moran Eye Center, University of Utah, Salt Lake City, UT, United States Retinopathy of prematurity (ROP) is a leading cause of blindness in children worldwide. Blindness can occur from retinal detachment caused by pathologic retinal angiogenesis into the vitreous, termed intravitreal neovascularization (IVNV). Although agents that interfere with the bioactivity of vascular endothelial growth factor (VEGF) are now used to treat IVNV, concerns exist regarding the identiﬁcation of optimal doses of anti-VEGF for individual infants and the effect of broad VEGF inhibition on physiologic angiogenesis in external organs or in the retina of a preterm infant. Therefore, it is important to understand VEGF signaling in both physiologic and pathologic angiogenesis in the retina. In this manuscript, we review the role of receptors that interact with VEGF in oxygen-induced retinopathy (OIR) models that represent features of ROP pathology. Speciﬁcally, we discuss our work regarding the regulation of VEGFR2 signaling in retinal endothelial cells to not only reduce severe ROP but also facilitate physiologic retinal vascular and neuronal development. INTRODUCTION Retinopathy of prematurity (ROP) remains a leading cause of blindness in children worldwide despite advances in neonatal care (1). The pathophysiology of ROP is described by a two-phase hypothesis that has been reﬁned with the ability to save extremely premature infants (2). In Phase I ROP, intraretinal vascularization is compromised, and ongoing physiologic vascular development is delayed leading to areas of hypoxic retina. In Phase II ROP, also classiﬁed as Stage 3 ROP (3), aberrant retinal angiogenesis grows into the vitreous and is termed intravitreal neovascularization (IVNV). IVNV leads to blindness from retinal detachment that is not, or cannot be, treated (4, 5). Currently, Phase II ROP is treated with methods to ablate the peripheral avascular retina, often with laser (6), or with intravitreal agents that interfere with the bioactivity of vascular endothelial growth factor (VEGF) (7–11). However, broad inhibition of VEGF in preterm infants might interfere with physiologic angiogenesis in external organs or in the developing retina where it can lead to persistent avascular retina and recurrent IVNV (9). Understanding VEGF-mediated molecular Received: 16 October 2021 Accepted: 10 November 2021 Published: 09 December 2021 Keywords: ROP, OIR, VEGF, VEGFRs, VEGF receptors, neuropilins Citation: Ramshekar A and Hartnett ME (2021) Vascular Endothelial Growth Factor Signaling in Models of Oxygen-Induced Retinopathy: Insights Into Mechanisms of Pathology in Retinopathy of Prematurity. Front. Pediatr. 9:796143. doi: 10.3389/fped.2021.796143 December 2021 \| Volume 9 \| Article 796143 Frontiers in Pediatrics \| www.frontiersin.org 1 Ramshekar and Hartnett VEGF Signaling in OIR neutralizing antibodies to rat VEGFA compared with IgG signiﬁcantly reduced IVNV in a dose-dependent manner without interfering with physiologic retinal vascular development at p18 in rat pups. However, IVNV and avascular retina area within the vascularized retina were signiﬁcantly increased at p25 in rat pups that received an eﬀective dose of anti-VEGFA (25). A VEGF-Trap, which binds VEGFA and PlGFs, was compared with a human Fc control after intravitreal injection at p8 in beagle pups. At p21, both IVNV and physiologic retinal vascular development were reduced at high doses of the VEGF- Trap compared with control. However, the lowest dose (5 µg) of the VEGF-Trap reduced IVNV but not physiologic retinal vascular development (26). Taken together, these studies provide experimental evidence that anti-VEGF agents can interfere with physiologic retinal vascular development, compromise already developed retinal vasculature, and lead to recurrent IVNV at certain doses. Therefore, studies were warranted to reﬁne the dose of anti-VEGF agents that would inhibit IVNV and permit suﬃcient VEGF expression at a concentration that allows physiologic vascular development of the peripheral retina. In support of this notion, Müller cells or astrocytes in the retina were demonstrated to overproduce VEGFA implicated in the development of IVNV in the murine OIR model (27–29). In rat pups raised in the OIR model, novel approaches to knock down VEGFA or VEGFA164 in Müller cells by subretinal introduction of lentiviral vectors that contained a CD44 promoter upstream of an miR-30-based shRNA cassette signiﬁcantly reduced IVNV at p18 (30, 31) without recurrence at p32 (32). However, lentiviral- mediated knockdown of Müller cell-derived VEGFA thinned the retinal outer nuclear layer compared with knockdown of Müller cell-derived VEGFA164 by lentiviral vectors (30). Although the data supported the hypothesis that an optimal anti-VEGF dose will not interfere with physiologic vascular development of the peripheral retina, identifying this dose in infants might be challenging due to variation of pathology among individual infants or eyes (33). Nonetheless, the data support the involvement of VEGFA in the pathophysiology of ROP and physiologic development of retinal vasculature, neurons, and glia. Citation: In this article, we discuss VEGFA signaling through diﬀerent receptors in models of ROP to identify mechanisms involved in the Phases of ROP pathology and provide insights into novel therapeutic approaches for ROP that overcome limitations in identifying optimal doses of antiangiogenic agents for individual infants. mechanisms involved in IVNV and physiologic vascular development of the peripheral retina is important to identify safe and eﬀective treatment targets. g To understand the role of VEGF in the pathophysiology of ROP, studies were conducted using animal models of oxygen- induced retinopathy (OIR) that recapitulate features of ROP pathology in preterm infants. The most common models were in mouse, rat, and beagle (5). The models diﬀer based on the extent of inner vascular plexus coverage to the ora serrata at the time animals are placed into the model, oxygen levels, duration of exposure to oxygen, the age when animals are placed into the model, and the features of ROP represented by in the model. In the murine OIR model, mice are born and raised in room air until postnatal day (p)7 when intraretinal vascularization of the inner plexus extends to the ora serrata. At p7, mice are placed into 75% oxygen for 5 days, which causes hyperoxia-induced compromise of the developed inner plexus in the central retina surrounding the optic nerve (vaso- obliteration). Mice are returned to room air and develop preretinal neovascular tufts (IVNV) at the junction of the vascular and avascular retina at p17 (Phase II) (12). In the rat model, newborn rat pups with almost no retinal vascular development are exposed to oxygen extremes that ﬂuctuate between 50% and 10% every 24 h for 14 days. At p14, rats have compromised physiologic vascularity and delayed physiologic retinal vascular development (Phase 1). Pups are placed into room air and develop IVNV at p18-20 (Phase II) (13). Although the mouse model is often used for ease of genetic manipulation, the rat OIR model best represents human ROP based on oxygen stresses similar to those in preterm infants (ﬂuctuations in oxygen and changes in extremes of arterial oxygen), similar appearing Phases in ROP (Phase I compromise in physiologic vascularization and delay in physiologic vascular development of the peripheral retina at p14, and Phase II IVNV, vascular tortuosity, and vascular dilation at p18-20), and extrauterine growth restriction (Figure 1). Citation: In the beagle OIR model, newborn pups at p1 are placed into 100% oxygen for 4 days and, at p5, are returned to room air. The beagle OIR model develops delayed physiologic retinal vascular development and compromised physiologic vascularity (Phase I) and IVNV (Phase II) that have been measured at p15 and observed until p45 (14–16). This OIR model is useful to assess pharmacologic treatments due to increased eye size in beagles compared with rodents. There are ﬁve members of the VEGF family: VEGFA, placental growth factors (PlGFs), VEGFB, VEGFC, and VEGFD (17). Studying the role of VEGF in the murine OIR model is diﬃcult since a single allele knockout of VEGF or VEGF receptors (VEGFRs) is lethal in mice (18–20). Although transgenic mice lacking VEGFB (Vegfb−/−) are viable, no diﬀerence was observed in IVNV compared with littermate wild-type mice (21). In rat pups raised in OIR compared with room air, retinal VEGFA protein was signiﬁcantly increased and, mainly, VEGFA splice variant 164 (VEGFA164) mRNA at p14 and p18 (22–24). These ﬁndings implicated VEGFA in both physiologic retinal vascular development and IVNV. Therefore, broad inhibition of VEGFA was predicted to reduce both. Surprisingly, intravitreal Frontiers in Pediatrics \| www.frontiersin.org The Role of Vascular Endothelial Growth Factor Receptor 1 in Models of Retinopathy of Prematurity of retinal sections from mice in the OIR model demonstrated colocalization of von Willebrand factor-labeled IVNV and VEGFR2, but not VEGFR1, at p19 (36). Retinal lysates from p18 rat pups raised in the OIR model had increased VEGFR2 mRNA, but not VEGFR1 mRNA, compared with p18 pups raised in room air (24). Immunostaining of retinal sections from rats raised in OIR demonstrated immunolabeling of VEGFR1 and VEGFR2 in areas of IVNV at p20 (37). Colocalization of VEGFR2 and von Willebrand factor-stained IVNV was also observed in retinal sections from p15 dogs raised in OIR (16). Speciﬁcally, immunostaining of phosphorylated VEGFR2 was reduced in retinal sections from p13 rats that were raised in rat OIR and treated with intravitreal antibodies against VEGFA compared with IgG (23). These ﬁndings primarily implicated VEGFR2 in the pathophysiology of ROP; however, this review will summarize studies regarding the role of VEGFR1 and VEGFR2 in models of ROP. of retinal sections from mice in the OIR model demonstrated colocalization of von Willebrand factor-labeled IVNV and VEGFR2, but not VEGFR1, at p19 (36). Retinal lysates from p18 rat pups raised in the OIR model had increased VEGFR2 mRNA, but not VEGFR1 mRNA, compared with p18 pups raised in room air (24). Immunostaining of retinal sections from rats raised in OIR demonstrated immunolabeling of VEGFR1 and VEGFR2 in areas of IVNV at p20 (37). Colocalization of VEGFR2 and von Willebrand factor-stained IVNV was also observed in retinal sections from p15 dogs raised in OIR (16). Speciﬁcally, immunostaining of phosphorylated VEGFR2 was reduced in retinal sections from p13 rats that were raised in rat OIR and treated with intravitreal antibodies against VEGFA compared with IgG (23). These ﬁndings primarily implicated VEGFR2 in the pathophysiology of ROP; however, this review will summarize studies regarding the role of VEGFR1 and VEGFR2 in models of ROP. Intraperitoneal administration of antibodies against VEGFR1 compared with IgG in mice reduced IVNV in mice placed in OIR (38, 39). However, intravitreal PlGF1, a VEGFR1- speciﬁc ligand, resulted in no diﬀerence in IVNV compared with buﬀered salt solution control even though previous investigators reported reduced IVNV after intravitreal neutralizing antibody to VEGFR1 (40). The disparity in studies might be because PlGF1 does not bind VEGFR2 monomers (41), and VEGFR2- related signaling is important in IVNV (see The role of vascular endothelial growth factor receptor 2 in models of retinopathy of prematurity section). THE ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS IN MODELS OF RETINOPATHY OF PREMATURITY VEGF members bind to VEGF receptors (VEGFRs), which induce receptor homodimerization or heterodimerization and activation through autophosphorylation of the tyrosine residues in the receptor intracellular domains (34). There are three subtypes of VEGFRs, but VEGFA binds VEGFR1 or VEGFR2 to elicit biologic functions (35). Immunohistochemical staining December 2021 \| Volume 9 \| Article 796143 2 Ramshekar and Hartnett VEGF Signaling in OIR FIGURE 1 \| Schematic representation of similarities between human retinopathy of prematurity (ROP) and oxygen-induced retinopathy (OIR) models. Human ROP is described by a two-phase hypothesis (row 1). In Phase I, events surrounding preterm birth (i.e., lack of maternally derived factors, relative hyperoxia, repeated oxygen ﬂuctuations, poor infant growth, etc.) cause a delay in physiologic retinal vascular development (PRVD) and compromise to already developed vessels (compromised physiologic vascularity). In Phase II, the hypoxic avascular retina releases pro-angiogenic factors that promote aberrant retinal angiogenesis into the vitreous termed intravitreal neovascularization (IVNV). The murine OIR model (row 2) recapitulates Phase I compromised physiologic vascularity and has been termed vaso-obliteration at p12, and Phase II IVNV at p17. The rat OIR model (row 3) recapitulates Phase I delay in PRVD to the peripheral retina and compromised physiologic vascularity at p14, and Phase II IVNV and vessel tortuosity and dilation at p18-20. Created with Biorender.com. FIGURE 1 \| Schematic representation of similarities between human retinopathy of prematurity (ROP) and oxygen-induced retinopathy (OIR) models. Human ROP is described by a two-phase hypothesis (row 1). In Phase I, events surrounding preterm birth (i.e., lack of maternally derived factors, relative hyperoxia, repeated oxygen ﬂuctuations, poor infant growth, etc.) cause a delay in physiologic retinal vascular development (PRVD) and compromise to already developed vessels (compromised physiologic vascularity). In Phase II, the hypoxic avascular retina releases pro-angiogenic factors that promote aberrant retinal angiogenesis into the vitreous termed intravitreal neovascularization (IVNV). The murine OIR model (row 2) recapitulates Phase I compromised physiologic vascularity and has been termed vaso-obliteration at p12, and Phase II IVNV at p17. The rat OIR model (row 3) recapitulates Phase I delay in PRVD to the peripheral retina and compromised physiologic vascularity at p14, and Phase II IVNV and vessel tortuosity and dilation at p18-20. Created with Biorender.com. The Role of Vascular Endothelial Growth Factor Receptor 1 in Models of Retinopathy of Prematurity In support of this notion, Zeng et al. observed disordered divisions of mouse embryonic stem cell-derived vessels from VEGFR1 knockout mice (ﬂt1−/−) (42). VEGFR1 acts as a decoy receptor, and when knocked December 2021 \| Volume 9 \| Article 796143 Frontiers in Pediatrics \| www.frontiersin.org 3 Ramshekar and Hartnett VEGF Signaling in OIR out, it does not bind to VEGF, which permits more VEGF to trigger signaling through VEGFR2 (43). In line with this thinking, rescue of VEGFR1 expression in ﬂt1−/−embryonic stem cell-derived vessels, with a transgene that expressed soluble VEGFR1 under the guidance of a PECAM promoter, reduced randomized divisions of endothelial cells and increased ordered divisions (42). Similarly, in the rat OIR model, pups treated with intravitreal anti-VEGFA antibodies had signiﬁcantly more vascular cell divisions that favored vascular extension rather than widening (44). The studies provided strong evidence that regulation of VEGFR2 is important in orienting dividing endothelial cells and supports the hypothesis that ordered divisions extend peripheral vascular development that occurs in developing retina. The role of VEGFR1 activation in physiologic vascular development of the peripheral retina using a representative model of ROP remains unknown. in the dog OIR model (26) compared with respective controls. Taken together, the data support the thinking that VEGFA signaling is important for neural retinal structure and function, and normal retinal vascularization. Furthermore, regulation of VEGFR2 signaling in retinal endothelial cells accomplishes safe inhibition of IVNV while promoting physiologic retinal vascular development and retinal structure and function. The data also suggest that a certain dose or agent that regulates VEGF-mediated signaling triggered through VEGFR2 in retinal endothelial cells might be a possible therapeutic approach to inhibit IVNV, facilitate physiologic retinal vascular development, and reduce the likelihood of recurrent IVNV in ROP. THE ROLE OF NEUROPILINS IN MODELS OF RETINOPATHY OF PREMATURITY Originally identiﬁed in Xenopus tadpole nervous tissues (48) as receptors for semaphorins (49, 50), neuropilins are cell surface glycoproteins that bind to VEGF family members (51) and form complexes with VEGFRs as co-receptors (52). There are two isoforms of the protein, neuropilin 1 and neuropilin 2, and both have been demonstrated to interact with VEGFRs to trigger signaling induced by VEGFA. Also, VEGFA164 has been demonstrated to bind to neuropilin 1 and neuropilin 2 (53). Neuropilin 1 mRNA was increased in retinal lysates from mice placed in OIR compared with room air at p17 (54). Also at p17, retinal sections from mice placed in OIR demonstrated colocalization of neuropilin 1 mRNA with IVNV (55). Speciﬁcally, neuropilin 1 (54, 56) or neuropilin 2 (57) protein colocalized with IVNV. Furthermore, Budd et al. found signiﬁcantly increased neuropilin 1 and neuropilin 2 mRNA in rats raised in OIR compared with room air at p14 and p18 (58). The Role of Vascular Endothelial Growth Factor Receptor 2 in Models of Retinopathy of Prematurity p y y As indicated in the above studies (40, 42), evidence suggested a role for VEGFR2 in ROP. Further support was found in mice with signiﬁcantly reduced IVNV after gavage with an antagonist to VEGFRs and platelet-derived growth factor receptors (PDGFRs, PTK787) compared with selective PDGFR antagonists (CGP57148 or CGP53716) or vehicle control (45). Similarly, mice treated with a subcutaneous tyrosine kinase inhibitor (SU5416) had signiﬁcantly reduced IVNV. However, room air-raised mice treated with SU5416 compared with vehicle control had signiﬁcantly reduced intraretinal vascular extension of the inner plexus to the ora serrata and reduced total retinal thickness of the peripheral retina (46). OIR- raised dogs implanted with a pellet that released antibodies against VEGFR2 into the vitreous had signiﬁcantly reduced IVNV and delayed physiologic vascular development of the peripheral retina compared with pups implanted with a pellet that released IgG into the vitreous (16). Taken together, the data suggest that inhibition of VEGFR2 aﬀects both physiologic and pathologic retinal angiogenesis and retinal structure. Therefore, this approach might not be a safe therapy for ROP. In an eﬀort to regulate VEGFR2 signaling speciﬁcally in retinal endothelial cells, lentiviral vectors that expressed shRNA against VEGFR2 or luciferase control under the guidance of an endothelial-speciﬁc promoter, Cdh5, were tested in the rat OIR model. Knock down of VEGFR2 in endothelial cells by shRNA signiﬁcantly reduced IVNV and allowed more physiologic vascular development of the peripheral retina compared with littermate controls at p20. Furthermore, total retinal thickness near the optic nerve head was not thinned after lentiviral delivered Cdh5-targeted shRNA against VEGFR2 compared with littermate controls. There was also no diﬀerence in a- or b-wave amplitudes assessed by full- ﬁeld electroretinography in adult rats compared with littermate controls (47). These ﬁndings contrasted with earlier studies in which Müller cell-derived VEGFA knockdown by lentiviral vectors in the rat OIR led to retinal thinning, (32) and intravitreal VEGF-Trap delayed physiologic retinal vascular development Frontiers in Pediatrics \| www.frontiersin.org The Role of Neuropilin 1 in Models of Retinopathy of Prematurity / Neuropilin 1 knockout mice (Nrp1−/−) are embryonically lethal (59–61). Neutralizing neuropilin 1 with intravitreal antibody signiﬁcantly reduced IVNV compared with IgG in mouse OIR (55). Compared with littermate control mice that lacked Cre alleles, tamoxifen-inducible knock out of endothelial neuropilin 1 in a Cre-loxP mouse model reduced IVNV in mice in OIR and delayed intraretinal vascular development of the inner plexus in mice raised in room air (62). However, knock out of neuropilin 1 in myeloid lineage cells using LysM-Cre did not aﬀect intraretinal vascular development of the inner plexus in room air compared with mice that lacked the ﬂoxed Nrp1 alleles but still expressed LysM-Cre (63). These ﬁndings implicate endothelial neuropilin 1 not only in the development of IVNV but also in physiologic retinal vascular development. To understand mechanistically how neuropilin 1 regulates angiogenesis, transgenic mice that expressed a mutant neuropilin 1 that lacked the cytoplasmic domain of the receptor were generated (64). The cytoplasmic domain of neuropilin 1 has been reported to interact with VEGFR2 to enhance VEGFR2- mediated signaling (65–67). Therefore, expression of a mutant neuropilin 1 receptor that lacked the ability to interact with December 2021 \| Volume 9 \| Article 796143 4 Ramshekar and Hartnett VEGF Signaling in OIR VEGFR2 to trigger signaling might aﬀect intraretinal vascular development in mice. However, the study reported no diﬀerence in intraretinal vascular development of the inner plexus between room air raised mice that expressed a mutant neuropilin 1 and littermate control mice that expressed wild-type neuropilin 1 (64). To determine if VEGFA-binding neuropilin 1 was required for angiogenesis, transgenic mice that expressed a mutant neuropilin 1 with a point mutation in the VEGF- binding b1 domain (Nrp1Y297A/Y297A) were generated along with littermate wild-type controls. Nrp1Y297A/Y297A mice raised in room air had signiﬁcantly reduced intraretinal vascular extension of the inner plexus at p7 and reduced IVNV in OIR at p17 compared with age-controlled littermate wild-type mice (68). Taken together, these observations suggest that VEGF- binding endothelial neuropilin 1, but not the interaction between neuropilin 1 and VEGFR2, was required for intraretinal vascular development. However, further studies are required to determine the role of neuropilin 1 in physiologic vascular development of the peripheral retina and IVNV in translational models of ROP. molecules may aﬀect signaling in other cells in the retina and aﬀect function and structure or potentially leak into the circulation and aﬀect developing organs. The Role of Neuropilin 1 in Models of Retinopathy of Prematurity / However, the use of correct dose or agent suggests that reducing the bioactivity of VEGF may have value to permit some VEGF signaling important in physiologic vascular development of the peripheral retina (10, 69). An appropriate dose of anti-VEGF may regulate overactive VEGFR2 in retinal endothelial cells, which occurs with increased ligand concentration (23, 31), without abolishing VEGFR2 signaling in endothelial or other cells of the retina. Besides anti-VEGF, alternative approaches are being explored to prevent VEGF-mediated ROP occurrence and progression. Oxidative stresses (i.e., reactive oxygen species) have been implicated in VEGF-mediated IVNV in rodent models of ROP (70). Administration of antioxidants Cu/Zn superoxide dismutase (71) or vitamin E (72) in extremely low gestational age infants reduced the risk of ROP. However, side eﬀects related to vitamin E (73) preclude widespread use. Also, antioxidants may fail to access the intracellular signaling mechanisms leading to pathology or counteract beneﬁcial mechanisms of oxidative signaling. Therapeutic approaches have been considered to regulate hypoxia inducible factors, either stabilization with prolyl hydroxylase inhibitors in phase I (74, 75) or potential inhibition in phase II. It remains to be seen if the phases described in the two-phase hypothesis of ROP can be distinguished suﬃciently in an individual human infant. Another treatment approach is carefully monitoring oxygen tension at birth to prevent hyperoxia-induced damage to blood vessels and reduce oxygen ﬂuctuations that slow vascular growth to the peripheral retina (76). Additional experimental studies to regulate semaphorin/neuropilin signaling (77) might lead to future approaches in ROP. Overall, these approaches provide insights into possible therapeutic approaches to regulate VEGF-induced VEGFR2 signaling in ROP. The Role of Neuropilin 2 in Models of Retinopathy of Prematurity p y y Neuropilin 2 knockout mice (Nrp2−/−) had signiﬁcantly reduced IVNV in OIR compared with littermate wild- type mice; however, neuropilin 2 mRNA was expressed in mice raised in room air from p0 to p7 (57). Therefore, it was postulated that Nrp2−/−mice would have reduced intraretinal vascular development compared with littermate controls. However, there was no diﬀerence in inner plexus vascular density between Nrp2−/− mice and littermate wild-type mice raised in room air and analyzed at p7 (57). Taken together, the data suggest that neuropilin 2 is involved in IVNV but not required for intraretinal vascular development. Further studies are warranted in OIR models to determine the eﬀect on regrowth after hyperoxia and physiologic vascular development of the peripheral retina before considering neuropilin 2 as a potential therapeutic target for ROP. AUTHOR CONTRIBUTIONS AR and MEH performed the literature searches and drafted and critically revised the manuscript. MEH provided funding support. All authors contributed to the article and approved the submitted version. DISCUSSION ROP is the leading cause of blindness and visual impairment in children worldwide. In severe cases of ROP, blindness can occur from retinal detachment caused by IVNV. Studies in OIR models that recapitulate aspects of human ROP have provided insights into VEGF signaling through VEGFRs and neuropilins in speciﬁc cell types. Experimental studies support the ﬁnding that regulating oversignaling through VEGFR2, especially in retinal endothelial cells, would not only reduce severe ROP but also facilitate normal vascular development. However, there is no suitable way to target endothelial VEGFR2 in premature infants yet. Broad inhibition of VEGF or VEGFR2 using intravitreal neutralizing antibodies or small Frontiers in Pediatrics \| www.frontiersin.org REFERENCES Revised indications for the treatment of retinopathy of prematurity: results of the early treatment for retinopathy of prematurity randomized trial. Arch Ophthalmol. (2003) 121:1684–94. doi: 10.1001/archopht.121.12.1684 25. McCloskey M, Wang H, Jiang Y, Smith GW, Strange J, Hartnett ME. 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(2011) 100:343–53. doi: 10.1159/000330174 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. December 2021 \| Volume 9 \| Article 796143 Frontiers in Pediatrics \| www.frontiersin.org 8
https://openalex.org/W2263188776	https://europepmc.org/articles/pmc4724953?pdf=render	English	null	Defining the optimal cut-off values for liver enzymes in diagnosing blunt liver injury	BMC research notes	2,016	cc-by	4,278	Abstract Background: Patients with blunt trauma to the liver have elevated levels of liver enzymes within a short time post injury, potentially useful in screening patients for computed tomography (CT). This study was performed to define the optimal cut-off values for serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in patients with blunt liver injury diagnosed with contrast enhanced multi detector-row CT (CE-MDCT). Methods: All patients admitted from May 2006 to July 2013 to Teikyo University Hospital Trauma and Critical Care Center, and who underwent abdominal CE-MDCT within 3 h after blunt trauma, were retrospectively enrolled. Using receiver operating characteristic (ROC) curve analysis, the optimal cut-off values for AST and ALT were defined, and sensitivity and specificity were calculated. Results: Of a total of 676 blunt trauma patients 64 patients were diagnosed with liver injury (Group LI+) and 612 patients without liver injury (Group LI−). Group LI+ and LI− were comparable for age, Revised Trauma Score, and Probability of survival. The groups differed in Injury Severity Score [median 21 (interquartile range 9–33) vs. 17 (9–26) (p < 0.01)]. Group LI+ had higher AST than LI− [276 (48–503) vs. 44 (16–73); p < 0.001] and higher ALT [240 (92–388) vs. 32 (16–49); p < 0.001]. Using ROC curve analysis, the optimal cut-off values for AST and ALT were set at 109 U/l and 97 U/l, respectively. Based on these values, AST ≥ 109 U/l had a sensitivity of 81 %, a specificity of 82 %, a positive predictive value of 32 %, and a negative predictive value of 98 %. The corresponding values for ALT ≥ 97 U/l were 78, 88, 41 and 98 %, respectively, and for the combination of AST ≥ 109 U/l and/or ALT ≥ 97 U/l were 84, 81, 32, 98 %, respectively. Conclusions: We have identified AST ≥ 109 U/l and ALT ≥ 97 U/l as optimal cut-off values in predicting the presence of liver injury, potentially useful as a screening tool for CT scan in patients otherwise eligible for observation only or as a transfer criterion to a facility with CT scan capability. Keywords: Blunt liver trauma, Liver transaminase, CE-MDCT, ROC curve analysis, Youden index hemodynamically normal(ized) will undergo CT if avail- able. Although CT has become the “gold standard” for detecting injuries to the intraabdominal solid organs, CT is not always present in every institution worldwide, even in high-income countries such as Japan. BMC Research Notes BMC Research Notes Koyama et al. BMC Res Notes (2016) 9:41 DOI 10.1186/s13104-016-1863-3 © 2016 Koyama et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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 Additionally, there is evidence demonstrating that CT scanning carries a risk of causing malignancies and thus should be avoided when possible [4]. Defining the optimal cut‑off values for liver enzymes in diagnosing blunt liver injury Tomohide Koyama1,3, Hirohisa Hamada2, Masamichi Nishida2, Paal A. Naess3, Christine Gaarder3 and Tetsuya Sakamoto1 © 2016 Koyama et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Correspondence: t‑koyama@office.nifty.jp 1 Department of Emergency Medicine, Teikyo University Hospital, 2‑11‑1 Kaga, Itabashi, Tokyo, Japan Full list of author information is available at the end of the article Backgroundh The liver is one of the most commonly injured abdomi- nal organs and is reported in approximately 5 % of all trauma patients [1, 2]. Since computed tomography (CT) was introduced in trauma evaluation in the early 1980s [3], patients with a history of significant trauma who are *Correspondence: t‑koyama@office.nifty.jp 1 Department of Emergency Medicine, Teikyo University Hospital, 2‑11‑1 Kaga, Itabashi, Tokyo, Japan Full list of author information is available at the end of the article Ultrasound has significant limitations as a diagnos- tic tool since the overall sensitivity is as low as 72 % for Koyama et al. BMC Res Notes (2016) 9:41 Page 2 of 6 Page 2 of 6 Page 2 of 6 detecting blunt liver injury based on detection of free fluid, parenchymal injury or both [5]. CT was performed using a 64-slice MDCT scanner (Aquilion 64, TSX-101A/HA, Toshiba Medical Systems Corp., Japan) with intravenous contrast material (Omni- paque 300 injection syringe, Daiichi Sankyo Company (Co.), Limited (Ltd.), Tokyo, Japan or Oypalomin 300 injection syringe, Fuji Pharma Co., Ltd., Toyama, Japan) unless the patient was known to suffer from chronic kid- ney disease. CT was performed using a 64-slice MDCT scanner (Aquilion 64, TSX-101A/HA, Toshiba Medical Systems Corp., Japan) with intravenous contrast material (Omni- paque 300 injection syringe, Daiichi Sankyo Company (Co.), Limited (Ltd.), Tokyo, Japan or Oypalomin 300 injection syringe, Fuji Pharma Co., Ltd., Toyama, Japan) unless the patient was known to suffer from chronic kid- ney disease. l On that background the serum biomarkers such as serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) have received attention as mark- ers of liver injury. Several previous studies have tried to define the cut-off value for AST and/or ALT in blunt liver injury [6–15]. However, the results have been conflicting. We infer that the variations might be related to biases such as the population studied, the detection method for liver injury, timing of blood sampling, and statistical analysis method.h The liver injury was defined from CE-MDCT scans based on Organ Injury Scale (OIS, 1994 revision) described by the American Association for the Surgery of Trauma [17]. Attending staff reviewed the CE-MDCT at the following morning conference, and consensus was reached. The purpose of this study was to establish optimal cut- off values for AST and ALT in patients with blunt liver injury. Backgroundh Such values could be potentially useful to indi- cate the need for CT scan in patients otherwise eligible for observation only or as a transfer criterion to a facility with CT scan capability. Statistical analysis was performed using the IBM SPSS Statistics version 22 for MacOSX [International Business Machines Corp., New York, United States of America (USA)] and the Microsoft Excel for Mac 2011 (Micro- soft Corp., Washington, USA). Categorical variables were presented as medians and underwent Chi square test. Continuous variables were presented as median with interquartile range (IQR), and subjected to the Mann– Whitney U test. All p values reported are two-sided, and p values <0.05 were considered to indicate statistical significance. Methods Based on the results from a previous study from our institution published in a Japanese journal [16], all blunt trauma patients admitted to Teikyo University Hospital Trauma and Critical Care Center who underwent initial evaluation with abdominal contrast enhanced (CE) multi detector-row computed tomography (MDCT) within 3 h after injury, were retrospectively enrolled between May 2006 and July 2013. This study was approved by Teikyo University Hospital Ethics Committee. gi Receiver operating characteristic (ROC) curve analy- sis was performed to define the optimal cut-off values for AST and ALT [18]. Two additional analysis methods were used to determine the optimal cut-off values objec- tively. The first method was ‘The closest to (0, 1) criteria’, in this paper called ‘upper-left (UL) index’, and represents the values at the shortest distance from the upper left corner to the ROC curve. The second was ‘the Youden index’, which describes the maximum vertical distance between the ROC curve and the diagonal or chance line [19]. After determining the optimal cut-off values for AST and ALT with these methods, sensitivity and speci- ficity were calculated. Admission data collected included the following: age, gender, mechanism of injury, Glasgow Coma Scale (GCS), and Revised Trauma Score (RTS). All patients included in the study were followed throughout their hospital stay. Injury Severity Score (ISS), Probability of survival (Ps), interventions (laparotomy and/or angioem- bolization (AE)), liver related complications, and mortal- ity were recorded.h The admission values of AST and ALT were measured using LABOSPECT 008 Automatic Analyzer or Clinical Analyzer Model 7600 (both Hitachi High- Technologies Corporation (Corp.), Tokyo, Japan). Discussion had other abdominal injuries, 12 had pelvic injury, 18 had spinal injury, and 20 had extremity injury. In the present study, blood samples were drawn imme- diately upon arrival and CT scans performed within 3 h post injury. Based on these strict criteria and the use of UL index and Youden index, we defined AST ≥ 109 U/l and ALT ≥ 97 U/l as the optimal cut-off values in pre- dicting the presence of blunt liver injury. In Group LI+, 11 (17.2 %) patients underwent AE and 5 (7.8 %) underwent laparotomy. A total of 5 (7.8 %) patients developed liver related complications. Two patients had biloma, one was treated with percutaneous drainage, and one resolved spontaneously. One patient had bile leakage treated with surgical drainage, one patient had a pseudo-aneurysm treated with AE, and one patient had cholecystitis treated with percutaneous tran- shepatic gallbladder drainage. Shadev et al. [12] used ROC curve analysis to define cut off values for AST and ALT in patients with blunt liver injury verified by ultrasound, diagnostic peritoneal lav- age, nuclear scanning, laparotomy or CT-scan. Moreo- ver, 50 % of the patients in the non-liver injury group were identified based on physical examination alone. The method for defining the optimal cut-off values for AST and ALT was not described. Five (7.8 %) patients in Group LI+ died from massive hemorrhage, none of them liver-related; three patients with pelvic fracture with retroperitoneal hematoma, and two patients with chest injury. In a study by Tian et al. [13] the cut-off values were set at AST ≥ 113 U/l and ALT ≥ 57 U/l by using ROC curve analysis. To identify the liver injury, CT and laparotomy Characteristics of this study population are presented in Table 1. Results During the study period, 1856 trauma patients were admitted. Of the 1643 patients with blunt trauma, 676 patients underwent abdominal CE-MDCT within 3 h after injury. Based on CE-MDCT scans, 64 patients were diagnosed with liver injury (Group LI+) and 612 patients without liver injury (Group LI−) (Fig. 1). Hemodynamically normal patients, on admission or after initial resuscitation, underwent CE-MDCT if at least one of the following criteria was fulfilled: (1) com- plaint of severe abdominal pain, (2) peritonism, (3) external signs of abdominal injuries, (4) presence of hematuria, melena or hematemesis, (5) abnormal radio- graphic findings commonly associated with abdominal injuries (intraperitoneal free air, lower rib fracture, pel- vic fracture, or lumbar fracture) (6) positive abdominal focused assessment with sonography in trauma (FAST), (7) acute anemia with hemoglobin <10 g/dl, (8) impaired consciousness due to suspected traumatic brain injury. Group LI+ consisted of nine patients with OIS grade I injuries, 30 patients with grade II injuries, 18 with grade III injuries, 6 with grade IV injuries, and 1 with grade V injury. In Group LI+, 9 (14.1 %) patients had isolated liver injury and 55 (85.9 %) patients had combined injuries. Of the 55 patients with combined injuries, 17 patients had head injury, 12 had facial injury, 45 had chest injury, 21 Page 3 of 6 Koyama et al. BMC Res Notes (2016) 9:41 Fig. 1 Flowchart of study population. CE-MDCT contrast enhanced multi detector-row computed tomography; LI+ with liver injury, LI− without liver injury RTS, and Ps. The groups differed in ISS [median 21 (IQR 9–33) vs. 17 (9–26); p < 0.01]. Group LI+ had higher AST than LI− [276 (48–503) vs. 44 (16–73); p < 0.001] and higher ALT [240 (92–388) vs. 32 (16–49); p < 0.001]. ROC curve analysis for AST and ALT was performed where the area under ROC curve (AUC) of AST was 0.88 (95 % confidence interval (CI) 0.83–0.92) and of ALT was 0.88 (95 % CI 0.83–0.94) (Fig. 2). With these analyses, the optimal cut-off values for AST was set at 109 U/l (UL index 0.26, Youden index 0.63) (Fig. 3) and ALT were set at 97 U/l (UL index 0.25, Youden index 0.67) (Fig. 4), and the calculated sensitivity and specificity based on these cut-off values are shown in Table 2. Results f Ten patients with AST < 109 U/l and ALT < 97 U/l had liver injury diagnosed on CE-MDCT; one OIS grade I injury, eight grade II, and one grade III. None of these ten patients required any interventions for their liver injury, and didn’t suffer any liver related complication or death. Fig. 1 Flowchart of study population. CE-MDCT contrast enhanced multi detector-row computed tomography; LI+ with liver injury, LI− without liver injury LI+ with liver injury, LI− without liver injury, AST aspartate aminotransferase, ALT alanine aminotransferase, ISS injury severity score, GCS revised trauma score, Ps probability of survival Discussion Group LI+ and LI− were comparable for age, Table 1 Characteristics of the study population Values are given as median (IQR) where not stated otherwise LI+ with liver injury, LI− without liver injury, AST aspartate aminotransferase, ALT alanine aminotransferase, ISS injury severity score, GCS Glasgow coma scale, RTS revised trauma score, Ps probability of survival All (n = 676) LI+ (n = 64) LI− (n = 612) p value AST 48 (31–106) 276 (48–503) 44 (16–73) <0.001 ALT 36 (22–69) 240 (92–388) 32 (16–49) <0.001 ISS 17 (9–26) 21 (9–33) 17 (9–26) <0.01 GCS 15 (13–15) 15 (14–15) 14 (13–15) 0.67 RTS 7.84 (6.90–7.84) 7.84 (7.37–7.84) 7.84 (7.37–7.84) 0.32 Ps 97.0 (89.5–99.2) 95.7 (88.1–100) 97.4 (92.9–100) 0.19 Age 46 (29–63) 40 (23–57) 46 (29–63) 0.17 Male gender, n (%) 462 (68.3) 36 (56.3) 426 (69.6) 0.03 Mortality, n (%) 58 (8.6) 5 (7.8) 53 (8.7) 0.82 Table 1 Characteristics of the study population Page 4 of 6 Koyama et al. BMC Res Notes (2016) 9:41 Fig. 2 ROC curve of AST and ALT. AST aspartate aminotransferase, ALT alanine aminotransferase, ROC receiver operating characteristic, AUC area under the curve Fig. 2 ROC curve of AST and ALT. AST aspartate aminotransferase, ALT alanine aminotransferase, ROC receiver operating characteristic, AUC area under the curve Fig. 2 ROC curve of AST and ALT. AST aspartate aminotransferase, ALT alanine aminotransferase, ROC receiver operating characteristic, AUC area under the curve of AST and ALT. AST aspartate aminotransferase, ALT alanine aminotransferase, ROC receiver operating characteristic, AUC area were used and blood samples were drawn up to 24 h after injury. Moreover, the values of AUC for AST and ALT and the method for defining the optimal cut-off values were not described in their study. Tan et al. [14] set the cut-off values for AST ≥ 83 U/l and ALT ≥ 64 U/l in a series of 99 patients of whom 55 patients were LI+ defined by CT and laparotomy. In a case–control study, Lee et al. [15] set the cut-off values Fig. 3 ROC curve of AST with UL index and Youden index. ROC receiver operating characteristic, AST aspartate aminotransferase, UL upper-left Fig. 4 ROC curve of ALT with UL index and Youden index. ROC receiver operating characteristic, ALT alanine aminotransferase, UL upper-left Tan et al. References 1. Croce MA, Fabian TC, Menke PG, Waddle-Smith L, Minard G, Kudsk KA, et al. Nonoperative management of blunt hepatic trauma is the treatment of choice for hemodynamically stable patients. Results of a prospective trial. Ann Surg. 1995;221(6):744–53. 2. Piper GL, Peitzman AB. Current management of hepatic trauma. Surg Clin North Am. 2010;90(4):775–85. 3. Federle MP, Goldberg HI, Kaiser JA, Moss AA, Jeffrey RB Jr, Mall JC. Evaluation of abdominal trauma by computed tomography. Radiology. 1981;138(3):637–44. As many as 10 LI+ patients presented with AST < 109 U/l and ALT < 97 U/l. However, they were all treated by observation alone and suffered no complica- tions. Thus, the cut-off values in the present study seem to represent clinically relevant thresholds for further diagnostics, especially in a remote and/or resource lim- ited institution, to decide whether a patient is eligible for observation alone without jeopardizing the patient’s safety or require closer monitoring and further investigations. 4. Brenner DJ, Hall EJ. Computed tomography–an increasing source of radiation exposure. N Engl J Med. 2007;357(22):2277–84. 5. Richards JR, McGahan JP, Pali MJ, Bohnen PA. Sonographic detection of blunt hepatic trauma: hemoperitoneum and parenchymal patterns of injury. J Trauma. 1999;47(6):1092–7. 6. Oldham KT, Guice KS, Kaufman RA, Martin LW, Noseworthy J. Blunt hepatic injury and elevated hepatic enzymes: a clinical correlation in children. J Pediatr Surg. 1984;19(4):457–61. 7. Hennes HM, Smith DS, Schneider K, Hegenbarth MA, Duma MA, Jona JZ. Elevated liver transaminase levels in children with blunt abdominal trauma: a predictor of liver injury. Pediatrics. 1990;86(1):87–90. 7. Hennes HM, Smith DS, Schneider K, Hegenbarth MA, Duma MA, Jona JZ. Elevated liver transaminase levels in children with blunt abdominal trauma: a predictor of liver injury. Pediatrics. 1990;86(1):87–90. In the aforementioned study of Hamada et al. [16] the optimal cut-off values for AST and ALT were defined to be 166 U/l and 130 U/l, respectively. We assume that the lower cut-off values for AST and ALT in the present study can be attributed to the higher detection capability of the MDCT compared to the one achievable in the sin- gle detector-row computed tomography [20–22] as used in the previous study. y 8. Puranik SR, Hayes JS, Long J, Mata M. Liver enzymes as predictors of liver damage due to blunt abdominal trauma in children. South Med J. 2002;95(2):203–6. 8. Puranik SR, Hayes JS, Long J, Mata M. Conclusions 14. Tan KK, Bang SL, Vijayan A, Chiu MT. Hepatic enzymes have a role in the diagnosis of hepatic injury after blunt abdominal trauma. Injury. 2009;40(9):978–83. In conclusion, we have identified AST ≥ 109 U/l and ALT ≥ 97 U/l as optimal cut-off values for predicting the presence of liver injury in blunt trauma, potentially use- ful as a screening tool for CT scan in patients otherwise eligible for observation only or as a transfer criterion to a facility with CT scan capability. 15. Lee WC, Kuo LC, Cheng YC, Chen CW, Lin YK, Lin TY, et al. Combination of white blood cell count with liver enzymes in the diagnosis of blunt liver laceration. Am J Emerg Med. 2010;28(9):1024–9. 16. Hamada H, Tagawa Y, Fujita T, Nishida M, Endo Y, Kobayashi K, et al. Cut-off values for AST and ALT as criteria for performing abdominal enhanced computed tomography (CT) in the diagnosis of blunt liver injury. J Jpn Assoc Acute Med. 2012;23:142–50 (Article in Japanese). TK, HH, MN, TS conceived the study. TK, HH collected and analyzed the data. TK, PAN, CG wrote the manuscript, which HH, MN, TS critically revised, and all authors read and approved the final manuscript. References Liver enzymes as predictors of liver damage due to blunt abdominal trauma in children. South Med J. 2002;95(2):203–6. 9. Karam O, La Scala G, Le Coultre C, Chardot C. Liver function tests in chil- dren with blunt abdominal traumas. Eur J Pediatr Surg. 2007;17(5):313–6.l 9. Karam O, La Scala G, Le Coultre C, Chardot C. Liver function tests in chil- dren with blunt abdominal traumas. Eur J Pediatr Surg. 2007;17(5):313–6.l 10. Stassen NA, Lukan JK, Carrillo EH, Spain DA, Norfleet LA, Miller FB, et al. Exami- nation of the role of abdominal computed tomography in the evaluation of victims of trauma with increased aspartate aminotransferase in the era of focused abdominal sonography for trauma. Surgery. 2002;132(4):642–6. 10. Stassen NA, Lukan JK, Carrillo EH, Spain DA, Norfleet LA, Miller FB, et al. Exami- nation of the role of abdominal computed tomography in the evaluation of victims of trauma with increased aspartate aminotransferase in the era of focused abdominal sonography for trauma. Surgery. 2002;132(4):642–6. There are some limitations to this study in addition to its retrospective nature. The number of LI+ patients was relatively small. Additional studies are required to verify whether the optimal cut-off values defined in a single urban center in Asia are applicable worldwide. 11. Srivastava AR, Kumar S, Agarwal GG, Ranjan P. Blunt abdominal injury: serum ALT-A marker of liver injury and a guide to assessment of its sever- ity. Injury. 2007;38(9):1069–74. 11. Srivastava AR, Kumar S, Agarwal GG, Ranjan P. Blunt abdominal injury: serum ALT-A marker of liver injury and a guide to assessment of its sever- ity. Injury. 2007;38(9):1069–74. y j y 12. Sahdev P, Garramone RR Jr, Schwartz RJ, Steelman SR, Jacobs LM. Evalu- ation of liver function tests in screening for intra-abdominal injuries. Ann Emerg Med. 1991;20(8):838–41. 12. Sahdev P, Garramone RR Jr, Schwartz RJ, Steelman SR, Jacobs LM. Evalu- ation of liver function tests in screening for intra-abdominal injuries. Ann Emerg Med. 1991;20(8):838–41. 13. Tian Z, Liu H, Su X, Fang Z, Dong Z, Yu C, et al. Role of elevated liver transaminase levels in the diagnosis of liver injury after blunt abdominal trauma. Exp Ther Med. 2012;4(2):255–60. 13. Tian Z, Liu H, Su X, Fang Z, Dong Z, Yu C, et al. Role of elevated liver transaminase levels in the diagnosis of liver injury after blunt abdominal trauma. Exp Ther Med. 2012;4(2):255–60. Acknowledgements We thank Teikyo University Medical Library for providing funds to support the open-access publication of this study. Received: 19 June 2015 Accepted: 14 January 2016 at AST ≥ 100 U/l and ALT ≥ 80 U/l. They compared 42 LI+ patients and 42 LI− patients based on findings on CT evaluation. Statistical analysis was done to deter- mine whether AST and ALT could predict the liver injury. However, in none of those studies any attempt to define the optimal cut-off values for AST and ALT were performed. Discussion [14] set the cut-off values for AST ≥ 83 U/l and ALT ≥ 64 U/l in a series of 99 patients of whom 55 ti t LI+ d fi d b CT d l t I Fig. 4 ROC curve of ALT with UL index and Youden index. ROC receiver operating characteristic, ALT alanine aminotransferase, UL upper-left Fig. 4 ROC curve of ALT with UL index and Youden index. ROC receiver operating characteristic, ALT alanine aminotransferase, UL upper-left Fig. 3 ROC curve of AST with UL index and Youden index. ROC receiver operating characteristic, AST aspartate aminotransferase, UL upper-left Fig. 3 ROC curve of AST with UL index and Youden index. ROC receiver operating characteristic, AST aspartate aminotransferase, UL upper-left were used and blood samples were drawn up to 24 h after injury. Moreover, the values of AUC for AST and ALT and the method for defining the optimal cut-off values were not described in their study. Tan et al. [14] set the cut-off values for AST ≥ 83 U/l and ALT ≥ 64 U/l in a series of 99 patients of whom 55 patients were LI+ defined by CT and laparotomy. In a case–control study, Lee et al. [15] set the cut-off values Tan et al. [14] set the cut-off values for AST ≥ 83 U/l and ALT ≥ 64 U/l in a series of 99 patients of whom 55 patients were LI+ defined by CT and laparotomy. In a case–control study, Lee et al. [15] set the cut-off values Page 5 of 6 Page 5 of 6 Koyama et al. BMC Res Notes (2016) 9:41 Table 2 Results of the optimal cut-off values of AST and ALT LI+ with liver injury, LI− without liver injury, AST aspartate aminotransferase, ALT alanine aminotransferase, PPV positive predictive value, NPV negative predictive value Cut-off values Sensitivity (%) Specificity (%) PPV (%) NPV (%) AST ≥ 109 81 82 32 98 ALT ≥ 97 78 88 41 98 AST ≥ 109 and/or ALT ≥ 97 84 81 32 98 Table 2 Results of the optimal cut-off values of AST and ALT Author details 1 1 Department of Emergency Medicine, Teikyo University Hospital, 2‑11‑1 Kaga, Itabashi, Tokyo, Japan. 2 Department of Emergency Medicine, Toranomon Hos- pital, Tokyo, Japan. 3 Department of Traumatology, Oslo University Hospital- Ulleval, Oslo, Norway. Competing interests The authors declare that they have no competing interests. Received: 19 June 2015 Accepted: 14 January 2016 Received: 19 June 2015 Accepted: 14 January 2016 Competing interests Competing interests The authors declare that they have no competing interests. LI+ with liver injury, LI− without liver injury, AST aspartate aminotransferase, ALT alanine aminotransferase, PPV positive predictive value, NPV negative predictive value 20. Shanmuganathan K. Multi-detector row CT imaging of blunt abdominal trauma. Semin Ultrasound CT MR. 2004;25(2):180–204. 21. Hammerstingl RM, Vogl TJ. Abdominal MDCT: protocols and contrast considerations. Eur Radiol. 2005;15(Suppl 5):E78–90. 22. Aschoff AJ. MDCT of the abdomen. Eur Radiol. 2006;16(Suppl 7):M54–7. 18. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology. 1982;143(1):29–36. 19. Perkins NJ, Schisterman EF. The inconsistency of “optimal” cutpoints obtained using two criteria based on the receiver operating characteristic curve. Am J Epidemiol. 2006;163(7):670–5. Koyama et al. BMC Res Notes (2016) 9:41 Authors’ contributions 17. Moore EE, Cogbill TH, Jurkovich GJ, Shackford SR, Malangoni MA, Cham- pion HR. Organ injury scaling: spleen and liver (1994 revision). J Trauma. 1995;38(3):323–4. Page 6 of 6 • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W4384130915	https://www.researchsquare.com/article/rs-2278755/latest.pdf	English	null	The impact of semi-upright position on severity of sleep disordered breathing in patients with obstructive sleep apnea: a two-arm, prospective, randomized controlled trial	BMC anesthesiology	2,023	cc-by	8,107	The Impact of Semi-upright Position on Severity of Sleep Disordered Breathing in Patients with Obstructive Sleep Apnea: A two-arm, prospective, randomized controlled trial Page 1/21 The Impact of Semi-upright Position on Severity of Sleep Disordered Breathing in Patients with Obstructive Sleep Apnea: A two-arm, prospective, randomized controlled trial Gincy Ann Lukachan Believers Church Medical College Hospital Azadeh Yadollahi KITE - Toronto Rehabilitation Institute, University Health Network, University of Toronto; Dennis Auckley MetroHealth Medical Center, Case Western Reserve University Bojan Gavrilovic KITE - Toronto Rehabilitation Institute, University Health Network, University of Toronto; John Matelski Biostatistics Research Unit, University Health Network, Toronto, Ontario, Canada Frances Chung Department of Anesthesia, Toronto Western Hospital, University Health Network, University of Toronto. Mandeep Singh (  mandeep.singh@uhn.ca ) Department of Anesthesia, Toronto Western Hospital, University Health Network, University of Toronto. Research Article Keywords: Obstructive sleep apnea, supine-related OSA, surgery, elevated position, positional therapy Posted Date: December 13th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2278755/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 BMC Anesthesiology on July 13th, 2023. See the published version at https://doi.org/10.1186/s12871-023-02193-y. Gincy Ann Lukachan Believers Church Medical College Hospital MetroHealth Medical Center, Case Western Reserve University Bojan Gavrilovic KITE - Toronto Rehabilitation Institute, University Health Network, University of Toronto; John Matelski Biostatistics Research Unit, University Health Network, Toronto, Ontario, Canada Frances Chung Department of Anesthesia, Toronto Western Hospital, University Health Network, University of Toronto. Mandeep Singh (  mandeep.singh@uhn.ca ) Department of Anesthesia, Toronto Western Hospital, University Health Network, University of Toronto. Background The severity of sleep-disordered breathing is known to worsen postoperatively and is associated with increased cardio-pulmonary complications and increased resource implications. In the general population, the semi-upright position has been used in the management of OSA. We hypothesized that the use of a semi-upright position versus a non-elevated position will reduce postoperative worsening of OSA in patients undergoing non-cardiac surgeries Study registration This trial was retrospectively registered in clinicaltrials.gov NCT02152202 on 02/06/2014. This trial was retrospectively registered in clinicaltrials.gov NCT02152202 o Methods This study was conducted as a prospective randomized controlled trial of perioperative patients, undergoing elective non-cardiac inpatient surgeries. Patients underwent a preoperative sleep study using a portable polysomnography device. Patients with OSA (apnea hypopnea index (AHI) >5 events/hr), underwent a sleep study on postoperative night 2 (N2) after being randomized into an intervention group (Group I): semi-upright position (30 to 45 degrees incline), or a control group (Group C) (zero degrees from horizontal). The primary outcome was postoperative AHI on N2. The secondary outcomes were obstructive apnea index (OAI), central apnea index (CAI), hypopnea index (HI), obstructive apnea hypopnea index (OAHI) and oxygenation parameters. Results: Thirty-five patients were included. Twenty-one patients were assigned to the Group 1 (females-14 (67%); mean age 65±12) while there were fourteen patients in the Group C (females-5 (36%); mean age 63±10). The semi-upright position resulted in a significant reduction in OAI in the intervention arm (Group C vs Group I postop AHI: 16.6 ± 19.0 vs 8.6 ± 11.2 events/hr; overall p = 0.01), but there were no significant differences in the overall AHI or other parameters between the two groups. Subgroup analysis of patients with “supine related OSA” revealed a decreasing trend in postoperative AHI with semi-upright position, but the sample size was too small to evaluate statistical significance. Conclusion: In patients with newly diagnosed OSA, the semi-upright position resulted in improvement in obstructive apneas, but not the overall AHI. DOI: https://doi.org/10.21203/rs.3.rs-2278755/v1 Version of Record: A version of this preprint was published at BMC Anesthesiology on July 13th, 2023. See the published version at https://doi.org/10.1186/s12871-023-02193-y. Page 1/21 Introduction Obstructive sleep apnea (OSA) is a common sleep-related breathing disorder, associated with increased morbidity and mortality in the general and surgical population(1,2) in the perioperative period.(3) It is an independent risk-factor for post-operative cardiac and respiratory complications(4–7) resulting in increased utilization of health care resources.(8) Page 2/21 Page 2/21 The screening and treatment of OSA is found to be cost effective on the lifetime horizon.(9) According to the current guidelines adult patients at risk of OSA should be screened preoperatively using validated tools such as STOP-Bang, P-SAP, Berlin, and ASA Check List.(10) The American Society of Anesthesiology (ASA) practice guidelines on the perioperative management of OSA advice to consider the initiation of continuous positive airway pressure (CPAP) therapy preoperatively in patients with newly detected severe OSA.(11), Despite improvement in OSA severity and oxygenation with CPAP, poor patient compliance has been a hurdle to their use in the perioperative period.(12,13) Other alternatives to OSA treatment, such as weight reduction,(14) custom-made orthodontic appliances(15), and surgery (orthodontic surgery,(16) uvulopalatopharyngeoplasty,(17) tonsillectomy,(18) or bariatric surgery(19)) are not feasible in the preoperative period. There is a need for alternative approaches for the management of OSA in surgical patients. Positional therapy could be a useful intervention in surgical patients with OSA in the perioperative period. (20) This option may be more feasible in the postoperative setting as it is cost-effective, easy to administer, and can be adjusted to allow patient comfort. In the general population, sleeping in the non- supine and elevated posture was found to be effective in reducing OSA severity.(21–23) The utility of positional therapy may be greater in patients with supine-related OSA. Supine-related OSA is defined as Apnea-hypopnea index (AHI) > 5 events/hr, and where the supine AHI was more than twice the AHI of the non-supine AHI, and the non-supine AHI was less than 5 events/hr.(24,25) The American Society of Anesthesiology practice guidelines on the perioperative management of OSA recognized that positional therapy may improve the AHI in patients with OSA, but acknowledged that the literature was “insufficient to evaluate the effects of positioning adult OSA patients in the postoperative setting”.(11) We hypothesized that the use of a semi-upright position versus a supine position will prevent postoperative worsening of OSA in patients undergoing non-cardiac surgeries. Study design This was a two-arm, prospective, randomized controlled, proof of concept trial. The intervention was patient positioning in a semi-upright position (Group I: intervention, head-end elevation 30 to 45 degrees from horizontal), compared to supine position (Group C: control). Introduction The objective of the study was to determine whether a semi-upright versus supine position while asleep helps decrease the postoperative worsening of AHI in surgical patients with newly diagnosed OSA. The secondary objective was to study the impact of the semi-upright position in a subgroup of patients with supine-related OSA. Patient Recruitment, Intervention and Follow-up Portable PSG was performed using a 10-channel portable PSG device (Embletta X100; Embla, Broomfield, CO). The PSG was obtained preoperatively (preop) at home and on postoperatively (postop) on N 2. (27) The Embletta X100 is a level 2 diagnostic tool for OSA and has been validated against laboratory PSG. (28) The PSG recording montage comprised two electroencephalographic channels (C3 and C4), left or right electro-oculogram, chin muscle electromyogram, nasal cannula (pressure), thoracic and abdominal respiratory effort bands, body-position sensor, and pulse oximetry. At bedtime, the portable PSG device was connected to the patient by a PSG technician at their home. Patients were taught how to disconnect the device, which was picked up by the same sleep technician the following morning. The portable PSG recording was scored by a certified PSG technologist who was supervised by a sleep physician. Apneas were defined as a reduction in airflow from intranasal pressure of at least 90% for 10 seconds or longer, and hypopneas as reduction in flow of at least 30% for 10 seconds or longer, associated with ≥ 4% oxygen desaturation.(29) Apneas and hypopneas were classified as either obstructive (presence of breathing effort) or central (absence of breathing effort) events. Mixed apneas were classified for events that began as central for at least 10 seconds and ended as obstructive, with a minimum of three obstructive efforts. AHI was the average number of apnea and hypopnea episodes per hour of recording. Apnea index was calculated as the average number of apnea episodes per hour. Hypopnea index was the average number of hypopnea episodes per hour. The secondary outcomes were obstructive apnea index (OAI), calculated as the total number of obstructive apneas divided total sleep time (TST); central apnea index (CAI) calculated as the total number of central apneas per hour; obstructive apnea hypopnea index (OAHI), calculated as the total number of obstructive apneas and hypopneas per hour; oxygen desaturation index (ODI), number of events with oxygen desaturation below 4% threshold per hour, and CT90, cumulative percentage of sleep duration with oxygen desaturation less than 90%. Randomization and allocation concealment Study setting This study was conducted at Toronto Western Hospital and Mount Sinai Hospital in Toronto, over a period of seven months. Institutional Review Board approval was obtained from both hospitals prior to start of Page 3/21 this study (University Health Network 11-0056AE and Mount Sinai Hospital 11-0021-E). This trial was registered at www.clincialtrials.gov (NCT02152202). The inclusion criteria of patients were adult patients, American Society of Anesthesiologists (ASA) physical status I to IV, undergoing elective inpatient non-cardiac surgery with newly diagnosed OSA. The exclusion criteria were: patients with OSA on treatment (continuous positive airway pressure (CPAP), oral appliance, or previous OSA surgery); known cervical, shoulder, spine abnormalities, and/or chronic pain predisposing to difficulty in maintaining a sitting position and specific types of surgery, such as hip or spine, where a sitting position would be contraindicated postoperatively. Patients were screened by using the STOP-Bang questionnaire.(26) Patients identified as high risk of OSA (STOP-Bang score of three or greater) were consented to undergo a home portable polysomnography (PSG) and OSA status was confirmed by an AHI over 5 events per hour. Perioperative anesthetic care and postoperative pain management A standardized balanced anesthetic technique was used in all patients per routine care. In general anesthesia (GA), patients received an induction dose of propofol, opioid (fentanyl and/or hydromorphone), an inhalational agent (sevoflurane or desflurane), and a muscle relaxant (rocuronium). The muscle relaxant was reversed with neostigmine and atropine. In regional anesthesia (RA), patients received spinal anesthetic and sedation using midazolam, fentanyl and a propofol infusion (20-150 mcg/kg/min). Use of intrathecal opioid (100 mcg preservative free morphine) was at the discretion of the anesthesiologist. Both groups received intravenous or oral narcotics in the postoperative period guided by the Acute Pain Service team, as per our institutional standard of care. Pain was evaluated on a score of 0–10, with 0 as no pain and 10 as the most excruciating pain. Intravenous morphine by patient-controlled analgesia was initiated when the verbal pain score was 4 or higher. The research assistant visited patients daily to assist them with application and removal of the portable PSG, collect data, and document adverse events during the hospital stay. Randomization and allocation concealment Page 4/21 Page 4/21 Page 4/21 Patients with OSA (defined as AHI > 5 events/hr), were randomized into two groups: Control or Intervention groups (computer generated blocks of 8) by the research analyst, who was not involved in group allocation or data collection during the study. Group allocation was concealed using sealed, opaque envelops, and patients were assigned to their group following surgery. In the Control group (Group C), there was no bed elevation, and patients were positioned at bedtime with no head elevation or bed angle to zero degrees from the horizontal. In the Intervention group (Group I), patients were positioned at bedtime in a semi-upright position with bed elevated to 30 to 45 degrees from horizontal. The bed angle was measured by a research assistant using an in-built bed angle monitor, or a goniometer, wherever applicable. The bed angle measurements were performed at night and in the morning to monitor compliance with the allocated bed position. Patients had the option to request changing the bed angle to facilitate recovery from surgery in view of pain and discomfort. Target sample size The primary outcome of our study was AHI on postop N2. There was no previous research from the perioperative setting evaluating the impact of body positioning. Previous studies in the general population found that the mean change in AHI between the upright position (6 ± 12 events/hr), and no head elevation (29 ± 6 events/hr), respectively.(23,30) The original sample size calculated in the protocol was 28 in each arm calculated after taking a minimal clinically significant difference (MCSD) of an effect size (change in AHI) of at least 10 events per hour from baseline, a power of 0.9 and a standard deviation of 10, after adjusting for an estimated drop-out, and loss of follow up to a total of 20%. However, the study was terminated early due to concerns with funding, and a final sample size of 32 patients was obtained, which had sufficient power of 0.8, with type 1 error of 0.05. It was decided to proceed with analysis of the data by the senior authors. Page 5/21 Page 5/21 Statistical analysis Analyses were performed using the SAS 9.2 statistical software for Windows (SAS Institute, Cary, NC) or R (version 3.1.1)(31). The analysis was blinded to allocation until the completion of data accrual period. Because of patient preference and deviation from assignment of intervention, a per-protocol analysis was performed for this study, where patients were analyzed based on the bed angle monitor reading noted in the morning following their PSG to show no deviation from protocol. An intention-to-treat analysis was performed as sensitivity analysis, meaning that all participants were analyzed in the group to which they were randomized. Baseline demographic variables are summarized for the entire study population and by treatment group using standard bivariate methods, as implemented in R package tableone(32). For each variable we include a standardized mean difference, along with a p-value against the null hypothesis of equality between groups. Continuous variables were compared using two-tailed, paired t-tests for variables with normally distributed data and Wilcoxon signed rank test for variables with non-normally distributed data. Pre-defined linear regression was performed for the primary and secondary outcomes, with preop AHI and supine-related OSA as covariates. Supine-related OSA was defined as AHI > 5 events/hr, and where the supine AHI was more than twice the AHI of the non-supine AHI, and the non-supine AHI was less than 5 events/hr. (24) A two-sided p value < 0.05 was considered significant and controlled for repeated observations, wherever applicable. Study population Patient recruitment and flow is summarized in Figure 1, based on the CONSORT recommendations. A total of 635 patients were screened preoperatively, with 164 patients giving consent, of which 135 patients completed home PSG study. Eighty-three patients with OSA (AHI > 5 events/hr) were randomized, Group C: 41 and Group I: 42. During the study, six patients in Group C requested to change position to semi-upright position and were allocated to Group I. Complete postoperative N2 PSG data were obtained from 15 and 24 patients for Group C and I, respectively. This was partly because of patient refusal to undergo the PSG postoperatively while recovering from surgery, primarily due to postoperative pain and discomfort. Four patients were excluded as they required oxygen supplementation. Per protocol analysis was done for 14 patients in Group C and 21 patients in Group I. The baseline characteristics for PP and ITT analysis are presented in Table 1, and supplementary table 1, respectively. While randomization led to a more balanced distribution of baseline demographic variables, deviation of protocol resulted in disturbances for the PP analysis where control group had higher neck circumference and lower OAI and CT 90 values (SMD>0.8). The preop PSG (Table 1) data showed no difference in AHI, OAHI, HI between Group C and Group I. The baseline OAI was significantly higher (11.7 ± 9.2 vs. 6.0 ± 3.6 events per hour; p=0.01) while CAI was Page 6/21 significantly lower (0.7 ± 1.4 vs 3.7 ± 9.5; p=0.04) in Group I than Group C. The CT90 was significantly higher (3.8 (0.4 - 6.1) % vs. 0.7 (0.1 - 1.2) %; p=0.04) and the lowest SaO2 was significantly lower (79.1 ± 6.1% vs 83.2 ± 5.0 %; p= 0.04) in Group I vs Group C. significantly lower (0.7 ± 1.4 vs 3.7 ± 9.5; p=0.04) in Group I than Group C. The CT90 was significantly higher (3.8 (0.4 - 6.1) % vs. 0.7 (0.1 - 1.2) %; p=0.04) and the lowest SaO2 was significantly lower (79.1 ± 6.1% vs 83.2 ± 5.0 %; p= 0.04) in Group I vs Group C. Primary outcome. Comparing postop N2 vs preop baseline, the AHI increased in Group C while it decreased in Group I (Table 2). The differences were not significant within the groups or between groups. Study population (Group C: postop AHI vs preop AHI: 28.4 ± 28.1 vs 18.1 ± 13.3 events/hr, p=0.33; Group I: postop AHI vs preop AHI: 20.8 ± 23.8 vs 21.4 ± 13.1; p=0.36); overall p = 0.15. (Fig. 2A) Comparing postop N2 vs preop parameters, the changes in OAI within the two groups were not significant but there was an overall significant change between the two groups (Group C vs Group I: postop AHI 16.6 ± 19.0 vs 8.6 ± 11.2 events/hr); (overall p = 0.01) (Fig. 2B). There were no significant differences in CAI, HI, OAHI within the two groups and between groups (Table 2). Among the oxygenation parameters, CT90 (Supplementary fig. 3) significantly increased postoperatively in both groups (Group C: postop CT90 vs preop : 4.4 ± 4.9% vs 1.3 ± 2.0% , p = 0.003; Group I: postop CT90 vs preop : 15.7 ± 19.8% vs 4.4 ± 4.9% ; p = 0.04), and the average SaO2 were significantly decreased in the postoperative period for both groups (Group C: postop average SaO2 vs preop : 90.0 ± 3.6% vs 93.4 ± 1.3% , p=0.01; Group I: postop average SaO2 vs preop : 90.3 ± 3.8% vs 93.1 ± 2.4% , p=0.002). However, between group comparison did not show a significant difference (Table 2). The other parameters were not significantly different from preop to postop and between the two groups (Table 2). Subgroup analysis. The impact of body position was examined in patients classified as “supine-related OSA” (n=8; Group C: 5 patients, and Group I: 3 patients). There was greater reduction in mean AHI in Group I (postop AHI vs preop AHI: 6.0 ± 3.0 vs 24.3 ± 13.9 events/hr) than in Group C (Fig. 3), but the sample size was too small to evaluate statistical significance. Discussion This is a novel study in the perioperative setting to evaluate the efficacy of semi-upright position postoperatively for management of newly diagnosed OSA. We found that it is feasible to institute positional therapy in the form of semi-upright position in the postoperative period for OSA patients. The elevated position resulted in a significant reduction in OAI by eight events per hour, but not the AHI, CAI, HI, and OAHI. The lack of positive results in AHI may be due to patients in the intervention group had significantly worse OAI, lower SaO2, and higher CT 90 preoperatively. Nevertheless, in the surgical patients with supine-related OSA, we were able to demonstrate the effectiveness of semi-upright position as the mean AHI decreased by 18 events per hour. Although positive airway pressure (PAP) is the mainstay of treatment for moderate to severe OSA, perioperative adherence has been poor as studies have demonstrated only 34% CPAP adherence(13) and Page 7/21 45% auto-titrated CPAP adherence(33) in patients with newly diagnosed OSA treated with PAP therapy before surgery. This suggests a need for alternative therapies for these patients. We found that positional therapy is a feasible alternative treatment option for OSA patients in the perioperative setting especially in those with supine related OSA. These findings could be explained by the close association of upper airway collapsibility with body, head, and neck positioning.(24) Previous work has suggested that the semi-upright position significantly enlarges the upper airway dimensions.(34),(35) The mean upper airway volume was greater with 44° head elevation compared to supine position.(35) Mild elevations of the head of the bed by 7.5° were associated with reductions in the AHI and improvements in oxygen parameters.(36) In a randomized crossover study of 30 postpartum women with OSA in the post-anesthesia care unit, 45° elevation of the upper body caused a significant reduction in AHI compared to non-elevated position.(37) A recently published randomized crossover trial among perioperative patients with moderate to severe OSA, compared high-flow nasal cannula (20 l/min with 40% oxygen concentration) with or without 30-degree head-of-bed elevation.(38) Patients were assessed with modified apnea hypopnea index, based exclusively on the airflow signal without arterial oxygen saturation criteria. Discussion High-flow nasal cannula caused significant improvement in OSA independently, with an additive effect when combined with 30-degree head-of-bed elevation (compared to Control flow- based AHI reduced by 10.9 (95% CI, 1 to 21) events · h–1, P = 0.028; and 23 (95% CI, 13 to 32) events · h– 1, P < 0.001 respectively). Body position may affect factors such as upper-airway passive collapsibility, airway dilator muscle activity, loop gain, and arousal threshold in patients with OSA.(39) These factors may play a role in how elevating th upper body can reduce OSA severity. In patients with OSA, pharyngeal critical closing pressure is higher in the supine position than lateral position.(40) which may be related to reduction in functional residual capacity.(41) An increase in the diaphragmatic descent during the respiratory cycle leads to an increase in lung volume and thus an increase in longitudinal traction on the upper airway which increase upper airway caliber during sleep and anesthesia.(42–44) In addition, rostral fluid shift may worsen OSA severity and thus consolidate the effects of gravity on the propensity of OSA as demonstrated in healthy men(45),(46) and non-obese men.(47) A retrospective study on OSA patients with upper airway surgery found that the prevalence of positional OSA increased from 26 to 54% in those with persistent OSA at six months.(48) Among the non-responders to OSA surgery, almost 70% of patients were position dependent on preoperative PSG with no improvement at six months postoperatively.(49) This highlights the need to explore positional therapy, especially in those with positional OSA. In a systematic review of positional therapy for OSA, CPAP was better than positional therapy to lower the AHI, while positional therapy was better than inactive controls to lower the AHI and improved daytime sleepiness.(50) Long term compliance and treatment benefit from positional therapy needs to be determined by longitudinal studies, and compared to other modalities such as CPAP. Page 8/21 In the general population, patients with supine-related OSA may be a suitable phenotype to benefit from positional therapy.(24) Good initial control of the OSA severity has been demonstrated but there is a lack of long-term compliance and outcome data.(24) The supine-related OSA phenotype can be easily identified on the preoperative sleep study. Though our data was limited, we found that patients with supine-related OSA benefit from postoperative semi-upright position with a reduction in AHI. Discussion Thus, we recommend the incorporation of semi-upright positioning as a practical adjunct to the perioperative management of OSA patients. It would be useful in those at high risk of OSA, newly diagnosed OSA or CPAP nonadherent patients. Further studies on custom-made pillows (to allow for elevated head position, or lateral position), tennis ball t-shirt,(21) or body position alarm devices (51) need to be done. There are several limitations to our study. First, our study had a small sample size with limited numbers in patients with supine-related OSA. Second, there can be variability in how the body position is reported and scored on the PSG. We used an accelerometer attached to the portable PSG which was placed on the patient′s chest. In-built automatic position sensors define body position as a categorical variable rather than a continuous variable, and may not reflect the physiological impact of various body positions on the collapsibility of the upper airway.(24) Third, head and neck position can independently influence the AHI. (52) Recording trunk position does not account for the effect of head and neck on upper-airway collapsibility and the impact on OSA severity.(53,54) However, we were able to show that the elevated position resulted in a significant reduction in OAI by eight events per hour. In those with supine-related OSA, we found a non-significant decrease in the mean AHI by 18 events per hour. Conclusion We found that the semi-upright position compared to supine position reduced postoperative OAI, indicating reduction in upper airway collapsibility and obstructive apneas. There was a decreasing trend in postoperative AHI in patients with supine-related OSA. Further studies on postoperative positional therapy are needed. Ethics approval and consent to participate Ethics approval for this study was provided by Research Ethics Board at Toronto Western Hospital (Approval no. 14–8710.0) and Mount Sinai Hospital (Approval no. 11-0021-E) on June 23, 2011 and June 22, 2011 respectively. The study was registered in clinicaltrials.gov NCT02152202 on 02/06/2014. The study was also done as per the declaration of Helsinki. The benefits and purposes of the study were explained to the patients, and each participant provided written, informed consent. Confidentiality was maintained at all levels of the study by avoiding identifiers and using codes to identify patients. Participants’ involvement in the study was voluntary. Participants who did not wish to participate in the study or who wished to withdraw at any time were informed that they could do so without restriction. Consent for publication Consent for publication Page 9/21 Not applicable. Not applicable. Anesthesiology and Pain Medicine at the University of Toronto. Anesthesiology and Pain Medicine at the University of Toronto. Frances Chung reports having received research support from the University Health Network Foundation (Toronto, ON, Canada) and the Ontario Ministry of Health and Long-Term Care, royalties from UpToDate Inc., and consultant fees from the Takeda Pharmaceutical Company Limited. She is a developer of the STOP-Bang questionnaire (proprietary to the University Health Network). Availability of data and materials All data and materials in this manuscript are available from the corresponding author on reasonable request. Competing interests Frances Chung holds the ResMed Research Chair of Anesthesia, sleep and perioperative medicine, consultant to Takeda Pharma. STOP-Bang questionnaire proprietary to University Health Network. Mandeep Singh (MS) currently holds the Canadian Anesthesiologists’ Society Career Scientist Grant and a Merit award from the Department of Anesthesiology and Pain Medicine, University of Toronto (Toronto, Canada) to support academic time. MS also serves on the Medical Advisory Board of Hypersomnia Foundation (Atlanta, GA) on a voluntary basis. Gincy A Lukachan, Azadeh Yadollahi, Dennis Auckley, Bojan Gavrilovic, John Matelski: No competing interests Gincy A Lukachan, Azadeh Yadollahi, Dennis Auckley, Bojan Gavrilovic, John Matelski: No competing interests Authors' contributions FC and MS conceptualized the study, acquired funding, designed the methodology, supervised and oversaw the trial, assisted with writing the original draft, reviewed and edited the final manuscript. GAL, AY, DA and JM assisted with data analysis, drafting and revising the article. BG assisted with data collection, data analysis, drafting and revising the article. The author(s) read and approved the final manuscript. Frances Chung and Mandeep Singh shared senior authorship for this study. Funding This study was supported by Departmental funds. This study was supported by Departmental funds. Mandeep Singh is supported by a Canadian Anesthesiologists’ Society Career Scientist Award and by the Merit Awards Program from the Department of Mandeep Singh is supported by a Canadian Anesthesiologists’ Society Career Scientist Award and by the Merit Awards Program from the Department of Anesthesiology and Pain Medicine at the University of Toronto. Corresponding author Correspondence to Mandeep Singh. Correspondence to Mandeep Singh. Authors and Affiliations Authors and Affiliations Department of Anesthesia, Toronto Western Hospital, University Health Network, University of Toronto. Frances Chung and Mandeep Singh Department of Anesthesia, Believers Church Medical College Hospital, Thiruvalla, Kerala, India Gincy A Lukachan KITE - Toronto Rehabilitation Institute, University Health Network, University of Toronto Azadeh Yadollahi and Bojan Gavrilovic Division of Pulmonary, Critical Care and Sleep Medicine, MetroHealth Medical Center, Case Western Reserve University, Cleveland, Ohio Dennis Auckley Biostatistics Research Unit, University Health Network, Toronto, Ontario, Canada John Matelski Corresponding author Correspondence to Mandeep Singh. References Department of Anesthesia, Toronto Western Hospital, University Health Network, University of Toronto. Frances Chung and Mandeep Singh Acknowledgements Page 10/21 Page 10/21 We acknowledge the help of Islam Sazzadual, MSc, Babak Amirshahi, MD, Hoda Fazel MD and Hisham Elsaid MD for conduct of the study. We thank Pu Liao MD for database management, Yuming Sun, MD for scoring polysomnography. 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Effect of body posture on pharyngeal shape and size in adults with and without obstructive sleep apnea. Sleep. Page 14/21 Page 14/21 Table 1. Patient demographics across the two groups (Per-protocol). Table 1. Patient demographics across the two groups (Per-protocol). Page 15/21 Control (n=14) Intervention (n=21) P value Standardized Mean Difference Age (years) 63±10 65±12 0.60 0.188 BMI (kg/m2) 32.8±4 35.2±7 0.26 0.420 Neck Circumference (cm) 43.3±3 41.0±3 0.04 0.752 Gender F/M 5/9 14/7 0.09 0.651 STOP-Bang score 4.5±1 4.5±1 0.95 0.020 Co-morbidity Hypertension 7 12 0.74 0.144 Gastroesophageal reflux 2 7 0.26 0.459 Diabetes Mellitus 1 6 0.20 0.583 Smoker 2 2 1.00 0.147 Asthma 1 3 0.64 0.232 COPD 1 1 1.00 0.101 CAD 3 0 0.06 0.739 Type of surgery 0.65 0.707 Orthopedic 8 12 General 5 6 Gynecology 0 2 Urology 1 0 Type of Anesthesia 1.00 0.048 General 7 10 Spinal 7 11 ASA Status 0.16 0.589 II 8 7 III 5 14 Total amount of opioids (in mg IV morphine equivalents) 44.4] 44.4] 1 48 h ] 1st 72 h 25.8 [14.2 - 60.50] 41.4 [25.0 - 81.5] 0.29 0.259 Preoperative sleep study data between the two groups AHI 18.1 ± 13.3 21.4 ± 13.1 0.48 0.246 OAI 6.1 ± 3.7 11.7 ± 9.2 0.04 0.807 OAHI 14.6 ± 7.2 20.7 ± 12.6 0.11 0.593 CAI 3.7 ± 9.5 0.7 ± 1.4 0.16 0.444 HI 8.6 ± 5.2 9.0 ± 7.3 0.84 0.071 ODI 15.5 [13.5 - 23.7] 17.9 [12.1 - 39.1] 0.57 0.281 CT90 0.7 [0.1 - 1.2] 3.8 [0.4 – 6.1] 0.04 0.804 Average SaO2 93.4 ± 1.27 93.1 ± 2.4 0.71 0.137 Lowest SaO2 83.2 ± 5.0 79.1 ± 6.1 0.04 0.748 41.4 [25.0 - 81.5] 0.29 0.259 Preoperative sleep study data between the two groups Data are expressed as mean (SD) or median (interquartile range IQR) where appropriate. Standardized Mean Difference was calculated using R package tableone Data are expressed as mean (SD) or median (interquartile range IQR) where appropriate. Standardized Mean Difference was calculated using R package tableone BMI - body mass index; COPD – chronic obstructive pulmonary disease; CAD – coronary artery disease; OR – operating room; h: hours. BMI - body mass index; COPD – chronic obstructive pulmonary disease; CAD – coronary artery disease; OR – operating room; h: hours. Table 1. Patient demographics across the two groups (Per-protocol). AHI: Apnea-Hypopnea Index; Apnea index: average number of apnea episodes per hour; Arousal \index: number of arousals × 60 / Total sleep time; CAI: Central Apnea Index: total number of central apneas per hour; CT90: cumulative percentage of Total sleep time with oxygen desaturation below 90%; HI: Hypopnea index, average number of hypopnea episodes per hour; OAHI: Obstructive Apnea Hypopnea Index, total number of obstructive apneas and hypopneas per hour; OAI: Obstructive Apnea Index: total number of obstructive apneas divided by Total sleep time; ODI: Oxygen Desaturation Index, number of events with oxygen desaturation below 4% threshold in one hour; REM%: Time spent in rapid eye movement stage of sleep; SaO2: saturation of oxygen in hemoglobin Table 2: Postoperative sleep-related outcomes in both groups Table 2: Postoperative sleep-related outcomes in both groups AHI: Apnea-Hypopnea Index; Apnea index: average number of apnea episodes per hour; CAI: Central Apnea Index: total number of central apneas per hour; CT90: cumulative percentage of total sleep time with oxygen desaturation below 90%; HI: Hypopnea index, average number of hypopnea episodes per hour; OAHI: Obstructive Apnea Hypopnea Index, total number of obstructive apneas and hypopneas per hour; OAI: Obstructive Apnea Index: total number of obstructive apneas divided sleep time; ODI: Oxygen Desaturation Index, number of events with oxygen desaturation below 4% threshold in one hour; SaO2: saturation of oxygen in hemoglobin. Table 2: Postoperative sleep-related outcomes in both groups Page 17/21 Control (n = 14) Intervention (n = 21) Between group comparison (ANCOVA) Variable Pre-op Post-op p- value Pre-op Post-op p- value p-value AHI 18.1 ± 13.3 28.4 ± 28.1 0.33 21.4 ± 13.1 20.8 ± 23.8 0.36 0.15 OAI 6.0 ± 3.6 16.6 ± 19.0 0.09 11.7 ± 9.2 8.6 ± 11.2 0.13 0.01* CAI 3.7 ± 9.5 3.6 ± 4.9 0.35 0.7 ± 1.4 1.3 ± 3.5 0.79 0.11 HI 8.6 ± 5.2 8.6 ± 8.4 0.9 9.0 ± 7.3 11.3 ± 11.4 0.34 0.49 OAHI 14.6 ± 7.2 25.2 ± 24.2 0.14 20.7 ± 12.6 19.8 ± 21.5 0.54 0.09 ODI 19.3 ± 12.5 26.2 ± 24.7 0.39 23.1 ± 14.3 26.1 ± 27.1 0.66 0.55 CT90 1.3 ± 2.0 28.1 ± 35.4 0.003* 4.4 ± 4.9 15.7 ± 19.8 0.04* 0.11 Average SaO2 93.4 ± 1.3 90.0 ± 3.6 0.01* 93.1 ± 2.4 90.3 ± 3.8 0.002* 0.40 Lowest SaO2 83.2 ± 5.0 75.0 ± 21.2 0.26 79.0 ± 6.1 77.8 ± 9.9 0.79 0.91 Supine % 44.5 ± 30.3 86.8 ± 20.4 <0.001 43.0 ± 35.6 66.2 ± 42.4 0.06 0.40 AHI: Apnea-Hypopnea Index; Apnea index: average number of apnea episodes per hour; CAI: Central Apnea Index: total number of central apneas per hour; CT90: cumulative percentage of total sleep time with oxygen desaturation below 90%; HI: Hypopnea index, average number of hypopnea episodes per hour; OAHI: Obstructive Apnea Hypopnea Index, total number of obstructive apneas and hypopneas per hour; OAI: Obstructive Apnea Index: total number of obstructive apneas divided sleep time; ODI: Oxygen Desaturation Index, number of events with oxygen desaturation below 4% threshold in one hour; SaO2: saturation of oxygen in hemoglobin. Control (n = 14) AHI: Apnea-Hypopnea Index; Apnea index: average number of apnea episodes per hour; CAI: Central Apnea Index: total number of central apneas per hour; CT90: cumulative percentage of total sleep time with oxygen desaturation below 90%; HI: Hypopnea index, average number of hypopnea episodes per hour; OAHI: Obstructive Apnea Hypopnea Index, total number of obstructive apneas and hypopneas per hour; OAI: Obstructive Apnea Index: total number of obstructive apneas divided sleep time; ODI: Oxygen Desaturation Index, number of events with oxygen desaturation below 4% threshold in one hour; SaO2: saturation of oxygen in hemoglobin. Figures Page 18/21 Page 18/21 Page 19/21 Figure 1 Participant flow in the study Figure 1 Participant flow in the study Participant flow in the study Page 19/21 Page 19/21 Page 19/21 Figure 3 The effect of semi-upright body position on the apnea-hypopnea index (AHI) in patients with supine- related OSA (n=10) Figure 2 A. The effect of semi-upright position on apnea-hypopnea index in the two groups per-protocol analysis. B. The effect of semi-upright position on obstructive index in the two groups per-protocol analysis. Page 20/21 Page 20/21 Figure 3 Supplementarymaterial.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Supplementarymaterial.docx Page 21/21
https://openalex.org/W2118223495	https://zenodo.org/records/1554153/files/article.pdf	English	null	Notes on the Phonology of Southern Kurmanji	The journal of the Royal Asiatic Society of Great Britain & Ireland/Journal of the Royal Asiatic Society of Great Britain & Ireland	1,922	public-domain	15,152	1 Justi, Introduction to Kurdische Grammatik. 2 Darmesteter, Etudes Iraniennes, vol. ii, p. 89. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the Notes on the Phonology of Southern Kurmanji j BY E. B. SOANE rPHE allocation of its proper place among Iranian languages to Kurmanji (Kurdish) has been rendered well-nigh impossible up to the present by the lack of material available for examination. The most careful Orientalists tend to consider it a non-Persian language, notably Justi, Darmesteter, and Socin (in the Grundriss der Iranischen Philologie). The first emphatically states that it is in no way a degraded New Persian, and that a description of the peculiarities of Iranian speech would not be exhaustive if the phonetics and etymology of Kurdish be disregarded.1 The second, confronted with many apparent phonological incongruities, leaves it an open question, but places importance upon the preservation of the Avestic z, which both in Old Persian and New Persian is represented by d in certain conditions. Examples of this will be seen in the notes below. Darmesteter furthermore lays stress upon the necessity of studying the material of a single dialect in order to avoid confusion. I have carefully excluded from the following notes consideration of any but the great southern section of Kurmanji. As bearing upon the apparent incongruities mentioned above I would quote a speculation of Darmesteter which study of the language has now proved to me to have been of the greatest importance. He says: " Ici se pose la question inextricable des emprunts au persan: l'identite de forme entre un mot persan et un mot kurde est, en g6neral, un indice que le mot kurde est emprunt6." 2 192 THE PHONOLOGY OF SOUTHERN KUEMANJI i In the section on Kurdish in the Grundriss der Iranischen Philologie, Socin prefaces his general remarks with the state- ment that Kurdish does not stand either to Pehlevi or New Persian in the relation of " sister-dialect", but that there is something remoter in the relationship. He further con- sidered that Kurdish does not come from Old Persian, and advised the comparison of the former with New Persian, in order to determine in which particulars it exhibits older or younger word-forms. I hope that the following notes may throw some new light upon the subject, and serve as a preliminary step to further investigation of a widely-spoken language which has hitherto suffered neglect because of our ignorance of it. I. SHORT a 1. Kj. a = Av. a, Phi. a, NP. a. waft, snow: Av. vafra, Phi. vafr, NP. 6CM/. pas, small cattle: Av. pasu-, Phi. pah, NP. —. waw, a common mountain tree : Av. vand, Phi. van, NP. —. hakar, aka, hagar, if : Av. ha-kara, Phi., NP. agar. hamdr, a store-room Av. ham\/bere, Phi., NP. ambdr. angust, finger: Av. angushta, Phi. angust, NP. angusht. aica, that: Av. awa, Phi. ava, NP. —. han-, verbal prefix : Av. han-, Phi., NP. an-, waraz, boar: Av. vardza-, Phi. vardz, NP. gurdz. asr, tears: Av. am/, Phi., NP. ars. az, aze, I : Av. azem, PH., NP. —. 1. Kj. a = Av. a, Phi. a, NP. a. waft, snow: Av. vafra, Phi. vafr, NP. 6CM/. pas, small cattle: Av. pasu-, Phi. pah, NP. —. waw, a common mountain tree : Av. vand, Phi. van, NP. —. hakar, aka, hagar, if : Av. ha-kara, Phi., NP. agar. hamdr, a store-room Av. ham\/bere, Phi., NP. ambdr. angust, finger: Av. angushta, Phi. angust, NP. angusht. aica, that: Av. awa, Phi. ava, NP. —. han-, verbal prefix : Av. han-, Phi., NP. an-, waraz, boar: Av. vardza-, Phi. vardz, NP. gurdz. asr, tears: Av. am/, Phi., NP. ars. az, aze, I : Av. azem, PH., NP. —. 2. Kj. a = Av. a, Phi. a, NP. a. 6ar, loose, free: Av. fra-, Phi. frdj, NP. ^/miz. maw, appertaining to: Av. manah-, Phi., NP. -man. awand, that so much : Av. avavant, Phi. hdvant, NP. —. 3. Kj. a = Av. ae, Phi. i, NP. i. am, this: Av. mm, Phi. ima, NP. im-. 4. Kj. a = Av. ai, Phi. a, NP. a. ^ar, mountain : Av. gairi-, Phi., NP. —•. kani, maiden : Av. kaine, Phi. kanik, NP. kani[z. Av. kaine, Phi. kanik, NP. kani[z. 5. Kj. a = Av. a, Phi. a, NP. a. a, to: Av. a, Phi. a, NP. —. \/aparmu, trust: Av. d-\/parmu, Phi., NP. •—. danik, a pip: Av. ddno-, Phi. ddnak, NP. ddnah. ga, time, place: Av. ^aiw-, Phi. (/as, NP. gah. pata, a pen for animals : Av. pdta-, Phi., NP. —. Notes on the Phonology of Southern Kurmanji I venture to think that sufficient individuality will be seen to invite further research, for though, as might be expected, Kurmanji is in consonance with Pablevi and New Persian in many of its differences and developments from Avestic, it has quite enough characteristics of its own to render its description as a dialect of one of the two younger languages very hazardous. Of its well-preserved grammar there is not space to treat here, nor of its peculiar affinities with Pushto, Baluchi, and Ossethian. I am aware that I have not done full justice to the vowel sounds, which require further subdivision, but the subject is a very large one, which demands separate treatment. Abbreviations used are :— Abbreviations used are :— Av. == Avestic. Phi. = Pehlevi. NP. = New Persian. Skt. = Sanskrit. Tk. = Turkish. Ar. = Arabic. Kj. = Kurmanji. OP. = Old Persian. Av. == Avestic. Phi. = Pehlevi. Av. == Avestic. NP. = New Persian. Tk. = Turkish. Kj. = Kurmanji. Kj. = Kurmanji. OP. = Old Persian. VOWELS Short: a e i o u. Long : a e % o u u. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THE PHONOLOGY OF SOUTHERN KURMANJI 193 II. LONG a yasA, after, behind: Av. pascha, Phi., NP. pas. «a>, cold : Av. sareta-, Phi. sart, NP. sard. 26. Kj. a = Av. a, Phi. a, NP. a. yasA, after, behind: Av. pascha, Phi., NP. pas. «a>, cold : Av. sareta-, Phi. sart, NP. sard. 3. Kj. a = Av. a, Phi. a, NP. a. judn, young: Av. yuvan-, Phi. yovdn, NP. javdn. Jcdni, a spring: Av. Jchan-, Phi. Tchdnik, NP. Tchdni. ferd, broad Av. fraOah, Phi. /ra^, NP. firdhh. Jiazhdr, miserable : Av. -V/ZKM", Phi. zhdr, NP. zar. mas?, fish : Av. masya-, Phi. mahik, NP. 4. Kj. a = Av. a0, Phi. ah, NP. aft. zMr, poison : Av. ja8ra, Phi., NP. zahr. pan, broad : Av. padan, Phi. pahan, ~NP. pahn. j , jdsh, a donkey foal: Ar. jahsh. Tcdrez, a conduit: NP Mhriz. II. LONG a Pronunciation very much lighter than the NP. a, practically the same as in English " half ", " father ". Pronunciation very much lighter than the NP. a, practically the same as in English " half ", " father ". 1. Kj. a = Av. a, Phi. a, NP. a. j as-, gazelle: Av. dsu-, Phi. ahuk, NP. ahu. mal, House, family: Av. nmdna-, Phi. man, NP. man. pdnia, heel: Av. pdshna-, Phi. pdshnak, NP. pdshna. dst, alignment: Av. rdsta-, Phi. rdst, NP. rdsta. dgir, fire : Av. dtar-, Phi JRAS. APRIL 1922. 13 as-, gazelle: Av. dsu-, Phi. ahuk, NP. ahu. mal, House, family: Av. nmdna-, Phi. man, NP. man. pdnia, heel: Av. pdshna-, Phi. pdshnak, NP. pdshna. dst, alignment: Av. rdsta-, Phi. rdst, NP. rdsta. dgir, fire : Av. dtar-, Phi JRAS. APRIL 1922. 13 13 https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THE PHONOLOGY OF SOUTHERN KURMANJI 194 dtdr, NP. dSar. bra, brother: Av. brdtar-, Phi. brat, NP. barddar. wd, wind: Av. vdta-, Phi. vat, NP. bad. hdwin, summer: Av. hdmin-, Phi. hdniin, NP. —. bd, certainly: Av. 6a-, Phi., NP. —. Tear, relatives, people: Av. kdra- Phi., NP. —. -\Zdzhu, drive, urge: Av. d-\/zi, az, Phi., NP. —. dtdr, NP. dSar. bra, brother: Av. brdtar-, Phi. brat, NP. barddar. wd, wind: Av. vdta-, Phi. vat, NP. bad. hdwin, summer: Av. hdmin-, Phi. hdniin, NP. —. bd, certainly: Av. 6a-, Phi., NP. —. Tear, relatives, people: Av. kdra-, Phi., NP. —. -\Zdzhu, drive, urge: Av. d-\/zi, az, Phi., NP. —. 2. Kj. d = Av. a, Phi. —, NP. —. dwd, down: Av. ava (seen in Phi. and NP. only as pre- fixial 5 to verbal roots). \/ndsh, become interred: Av. •yjnash. 2a. Kj. d = Av. a, Phi. a, NP. —. hawdr, an encampment or camping ground : Av. vara-, Phi. var, NP. —. hawdr, an encampment or camping ground : Av. vara-, Phi. var, NP. —. 26. Kj. a = Av. a, Phi. a, NP. a. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THE PHONOLOGY OF SOUTHERN KURMANJI 195 2. Kj. e = Av. ai, Phi. ay, NP. a, ay. V"e, come : Av. \/d-i, Phi. ^/ay, NP. \/d, ay. 3. Kj. e = Av. a, Phi. a, NP. a, u. est, bone : Av. asta-, Phi. as<, NP. ust (in ustakhioan): nezik, near : Av. nazda-, Phi., NP. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the ITI. SHORT e. 1. Kj. e = Av. a, Phi. «, NP. u. 1. Kj. e = Av. a, Phi. «, NP. u. dahem, tenth : Av. dasdma, Phi., NP. dahum* berz, high : Av. bdraz, Phi., NP. burz. perd, bridge: Av. paratu-, Phi. puhr puhl NP pul dahem, tenth : Av. dasdma, Phi., NP. dahum berz, high : Av. bdraz, Phi., NP. burz. perd, bridge: Av. paratu-, Phi. puhr, puhl, NP. pul. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THE PHONOLOGY OF SOUTHERN KURMANJI IV. LONG e Pure vowel sound as e1 in French bete, not ai diphthong. 1. Kj. e = Av. e, Phi. e, NP. e. e»na, we : Av. ehmd, Phi. emd, NP. —. eivdre, evening: Av. —, Phi. ewdrak, NP. eivdr. e»na, we : Av. ehmd, Phi. emd, NP. —. eivdre, evening: e»na, we : Av. ehmd, Phi. emd, NP. —. eivdre, evening: Av. —, Phi. ewdrak, NP. eivdr. Av. —, Phi. ewdrak, NP. eivdr. 2. Kj. e = Av. at, Phi. a, NP. a. peri, fairy: Av. pairika-, Phi. ^an&, NP. pan. pe, to : Av. pai<i, Phi. pat-, NP. 6a. &eM, girl: Av. Jcaine, PhL kanik, NP. &am 2. 3. Kj. e == Av. ai, Phi. at, NP. a. ner, male : Av. ndirya-, Phi. ndirik, NP. war. 4. Kj. e = Av. a, Phi. a, a, NP. a, a. JTter^, turf: Av. maregha-, Phi. —, NP. ? margh. pere, yester: Av. paroayar, Phi. parer, NP. parir-. stet\ star: Av. star-, -Phi. star, NP. sitar. /hezli, speak : Av. vach,- Phl. w;. NP. —. kerd, knife : Av. fozrata, Phi. kdrt, NP. Mrc?. jezAw, festival: Av. yasna-, Phi. yashn, NP. jashn. kel, cultivation: Av. harsh-, Phi. Mr, NP. kdr. pe, foot: Av. pad-, Phi. JJOT, NP. pa. meshik, brain : Av. mazga-, Phi. wiazgr, NP. maghz. 5. Kj .e = Av. a, Phi. », NP. i. \/hel, refrain, permit, leave alone : Av. \/harz, PhL, NP. 6. Kj. e == Av. a, Phi. «, NP. a, d. drezh, long: Av. drdjo-, Phi. oVo/, NP. dirdz. chesht, a meal: Av. ?, Skt. Rehash, Phi. cM«^, NP. chasht. \/ena, •\/hena, bring: Av. d-^/nay, Phi. —-, NP. —. THE PHONOLOGY OF SOUTHERN KUEMANJI 196 khue, owner : Av. khvada, Phi. khutm, NP. khuda. -en, the agent: Av. ?, Phi., NP. -an. khue, owner : Av. khvada, Phi. khutm, NP. khuda. -en, the agent: Av. ?, Phi., NP. -an. 6a. Kj. e = a in loan-words. hire, hire, rent: Ar. kird. kteb, book : Ar. kitab. 7. Kj. e = Av. a, Phi. 6, NP. il. pest, skin, bark : Av. pasto-, Phi. j»ds£, NP. pust. 8. Kj. e = Av. oe, ad, Phi. e, NP. i. ben, nose: Av. vaana-, Phi. w?m, NP. 6Tm. Mm, pus, mucus: Av. hadm-, Phi. khem, NP. Mlm. e, terminal indefinite article : Av. aeva, Phi. ev, NP. i. netva, vicinity : Av. nadma, Phi. nemak, NP. —. 9. IV. LONG e Kj. e = Av. ao, Phi. a, NP. —. •\/ezh, speak : Av. •y/aoj, aoghzh, Phi. •s/naj, NP. —. 9 j v , , •\/ezh, speak : Av. •y/aoj, aoghzh, Phi. •s/naj, NP. —. 10. Kj. e = Av. ao (i), Phi. o, 6, NP. o, u. hez, hezh, strength : Av. aoja-, Phi. qj, NP. —. ben, smell: Av. baoidhi-, Phi. 6orf, NP. bo, boi. mer, ant: Av. maoiri-, Phi. mor, NP. mur. 11. Kj. e = Av. CM, Phi. o, NP. M. &ef, mountain : Av. kaufa-, Phi. kof, NP. &«&. &ef, mountain : Av. kaufa-, Phi. kof, NP. &«&. 12. Kj. e = Av. e, Phi. 6, NP. w. rewT, fox : Av. revish-, Phi. ro&as, NP. rubah. rewT, fox : Av. revish-, Phi. ro&as, NP. rubah. 13. Kj. e = Av. id, Phi. eS, NP. —. era, hera, here : Av. iOra, Phi. eSar, NP. —. 14. Kj. e = Av. ya, Phi. ya, NP. ja. esh, pain, affliction : Av. yaska-, Phi. yos&, NP. jask. 15. Kj. e = o in loan-word. helddsh, travelling companion : Tk. yoldash. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the V. SHORT i 1. Kj. i = Av. i, Phi. i, NP. —. j , , <7isAf, all: Av. vispo-, Phi. vis, nizm, low : Av. nitem-, nisma, Phi. —. 2. Kj. i = Av. a, Phi. a, NP. a. mm, dampness : Av. namna-, Phi., NP. nam. min, me: 2. Kj. i = Av. a, Phi. a, NP. a. mm, dampness : Av. namna-, Phi., NP. nam. min, me: https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THE PHONOLOGY OF SOUTHERN KURMANJI 197 Av. mana, Phi., NP. man. Jcirdin, to do : Av. -\/kar, Phi. kartan, NP. Jcardan. Av. mana, Phi., NP. man. Jcirdin, to do : Av. -\/kar, Phi kartan, NP. Jcardan. 3. Kj. i = Av. a, Phi. a, NP. u. 3. Kj. i = Av. a, Phi. a, NP. u. 3. Kj. i = Av. a, Phi. a, NP. u. giro, gullet: Av. garah-, Phi. garuk, NP. grwZw. gnaw : Av. \/^arJ Phi. \/karin, NP. —. j , , giro, gullet: Av. garah-, Phi. garuk, NP. grwZw. gnaw : Av. \/^arJ Phi. \/karin, NP. —. 4. Kj. i = Av. a, Phi. M, NP. w. 4. Kj. i = Av. a, Phi. M, NP. w. y/hir, cut: Av. -y^^ Phi., NP- V&Mr- Vmr> die: Av. \/mar, Phi., NP. \/murd. 5. Kj. i = Av. «, Phi. —, NP. —. girj, wrinkle : Av. \/garez, Phi., NP. —. 6. Kj. i = Av. a, Phi. u, NP. u. mirishk, hen: Av. mardgha-, Phi., NP. murgh. \/pirs ask : Av. ^ipms, Phi., NP. ^purs. mirishk, hen: Av. mardgha-, Phi., NP. murgh. \/pirs ask : Av. ^ipms, Phi., NP. ^purs. 7. Kj. • = Av. oa, Phi. e, NP. e. wM, mist: Av. masgha-, Phi., NP. megh 7. Kj. • = Av. oa, Phi. e, NP. e. 8. Kj. i = Av. u, Phi. M, NP. u. diz, thief : Av. duzhda-, Phi. dwzM, NP. duzd. dizhmin, enemy : Av. dushmainyu-, Pbl., NP. dushman. bin, bottom : Av. buna-, Phi., NP. bun. bizin, goat: Av. buja-, Phi. buj, NP. buz. mist, fist: Av. mushti-, Phi. mws£, musht, NP. musht. hishk, dry: Av. Jiushka-, Phi. khushka, NP. khushk. V. SHORT i jigd, separate : Av. ?/wfo-, Phi. _/wta&, NP. ,/wrfa. 9. Kj. i = Av. ae, Phi. e, NP. i. -ire, adjectival suffix: Av. -aena, Phi. -en, NP. -m. 10. Kj. i =-• Av. i, Phi. i, NP. ?. y/chin, pluck, cull: Av. y/chinv, Phi. •\/chln, NP. \/ c^ w - y/chin, pluck, cull: Av. y/chinv, Phi. •\/chln, NP. \/ c^ w - https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THE PHONOLOGY OF SOUTHERN KUEMANJI THE PHONOLOGY OF SOUTHERN KUEMANJI 198 % Kj. I = Av. i, Phi. i, NP. i. % Kj. I = Av. i, Phi. i, NP. i. shin, wailing: Av. khshim-, Phi. shin, NP. —. •\/zh\, live: Av. \/jiv, Phi. \/zw, NP. <\/z' shin, wailing: Av. khshim-, Phi. shin, NP. —. •\/zh\, live: Av. \/jiv, Phi. \/zw, NP. <\/z' 3. Kj. i = Av. i, Phi. —, NP. a. bianu, pretext: Av. m- \/dhd, Phi. vhdnak, NP. bah 4. Kj. I = Av. i, Phi. w, NP. ». ispi, louse : Av. s^'sA, Phi. shapush, NP. shapish. 5. Kj. 2 = Av. ae, Phi. i, e, NP. £. spi, white : Av. spaeta, Phi. spet, NP. safid. qizh, locks : Av. gaesa-, Phi. <?&s, NP. ^is. ^tm, fat: Av. paem-, Phi. jotm, NP. pi. shin, blue, green: Av. khshaena; Phi., NP. Jchashin. niw, half : -Av. naema-, Phi., NP. mm. 6. Kj. % = Av. a, Phi. a, NP. a. 6. Kj. % = Av. a, Phi. a, NP. a. -v/s^, being worthy: Av. <s/khshay, Phi., NP. -\Zshay. shiw, evening meal: Av. khshafniya, Phi., NP. sham. 6. Kj. % = Av. a, Phi. a, NP. a. -v/s^, being worthy: Av. <s/khshay, Phi., NP. -\Zshay. shiw, evening meal: Av. khshafniya, Phi., NP. sham. -v/s^, being worthy: Av. <s/khshay, Phi., NP. -\Zshay. shiw, evening meal: Av. khshafniya, Phi., NP. sham. VI. LONG %. 1. Kj. I = Av.», Phi.», NP. •. 6lr, reason, understanding : Av. nra, PH., NP. inr. shir, milk: Av. khslnra-, Phi., NP. sAlr. -\/di, see: Av. Phi., NP. 1. Kj. I = Av.», Phi.», NP. •. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the 2. Kj. o = Av. ae, Phi. ?, NP. o. josh, white heat of iron : Av. \/yaesh, Phi. ?, "NP.jdsh. \//og, bursting out: Ar. ju«i» 8. Kj. o = uh in loan-word. mor, seal: NP. muhr. 9. Kj. o = Phi. m, NP. u in loan-word. khosh, pleasant: Phi. khvash, NP. khush. 10. Kj. o = o in loan-words. <o^), cannon : NP. top. loz, dust: Tk. toz. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the VII. SHORT O y/duz, Phi., NP. V^os^- j Aos/t, reasoning : Av. ushi-, Phi., NP. hosh. y/dosh, milk : Av. y/duz, Phi., NP. V^os^- 7. Kj. o = Av. u, Phi. a/, NP. —. gora, large : Av. i<^ro-, Phi. gafra, NP. —. 7a. Kj. o = tt in loan-word. 7a. Kj. o = tt in loan-word. \//og, bursting out: Ar. ju«i» VII. SHORT O This vowel is not common, nor are there sufficient words of which the derivation is clear, for comparison. In the case of o " and ", the equivalent in Av., Phi., and NP. is u, while in the word mort " myrtle ", u is seen in the NP. murad. In "the word mohh " marrow of bones ", it appears to represent Av. a in mazga-, and Phi., NP. a in mazg and maghz respectively. It occurs in the words mor " goggle-eyed ", polk " pea, pod ", rong, a kind of felt, the etymology of none of which is clear. The word tor " anger, chagrin, offence " appears to have a NP. counterpart in tor. It also appears in the loan-words from Turkish, otrdq, qondgh, koniite. VIII. LONG 5 1. Kj. 6 = Av. a, Phi. ?, NP. a. dol, a dale : Av. darena-, Phi. ?, NP. darra. VIII. LONG 5 1. Kj. 6 = Av. a, Phi. ?, NP. a. dol, a dale : Av. darena-, Phi. ?, NP. darra. 2. Kj. o = Av. ae, Phi. ?, NP. o. josh, white heat of iron : Av. \/yaesh, Phi. ?, "NP.jdsh. 2. Kj. o = Av. ae, Phi. ?, NP. o. josh, white heat of iron : Av. \/yaesh, Phi. ?, "NP.jdsh. THE PHONOLOGY OF SOUTHERN KURITANJI 199 3. Kj. o = Av. ao, Phi. 5, NP. 5, u. ro, day : Av. raochaji-, Phi. roch, NP. roz, ruz. bor, brown : Av. ?, Phi. ?, NP. bur. dosh, a load for the back: Av. daosha-, Phi. dosh, NP. dmh. 4. Kj. o = Av. ara, Phi. ava, NP. aw. ora, there : Av. avaOra, Phi. —, NP. —. no, new : Av. nava, Phi. navaJc, NP. «.««>. 4a. Kj. o = Av. ? ava, Phi. o, NP. —. -ok, attributive suffix : Av. ? avaka-, Phi. o&, NP. —. 4a. Kj. o = Av. ? ava, Phi. o, NP. —. -ok, attributive suffix : Av. ? avaka-, Phi. o&, NP. —. 5. Kj. 5 = Av. aw, Phi. ?, NP. M. ^oza, a large jar: Av. kawza-, Phi. ?, NP. tea. ^oza, a large jar: Av. kawza-, Phi. ?, NP. tea. 6. Kj. o = Av. M, Phi. o, NP. o. 6. Kj. o = Av. M, Phi. o, NP. o. Aos/t, reasoning : Av. ushi-, Phi., NP. hosh. y/dosh, milk : Av. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the NP. —. zhuzhik, hedgehog : Av. duzhaka-, Phi. zhuzhak, NP. zhuzha. NP. —. zhuzhik, hedgehog : Av. duzhaka-, Phi. zhuzhak, NP. zhuzha. 3. Kj. u = Av. a, Phi. a, NP. o. \/wush, sway, wave: Av. \/vaza, Phi. s/vaz, NP. —. qurna, corner, border: Av. Jcarana-, Phi. kandrak, NP. kandr. mughdgh, a low-lying place : Av. magha-, Phi. magh, NP. maghdk. pursha, sleet: Av. parshva-, Phi. parashveh, NP. —. 4. Kj. M = Av. a, Phi. a, NP. «. jwrg', throat, gullet: Av. garah-, Phi. garok, galok, NP. ^WZM. 5. Kj. u = Av. ao, Phi. o, M, NP. w, M. (ZMSA, last night: Av. daosh-, Phi. cfosA, NP. dush. \/kush, (ZMSA, last night: Av. daosh-, Phi. cfosA, NP. dush. \/kush, kill: Av. \/kaosh, Phi. \/kush, NP. i/kush. (ZMSA, last night: Av. daosh-, Phi. cfosA, NP. dush. \/kush, kill: Av. \/kaosh, Phi. \/kush, NP. i/kush. 6. Kj. M = Av. M, Phi. «, NP. it. tu, thou : Av. tu, turn, Phi. <i«, NP. tu. 6. Kj. M = Av. M, Phi. «, NP. it. tu, thou : Av. tu, turn, Phi. <i«, NP. tu. 6. Kj. M = Av. M, Phi. «, NP. it. tu, thou : Av. tu, turn, Phi. <i«, NP. tu. 7. Kj. M = Av. e, Phi. «, NP. u. gurchik, kidney: Av. veretka-, Phi. gurtak, NP. gurda. 7. Kj. M = Av. e, Phi. «, NP. u. gurchik, kidney: Av. veretka-, Phi. gurtak, NP. gurda. gurg, wolf: Av. vehrka-, Phi. </«»#, NP. $w<7. 8. Kj. u = Av. t, Phi. —, NP. —. y'nus, sleep : Av. m- \/si, Phi. —, NP. —. 8. Kj. u = Av. t, Phi. —, NP. —. y'nus, sleep : Av. m- \/si, Phi. —, NP. —. IX. SHORT U 1. Kj. M = Av. M, Phi. M, NP. u. amust, finger : Av. angushta-, Phi. angust, NP. angusht. duwd, the rear, behind : Av. dum-, Phi. ?, NP. dum- (in 2. Kj. u = Av. M, Phi. v, u, NP. «. ^ , a fragment, small piece: Av. Jcutaka-, Phi. —, ^ , a fragment, small piece: Av. Jcutaka-, Phi. —, THE PHONOLOGY OF SOUTHERN KUBMANJI 200 X. LONG U 1. Kj. u = Av. M, Phi. M, NP. M, u. 1. Kj. u = Av. M, Phi. M, NP. M, u. gu, excrement: Av. gudha-, Phi. guh, NP. <7«A. anu-, now: Av. TO, Phi. nun, NP. aknun. azhnu, knee: Av. zhnu-, Phi. zdnuk, NP. zam?. au, quick : Av. y^'* Phi. ZM/, NP. zud. hum, earth: Av. bumi-, Phi. 6«m, NP. bum. \/aparmu, hope : Av. a- ^/parmu, Phi. —, NP. —. 2. Kj. u = Av. a, Phi. ak, NP. a. bianu, pretext: Av. —, Phi. vhdnak, NP. bahdna. zhingii, alive: Av. —, Phi. zivandak, NP. zinda. hamu, all: Av. hama, Phi. hamak, NP. Aawia. khdnu, house (probably loan-word): Phi. khdnak, NP. khana. THE PHONOLOGY OF SOUTHERN KURMANJX 201 3. Kj. u = Av. aw, Phi. aw, ap, NP. ab. haslur, thick, heavy, strong : Av. staivra-, Phi. stawr, stapr, NP. sitabr. j p haslur, thick, heavy, strong : Av. staivra-, Phi. stawr, stapr, NP. sitabr. 4. Kj. « = Av. ao, Phi. o, u, NP. 6, u. 4. Kj. « = Av. ao, Phi. o, u, NP. 6, u. ^/ru, grow: Av. \/raoS, Phi. V^> NP. \/ ro. run, light, refulgent: Av. raokhshna-, Phi. roshan, NP. roshan. run, butter: Av. raoghna-, Phi. rolcan, NP. roghan. -\/khurush, noise, confusion: Av. khraosh-, Phi. Jchrust, NP. khurosh. pitch, empty : Av. paosh-, Phi. —, NP. pwc^. 5. Kj. M = Av. av, Phi. av, NP. w. 5. Kj. M = Av. av, Phi. av, NP. w. "v/rfw, answer, retort: Av. \/dav, Phi. -\/dav, NP. —. VC^M, go : Av. -\Zshav, Phi. —, NP. \/shu. "v/rfw, answer, retort: Av. \/dav, Phi. -\/dav, NP. —. VC^M, go : Av. -\Zshav, Phi. —, NP. \/shu. 6. Kj. M = Av. u, Phi. M, NP. u. jut, a plough : Av. yukhta-, Vhl. yukht,'NP.juft. sur, red : Av. sukhra-, Phi. swMr, NP. surkh. suchar, porcupine: Av. sukuruna-, Phi. suleur, NP. sugur. 7. Kj. M = Av. M, Phi. M, o, NP. it, o. parasu, rib: Av. paresu-, Phi. pahluk, NP. paJdu. dru, lie: Av. i/druj, Phi. darog, NP. durogh. shu, husband : Av. shudra-, Phi. SACM, NP. SAM; S^O. kuderi, where: Av. &M0ra, Phi. ?, NP. —. 7. Kj. M = Av. M, Phi. M, o, NP. it, o. parasu, rib: Av. paresu-, Phi. pahluk, NP. paJdu. dru, lie: Av. i/druj, Phi. darog, NP. durogh. shu, husband : Av. shudra-, Phi. SACM, NP. X. LONG U SAM; S^O. kuderi, where: Av. &M0ra, Phi. ?, NP. —. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the 4. Kj. u = Av. va, Phi. vd, NP. wd. 4. Kj. u = Av. va, Phi. vd, NP. wd. j , , <\/khuin, read, sing: Av. -\/khvan, Phi. khvan, NP. •%/khwdn. <\/khuin, read, sing: Av. -\/khvan, Phi. khvan, NP. •%/khwdn. THE PHONOLOGY OF SOUTHERN KUKMANJI 202 THE PHONOLOGY OF SOUTHERN KUKMANJI X I . M SIMILAR TO FRENCH U Generally speaking this vowel shows the same equivalents in other languages as does u, and is sometimes interchangeable therewith. 1. Kj. u — Av. ao, Phi. 5, NP. u, aw. gue, ear : Av. gaosha-, Phi. gosh, NP. gush, suind, oath : Av. saokenta-, Phi. —, NP. sawgand. 1. Kj. u — Av. ao, Phi. 5, NP. u, aw. gue, ear : Av. gaosha-, Phi. gosh, NP. gue, ear : Av. gaosha-, Phi. gosh, NP. gush, suind, oath : Av. saokenta-, Phi. —, NP. sawgand. 2. Kj. u = Av. a, Phi. e, NP. i. tuzh, sharp : Av. taezha-, Phi. ft??', NP. <«: 3. Kj. u = Av. m, Phi. w, NP. M. khue, owner : Av. khvaddta-, Phi. khudai, NP. khudd. 3. Kj. u Av. m, Phi. w, NP. M. khue, owner : Av. khvaddta-, Phi. khudai, NP. khudd. khue, owner : Av. khvaddta-, Phi. khudai, NP. khudd. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the 1. do. In fraosA, the upper part of the side, the side of the bosom, do represents Av. aro with loss of r, for other examples of which see consonant tables, cf. Av. barozMa-. There does not appear to be any equivalent word in Phi. or NP. The diphthong do also occurs in blao, scattered, dispersed, which has no counterpart as a separate word in either Phi. or NP., of which the origin from Av. para-, OP. -para-, suggests itself. In kldo, a hat, an equivalent a is seen in NP. kuldh, from which it is quite possibly a loan-word. 2 In the word draosh, shining, glinting, ao represents NP. af in dirafsh. Draosh is probably a NP. loan-word, ao occurs in a number of words of which the etymology is not clear, and in loan-Words from Ar., when it usually represents aw, This only occurs in loan-words, as aiwdn, NP. aiudn ; aib, Ar. 'aib; ain. Ar. '«'». This combination is very common. In loan-words it represents original t, as in gielas, from NP. gilds, the sweet cherry. In the word kierd it represents Av. a, Phi., NP. a (see also e in this value). It occurs in many words as a result of the diminutive ela, being added to a word already ending in i or I, as pshiela, a cat = pshi-ela,; birsiela, unripe grapes = bir-t/si-ela. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the ANAPTYXIS ANAPTYXIS Unlike its neighbour, NP., Kurmanji displays a repugnance to much-vocalized words, and in many cases where NP. has ANAPTYXIS Unlike its neighbour, NP., Kurmanji displays a repugnance to much-vocalized words, and in many cases where NP. has THE PHONOLOGY OF SOUTHEEN KUEMANJI 203 introduced anaptyxis, Kj. has perpetuated the original form. This is most noticeable in the case of initial pairs of consonants. p Examples : drezh, Av. drdjo-, NP. dirdz. drii, Av. draogha-, NP. durvgh. ster, Av. star-, NP. sitdra. \/shka, Av. \/schan, Examples : drezh, Av. drdjo-, NP. dirdz. drii, Av. draogha-, NP. durvgh. ster, Av. star-, NP. sitdra. \/shka, Av. \/schan, NP. \/shik. spi, Av. spaeta-, NP. sifid. stun, Av. stilna-, NP. sutun, and many others. In all these examples Phi. does not show anaptyxis. NP. \/shik. spi, Av. spaeta-, NP. sifid. stun, Av. stilna-, NP. sutun, and many others. In all these examples Phi. does not show anaptyxis. Kj. rejects short vowels in initial positions before two consonants, or short vowels between them. Examples: •s/spdr, Av. us-\/par; sldm, Ar. a^—'I Jcteb, Ar. C_>UJ ; mgheri, NP. bukhdrl; \/bwdr, Av. vi-\/tar ; Jcteb, Ar. C_>UJ ; mgheri, NP. bukhdrl; \/bwdr, Av. vi-\/tar ; •\/kr, Av. \/kar; she, Ar. n^>, and numerous others. •\/kr, Av. \/kar; she, Ar. n^>, and numerous others. In many cases Kj. is not averse to a word of two or three consonants entirely unvocalized. Such are psr, a rent or tear ; pr, full; qng, anus ; sht, thing ; pchk, small; fshk, a. spark ; \/brzk, scorch ; and many others. XII. SEMI-VOWEL w 1. Kj.M) = Av. w, Phi./, NP./. wa, towards, against : Av. aiwi, Phi., NP. af- (in verb forms). wa, towards, against : Av. aiwi, Phi., NP. af- (in verb forms). 2. Kj. w = Av. w, Phi. w, NP. 6. , cloud : Av. awra, Phi. awr, NP. a&r. 3. Kj. w = . Av. tt,-, Phi. —, NP. —. chwdr, four : Av. chadwdr. 3. Kj. w = . Av. tt,-, Phi. —, NP. —. chwdr, four : Av. chadwdr. 4. Kj. w = Av. v, Phi. i;, NP. 6. ward, a patch of ploughing : Av. \/varz, Phi. mrs, NP. barz. vjach, twig, shoot: Av. \/vakhsli, Phi. vakhsh, NP. —. wdrdn, rain : Av. wra-, Phi. vdrdn, NP. bdrdn. wafr, snow : Av. vafra, Phi. va/r, NP. &ar/V wcm, iree: Av. vand-, Phi. , NP. —. wd, wind : Av» vata-, Phi. «a§, NP. 6ad. 204 THE PHONOLOGY OF SOUTHERN KUEMANJI UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THE PHONOLOGY OF SOUTHERN KURMANJI 205 11. Kj. w = Av. h, Phi. kh, NP. kh. 11. Kj. w = Av. h, Phi. kh, NP. kh. 11. Kj. w = Av. h, Phi. kh, NP. kh. wishk, dry : Av. hushka, Phi., NP. khushk. wash, pleasant: Av. hush, Phi. khvash, NP. khush. wishk, dry : Av. hushka, Phi., NP. khushk. wash, pleasant: wishk, dry : Av. hushka, Phi., NP. khushk. wash, pleasant: Av. hush, Phi. khvash, NP. khush. Av. hush, Phi. khvash, NP. khush. Initial w in wirch, a bear, Av. aresha-, may be a change from the h augment of hirch, which is also commonly heard ; similar to h to w in wishk (see above), Phi. and NP. both show khirs. -y/wis, wish, desire: invites comparison with Av. y/vas, \/vlst as apart from Av. hvdd, hvdst, and Phi., NP. y/khvdst. -y/wis, wish, desire: invites comparison with Av. y/vas, \/vlst as apart from Av. hvdd, hvdst, and Phi., NP. y/khvdst. In the word ward, small, broken small (which also occurs as hur, hurd), w appears to represent kh in Av. y/khvar, Phi. khurtak, and NP. khurda. In the word ward, small, broken small (which also occurs as hur, hurd), w appears to represent kh in Av. y/khvar, Phi. khurtak, and NP. khurda. 12. Kj. w as augment. icushtir, camel : Av. ushtra-, Phi. ushtr, NP. shutur. wuldkh, beast of burden: NP. uldgh. wutu, flat iron: NP. utii,. 13. Kj. w = Av. t, Phi. t, NP. d,h. 13. Kj. w = Av. t, Phi. t, NP. d,h. y/bwdr, crossing over: Av. vi- y/tar, Phi. \/vitar, NP. y/guhar. dwr, fire : Av. dtar, Phi. dtdr, NP. dBar. saw, hundred : Av. sata-, Phi. sat, NP. sad. The words bwdr and dwr are seen also as bgdr and ajir, and may be developments therefrom. See XVI, 6. THE PHONOLOGY OF SOUTHERN KUEMANJI THE PHONOLOGY OF SOUTHERN KUEMANJI 5. Kj. w = Av. v, Phi. v, NP. v. 5. Kj. w = Av. v, Phi. v, NP. v. dtw, devil: Av. daava-, Phi. dev, NP. div j dtw, devil: Av. daava-, Phi. dev, NP. div 6. Kj.w = Av. t>, Phi. v, NP. #. wendsa, violent offence, quarrel: Av. ?, Phi. vinos, NP, gundh. wer, circular motion: Av. -\/vered, Phi. \/vartr NP. -\/gard. Note also Kj. m'», lost, where NP. shows gum ; and dwr, fire, against Kj. dgir. 6. Kj.w = Av. t>, Phi. v, NP. #. , , q , , , gundh. wer, circular motion: Av. -\/vered, Phi. \/vartr NP. -\/gard. Note also Kj. m'», lost, where NP. shows gum ; and dwr, fire, against Kj. dgir. 7. Kj. w = Av. /, Phi./, NP. h. Jcew, mountain : Av. kaofa-, Phi. kof, NP. kuh 8. Kj.w = Av./,Phl./,NP./. ndwik, navel: Av. ndfah-, Phi. ndfak, NP. wa/. wer, over,, forward : Av./m-, Phl./ra-, NP./ar-. 8a. Kj. w = / i n loan-words. kawsh, footgear : NP. kafsh. kawgir, ladle : NP. kafglr. 9. Kj. w = Av. p, Phi. ,p,/, NP. b, p, v. aw, water: Av. dpo-, Phi, dp, NP. db. \/nwis, write: Av. ni-\/pish, Phi. nipishtan, NP. navishtan. •y/rew, go: Av. \/rap, Phi. i/raf, NP. ^/raf^rav. -wan, a guardian: Av. pdna, Phi. -\|)aw, NP. -6<m. y^a, flee: Av. ^Jpad, Phi. —, NP. —. 10. Kj. w = Av. m, Phi. JW, NP. m. chaw, eyes: Av. chashman-, Phi., NP. chashm. zdwd, bridegroom : Av. zdmdtdr-, Phi. ddmad, NP. ddmdd. hdwin summer: Av. hdmin, Phi. hdniin, NP. —. naw>, name: Av. ndman-, Phi., NP. warn, niw, half: Av. nadma, Phi. jiem, NP. s k \/nwd, display: Av. ni- s/md, Phi., NP. •\/nimd. nwezh, prayer: Av. nemah-, Phi. namdch, NP. namdz. This last word is probably a loan-word from Phi. or NP. 10a. Kj. w = m in modern loan-words. awin, safe : Ar. A.~«i. hawir, dough : Ar. ^Jt~. kwt, awin, safe : Ar. A.~«i. hawir, dough : Ar. Jt~. kwt, chestnut-coloured : Ar. O—«J . laqdw, bridle : NP. lagdm. chestnut-coloured : Ar. O—«J . laqdw, bridle : NP. lagdm chestnut-coloured : Ar. O—«J . laqdw, bridle : NP. lagdm. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. XIV. k 1. Kj. k = Av. , Phi. k, NP. k. kamar, rock: Av. kamard-, Phi., NP. kamar. -\/kan, dig, carve: Av. \/kan, Phi., NP. s/kan. ivishk, dry; Av. hushka, Phi., NP. khushk. tak, rutting: Av. taka-, Phi., NP. tak. 2. Kj. k = Av. ?, Phi. kh, NP. kh. kinn, to buy : Av. ?, Phi. kharitan, NP. kharidan. 3. Kj. £ = Av^,Phl. M,NP. M.- Mm, pus, temper: Av. haam, Phi. Mew, NP. Mtm. 4. Kj. £ = Av. kh, Phi. M, NP. M. MM, spring, source: Av. khan-, Phi. khdnik, NP. kharii. kar, ass : Av. Mara-, Phi., NP. Mar. y'femrf, laugh : Av. \/khvand, Phi., NP. <\/khand. 5. Kj. & = Av. cA, Phi. A, NP. k. kerd, spawn: Av. chidra, Phi., NP. —-. •s/shka, break: Av. -\/soha, Phi. ^shika, NP. \/shika. 6. Kj. k = Av. #, Phi. #, NP. #. kirch, wrinkle : Av. y/garez, Phi., NP. —. kisht-, finger : Av. angushta-, Phi. angust, NP. angusht. kirch, wrinkle : Av. y/garez, Phi., NP. —. kisht-, finger : Av. angushta-, Phi. angust, NP. angusht. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the XIII. y This letter is rare, and is usually seen only in loan-words as ydkhud, NP. ydkhud ; yasdkh, Tk. yasdkh. It appears as an This letter is rare, and is usually seen only in loan-words, as ydkhud, NP. ydkhud ; yasdkh, Tk. yasdkh. It appears as an augment in yakhsir, a prisoner of war : Ar. j ^ , but this is probably borrowed from Oriental Tk. yesir. probably borrowed from Oriental Tk. yesir. CONSONANTS Guttural k kh g gh q Palatal ch j Dental t d Labial p f b m Nasal n n ng CONSONANTS Guttural k kh g gh q Palatal ch j Dental t d Labial p f b m Nasal n n ng https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the 206 THE PHONOLOGY OF SOUTHERN KURMANJT Liquids Sibilant 8 "shz zh lant XV. M 1. Kj. kh = Av. Jb, Phi. khu, NP. MM. Mo, self : Av. hva-, Phi., NP. khud (Av. hvato). khezt relatives: Av. hvadtush-, Phi., NP. khvesh. khue, owner: Av.. hvaSdta, Phi. khutai, NP. khudd. khoshk, sister: Av. hvanhar, Phi., NP. khvdhar. khur, sun : Av. hvare-, Phi., NP. Mwr. The influence of the v in Aw is seen in most of the vowels- following the Kj. kh. THE PHONOLOGY OF SOUTHERN KUEMANJI 207 2. Kj. kh = Av. k, PH. k, NP. g. sikhur, a porcupine : Av. sukuruna-, Phi. mkur, NP. sugur. 3. Kj. kh =•-• gh in loan-words. 3. Kj. kh =•-• gh in loan-words. kham, sorrow : Ar. Je-. khalat, error : Ar. Jalc-. khanb, kham, sorrow : Ar. Je-. khalat, error : Ar. Jalc-. khanb, stranger : Ar. < > jz . khuncha, bud : NP. Asdc-. zdkh, stranger : Ar. < > jz . khuncha, bud : NP. Asdc-. zdkh, alum : Ar. NP. f-lj. bdkhcha, garden : NP. Aaii. khaml, body ornaments: Ar. J J » - khend, henna : AT. U>- . 5. .Kj. kh = Tk. and North Kj. q, Ar. q. 5. .Kj. kh = Tk. and North Kj. q, Ar. q. khanjila, a pretty, small child: Tk., North Kj. qenj. nakhti, ready money: Ar. >XJu. takhdir, fate:" Ar. yXu. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the XVI. g 1. Kj. g (initial) = Av. g, Phi. g, NP. g. ga, time, place : Av. gdtu-, Phi. gas, NP. gdh. gan, fetid i Av. gainti-, Phi. gand, NP. gwwZ. ganum, wheat: Av. gantaoma-, Phi. gantum, NP. gandum. germ, warm : Av. garema-, Phi., NP. <7GWTO. #er, hill: Av. gram, Phi. —, NP. —. gd, ox : Av. gdv-, Phi. gav, NP. ^av. gdw, pace : Av. gdmay Phi., NP. <jra»i. girifdn, pouch, pocket: Av. garewa-, Phi. garivpdn, NP. ginbdn. 2. Kj. # = Av. £, Phi. g, NP. gr. ' -<7er, a worker in, maker of : Av. -kara, Phi., NP. ^rar. 3. Kj.£ = Av. k, Phi. ?, NP. . <7dza, earthen pob: Av. kawza-, NP. ifcwza. 4. Kj. <7 = Av. u, Phi. 5-, NP. g. gurch, kidney : Av. veretka-, Phi. gurt, NP. gurda. y/gur, change: Av. y/varet, Phi. gash, NP. \/gdsh, gar. gurg,. THE PHONOLOGY OF SOUTHERN KUEMANJI 208 wolf: Av. vehrka-, Phi., NP. gurg. gund, village: (Skt. vrnda-), Phi. gund, NP. ghund. wolf: Av. vehrka-, Phi., NP. gurg. gund, village: (Skt. vrnda-), Phi. gund, NP. ghund. 5. Kj. g = Av. v, Phi. v, NP. g. \/gar, turn, wander: Av. ^varet, Phi. Vwi-rf, NP. -\/gard. gish-k, all: Av. vispo-, Phi. sisp, NP. —. 6. Kj. g = Av. «, Phi. «, NP. d, 8. dgir, fire : Av. dtar-, Phi. <Mr, NP. dBar. jigd, separate: Av. yuta-, Phi. jutdk, NP. y«da. reg, line, row : Av. areta-, Phi. —, NP. rcwfo. Compare also zerg, yellow, with NP. zard; tagbtr, loan- Compare also zerg, yellow, with NP. zard; tagbtr, loan- word from Ar. j\> <—v ; jndga, loan-word from NP. piada. word from Ar. _j\> <—v ; jndga, loan-word from NP. piada _j ; j g , p 7. K\.g = Av. gh, Phi. ?, NP. ^A. merg, turf, meadow : Av. maregha-, Phi. ?, NP. margh. _j ; j g , p 7. K\.g = Av. gh, Phi. ?, NP. ^A. merg, turf, meadow : Av. maregha-, Phi. ?, NP. margh. 8. Kj.^=,Av.(7a, Phl.y,NP.y. 7i l A i/ Phi NP ^ 8. Kj.^=,Av.(7a, Phl.y,NP.y. <7iaw, soul: Av. gai/a-, Phi., NP. ^aw. 8. Kj.^=,Av.(7a, Phl.y,NP.y. <7iaw, soul: Av. gai/a-, Phi., NP. ^aw. <7iaw, soul: Av. gai/a-, Phi., NP. ^aw. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the XVII. gh The sound is rarely heard except in loan-words, usually having been hardened to kh (which see). It appears in the place-name Mughdgh, a low-lying marshy place, which is a loan-word from NP. mughak. In the loan-word mgheri, it represents NP. kh in bukhdri. XVIII. q (Ar. i3) 1. Kj. q = Av. ft, Phi. , NP. ft. qurna, corner, edge : Av. karana-, Phi. kandrak, NP. kandr. qin, anger : Av. kasna-, Phi. ken, NP. kin. quz, vulva : Av. ?, Phi. ?, NP. kus. qng, anus : Av. ?, Phi., NP. jfe«». k is seen' unchanged in Awn, a hole. There are several cases where, although k is seen changed to q, the original form is still in use, as in pishqil, pishkil; qcdadar, kaladar; qol, kol; qdliq, kdlik. 1. Kj. q = Av. ft, Phi. , NP. ft. qurna, corner, edge : Av. karana-, Phi. kandrak, NP. kandr. qin, anger : Av. kasna-, Phi. ken, NP. kin. quz, vulva : Av. ?, Phi. ?, NP. kus. qng, anus : Av. ?, Phi., NP. jfe«». k is seen' unchanged in Awn, a hole. There are several cases where, although k is seen changed to q, the original form is still in use, as in pishqil, pishkil; qcdadar, kaladar; qol, kol; qdliq, kdlik. 2. Kj. ; = Av. g, Phi. g, NP. g. qurg, gullet: Av. garah-, Phi. garuk, NP. #W£M, yM, locks qurg, gullet: Av. garah-, Phi. garuk, NP. #W£M, yM, locks : THE PHONOLOGY OF SOUTHERN KUEMANJI 209 Av. gaesa-, PH. ges, NP. ges. qirch, wrinkle : Av. -\/garez-, Phi., NP. —. The last word is also seen as girj, chirch, and kirch. 3. Kj. q = kh in loan-words. 3. Kj. q = kh in loan-words. qira, dotard : Ar. I^»J>-. qirzhdnk, crab : NP. kharchang. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on XX. j jindu, alive : Av. y^', Phi. zivanddk, NP. zinda. areju, desire: Av. -\/arej, Phi. ar/, NP. arat. -jar, a place of: Av. ?, Phi. jar, NP. ear. 2. Kj.j=Av. y, Phi. y,j, NP.j. JM<, plough : Av. yukhta-, Phi. ywM<, NP. JM/(!. jezhn, festival: Av. yasna-, Phi. yashn, NP. jashn. jerg, liver: Av. yakar-, Phi. jakar, NP. j'tgar. jVya, separate : Av. «/wta-, Phi. jwia^, NP. judd. jiiin, to chew : Av. ?, Phi. jutan, NP. jdvidan. 3. Kj.y = Av. cA, Phi. cA, NP. z. roj, light, day : Av. raochah-, Phi. rocA, NP. ruz. ja, time : Av. gdtu-, Phi. gas, NP. #a&. ja, time : Av. gdtu-, Phi. gas, NP. #a&. 5. Kj. j = Av. ? (Skt. 0, HJ. —, NP. d. 5. Kj. j = Av. ? (Skt. 0, HJ. —, NP. d. janjar, a threshing appliance: Skt. yantrd-, Phi. —, NP. jandara. janjar, a threshing appliance: Skt. yantrd-, Phi. —, NP. jandara. THE PHONOLOGY OF SOUTHERN KURMANJI THE PHONOLOGY OF SOUTHERN KURMANJI 10. Kj. ch = Av. t, Phi. t, NP. d. gurch, kidney : Av. veretka-, Phi. gurtak, NP. gurd 11. Kj. ch = Av. ?, Phi. ?, NP. kh. chilm, mucus : Av. ?, Phi.?, NP. khilm. 12. Kj. ch = Av. ?, Phi. ?, NP. jfe. A, flea : Av. ?, PH. ?, NP. kek. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the XIX. ch XIX. ch 1. Kj. ch = Av. ch, PH. ch, NP. ch. 1. Kj. ch = Av. ch, PH. ch, NP. ch. chwdr, four : Av. chadwdr, PH., NP chwdr, four : Av. chadwdr, PH., NP. chahdr. chaw, eye: Av. chashman, PH., NP. chashm. \/che, cultivate, sow: Av. -\/chay, Phi. y'c7«, NP. \/ch%. char, face: Av. chiBra-, Phi. chihrak, NP. chihra. 2. Kj. eft = Av. sft, Phi. s, NP. s. hirch, mrch, bear : Av. aresha-, PH., NP. Mt>s. 3. Kj. ch = Av. sh, PH. sA, NP. sft. gochka, ear : Av. gaosha-, Phi., NP. ^rosft. 4. Kj. ch = Av. s, PH. s, NP. s. y/chri, call, cry : Av. yVM> Phi. Vsru, NP. ^/sure. pacA-, cattle : Av. pasu-, PH. —, NP. —. y/chri, call, cry : Av. yVM> Phi. Vsru, NP. ^/sure. pacA-, cattle : Av. pasu-, PH. —, NP. —. 5. Kj. ch = Av. s, PH. 2, NP. s. pirch, locks : Av. varesa-, PH. w z , NP. gares. 6. Kj. eft = Av. —, PH. sh, NP. sft. chaqdl, jackal: (Skt. srgdla-), Phi., NP. shaghdl. 7. Kj. cA = Av. 2, 8, PH. —, NP. —. chirch, wrinkles: Av. -\/gareS, garez. china, chin: Av. zanva- (cf. NP. zanakh). chirch, wrinkles: Av. -\/gareS, garez. china, chin: Av. zanva- (cf. NP. zanakh). 8. Kj. ch = Av. k, PH. £, NP. g. suchar, porcupine: Av. sukuruna-, PH. sukur, NP. sugur. cherd, shears: Av. ^/keret, PH., NP. —. 9. Kj.cA = Av.£ ;PH. —, NP. —. / chirch, wrinkles : Av. ^/garez, Phi., NP. —. JBAS. APRIL 1922. 14 14 https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the 210 nakhte, some : Ar. X&. 5. Kj. t = Av. cA, Phi. ch, NP. z. VsMi, burn : Av. \/such, Phi. soc, NP. y/soz. 6. Kj. * = Av. s, Phi. ?, NP. cA. yw<; empty, rotten, rubbish : Av. paosh-, Phi. ?, NP. puch. Nos. 5 and 6 are more fully exemplified in Northern Kj. 6. Kj. * = Av. s, Phi. ?, NP. cA Nos. 5 and 6 are more fully exemplified in Northern Kj. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the XXI. * l.Kj. t Av. , Phi. , NP. . (w, seeds : Av. taokhma-, Phi. tokhm, NP. tukhm. ^/tw, melt, flow : Av. -\/tach, Phi. -y/vitdkh (NP. gudakhtan). ster star: Av. star-, Phi. s£ara&, NP'. sitdra. twr, hatchet: Av. ?, Phi. tabrak, NP. tabar. jut, plough: Av. yukhta-, Phi. yukht, NP. _/«/<. -\/tdsh, carve, cut: Av. -\/tash, Phi. -\/tash, NP. -y/tarash. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the 211 THE PHONOLOGY OF SOUTHERN KURMANJI 2. Kj. t = Av. t, PH. I, NP. d. nwt, felt: Av. nimata-, Phi. namat, NP. namad 3. Kj. t = Av. d, Phi. rf, NP.d. tush, difficulty, obstruction: Av. duzh-, dush-, Phi. dush-, NP. dush-. 4. Kj. £ = fl! in loan-word. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the XXIII. p. p 1. Kj. p = Av. p, Phi. p, NP. p. •\/aparmu, trust, hope: Av. a- \Zparmii, Phi., NP. —. spl, white: Av. spaeta, Phi. spet, NP. safid. spar, trust, entrust: Av. us- \/par, Phi. \/spar, NP. \/sipar. pas, small cattle: Av. pasu-, Phi. ^aA, NP. —. parasu, ribs: Av. paresu-, Phi. pahluk, NP. pahlu. pert, bridge: Av. p&rdtu-, Phi. jmM, NP. pw£. pursha, sleet: Av. parshva-, Phi. parashveh, NP. —. •\/aparmu, trust, hope: Av. a- \Zparmii, Phi., NP. —. spl, white: Av. spaeta, Phi. spet, NP. safid. spar, trust, entrust: Av. us- \/par, Phi. \/spar, NP. \/sipar. pas, small cattle: Av. pasu-, Phi. ^aA, NP. —. parasu, ribs: Av. paresu-, Phi. pahluk, NP. pahlu. pert, bridge: Av. p&rdtu-, Phi. jmM, NP. pw£. pursha, sleet: Av. parshva-, Phi. parashveh, NP. —. 2. Kj.p = Av. p, Phi. p, NP. b. pai, to : Av. paiti, Phi. potf, NP. ha. 3. Kj. p = b in loan-words. pirinj, rice : NP. birinj. sharap, wine : Ar. word borrowed 3. Kj. p = b in loan-words. pirinj, rice : NP. birinj. sharap, wine : Ar. word borrowed through NP. sharab. Jchardp, bad : Ar. <^J\J>-. tapl, drum 3. Kj. p b in loan words. pirinj, rice : NP. birinj. sharap, wine : Ar. word borrowed through NP. sharab. Jchardp, bad : Ar. <^J\J>-. tapl, drum Ar. JJ». 4. Kj. p = Av. v, Phi. v, NP. g (see also Kj. b, 3 and 4). 4. Kj. p = Av. v, Phi. v, NP. g (see also Kj. b, 3 and 4). pirch, locks: Av. varesa-, Phi. mrs, NP. ^rars. \/ysr> break, apart: Av. vi- -y/sard, Phi. visastan, NP. gusistan \/pshew, being confused, perturbed : ? Av. vi- ^Jlchshiw, Phi., NP. —. 5. Kj. p = / i n loan-words. 5. Kj. p = / i n loan-words. 5. Kj. p = / i n loan-words. sipla, humble, mean: Ar. 4l/Lw. pata, the Ar. proper XXII. d 1. Kj. d = Av. d, Phi. d, NP. d. rfaw, tooth : Av. dantan, Phi. dantan, NP. danddn. danik pip: Av. ddno-, Phi. ddnak, NP. ddnah. derzi, needle: Av. i/darez, Phi. cfarz, NP. fai (tailor), y^f) ear : Av. V^ar, PH., NP. V»r- V<K give: Av. A,/^, PH., NP. \/da. 2. Kj. d = Av.;, Phi. z, NP. z. \/rfo, strike: Av. -\/jan, Phi., NP. -\/zan. Compare also Kj. y/doz, seek, find, with 'NV.justan from OP. -\/jad. \/rfo, strike: Av. -\/jan, Phi., NP. -\/zan. Compare also Kj. y/doz, seek, find, with 'NV.justan from OP. -\/jad. 3. In the following two words—dast, hand, dost, friend—lie one of the incongruities of Kj. As original z does not become d it would appear that dast and dost must (a) have been borrowed from PH. or NP- if Kj. is a non-Persian language, and supplanted the words zast and zost, which would have followed Av. zasta- and zaosha-, (b) be original words in a Persian language. 3. In the following two words—dast, hand, dost, friend—lie one of the incongruities of Kj. As original z does not become d it would appear that dast and dost must (a) have been borrowed from PH. or NP- if Kj. is a non-Persian language, and supplanted the words zast and zost, which would have followed Av. zasta- and zaosha-, (b) be original words in a Persian language. A further difficulty is, however, presented by the word ward, a cultivated plot, against which z is seen in Av. ^/varz, Phi. Vvarz, NP. THE PHONOLOGY OF SOUTHERN KURMA.NJI 212 XXV. / 1. K j . / = Av./,Phl./,NP./. ferd, broad, ample: Av. fradah, Phi. frdh, NP. ferdkh. i/frush, sell: Av. fra-\fvdkhsh, Phi. ferush, NP. feriish. ferd, broad, ample: Av. fradah, Phi. frdh, NP. ferdkh. i/frush, sell: Av. fra-\fvdkhsh, Phi. ferush, NP. feriish. 2. Kj./=Av./, PH./.NP.A. &\|f, mountain : Av. Jcaufa-, Phi. &o/, NP. &MA &\|f, mountain : Av. Jcaufa-, Phi. &o/, NP. &MA \| 3. Kj./ = Av. ?,Phl.p,NP./. V&qft fall: Av. V?a'» Phl- V^A NP. V^- 4. Kj. / = Av. p, Phi. p, NP. j9. y/er, fly : Av. parena-, Phi., NP."\/?w- y/er, fly : Av. parena-, Phi., NP."\/?w- 5. Kj./ = NP. hh. fenuk, cool: NP. Tchunuk. 5. Kj./ = NP. hh. 5. Kj./ = NP. hh. fenuk, cool: NP. Tchunuk. fenuk, cool: NP. Tchunuk. XXIV. 6 (6 is seldom seen except initially, changing to w medially and finally.) (6 is seldom seen except initially, changing to w medially and finally.) (6 is seldom seen except initially, changing to w medially and finally.) 1. Kj. b = Av. b, Phi. b, NP. b. 1. Kj. b = Av. b, Phi. b, NP. b. 1. Kj. b = Av. b, Phi. b, NP. b. j ba, certainly: Av. bd, Phi., NP. —. bar, breast, front: Av. para-, Phi., NP. —. berz, high : Av. bdrdz, Phi. burz, NP. —. bra", brother : Av. brdtar, Phi. bratar, NP. berdd ba, certainly: Av. bd, Phi., NP. —. bar, breast, front: Av. para-, Phi., NP. —. berz, high : Av. bdrdz, Phi. burz, NP. —. bra", brother : Av. brdtar, Phi. bratar, NP. berdd THE PHONOLOGY OF SOUTHERN KUEMANJI 213 bin, the bottom: Av. buna-, Phi., NP. bun. bizin, goat: Av. buja-, Phi. buj, NP. buz. 2. Kj. b = Av. v, Phi. v, NP. 6. 2. Kj. b = Av. v, Phi. v, NP. 6. 6a/r, snow : Av. vafra-, Phi. vafr, NP. 6ar/. 6ew, snout: Av. vaena, Phi. •yem&, NP. binl. bd, wind : Av. vdta-, Phi. w«, NP. bad. 3. Kj. 6 (i) = Av. v (i), Phi. i; (i), NP. #w. 3. Kj. 6 (i) = Av. v (i), Phi. i; (i), NP. #w. \/bwdr, passing over: Av. vi- i/tar, Phi. -y/vitar, NP. gudhar. ^/bzhdr, choosing from : Av. vi- \/char, Phi. •s/vchar NP. ^Jguzar. bindsa, quarrel, violent offence : (Skt. vi- •\/na?)> Phi. mwas, NP. gundh. bardz, hog : Av. vardza-, Phi. mraz, NP. gurdz. 4. Kj.6 = Av. ?, Phi. $r, NP. flr. Mrs, hunger : Av. ?, Phi. gursah, NP. <7«r 5. Kj.6 = Av./, Phi./, NP./. 6ar, cause, reason: Av. fra-, Phi. freh, NP. ^riA. ?>ar, loose, free : Av. fra-, Phi. frdj, NP. _/iraz. 6ar, cause, reason: Av. fra-, Phi. freh, NP. ^riA. ?>ar, loose, free : Av. fra-, Phi. frdj, NP. _/iraz. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the XXVI. m 1. Kj. m = Av. m, Phi. m, NP. m. mesh, fly: Av. makhshi-, Phi. magas, NP. magas. pirn, f t A Phi j (NP jt ) i d mesh, fly: Av. makhshi-, Phi. magas, NP. magas. pirn, fat: Av. poew»«»» Phi. jwm (NP. jtrc). nim, dampness: https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THK PHONOLOGY OF SOUTHERN KURMANJI 214 Av. namna-, Phi., NP. nam. maz, mas, large: Av. mas, Phi. mas, NP. mih. mdsi, fish : Av. masya-, Phi. mdhik, NP. main. Av. namna-, Phi., NP. nam. maz, mas, large: Av. mas, Phi. mas, NP. mih. mdsi, fish : Av. masya-, Phi. mdhik, NP. main. 2. Kj. TO = Av. ii, Phi. v, NP. 6. zmdn, tongue : Av. hizu-, Phi. MZW, NP. zaban. zmdn, tongue : Av. hizu-, Phi. MZW, NP. zaban. 3. Kj. TO = b in loan-words. ghumar, mist: Ar. ^l^. mghen, stove: NP. c£jli4. mhdna, pretext: NP. 4) l^j . 4. Kj. TO = Av. ng, Phi. ng, NP. w#. amust, finger : Av. angushta, Phi. angust, NP. angush amust, finger : Av. angushta, Phi. angust, NP. angush XXVII. n 1. Kj. » = Av. », Phi. w, NP. n. ganum, wheat: Av. gantaoma-, Phi. gantum, NP. gandum. chan, some : Av. chvant-, Phi. chvant, NP. chand. nawa, offspring: Av. wa^a, Phi. ndf, NP. navddah. ner, male: Av. nairya-, Phi., NP. new. m'zwi, low: Av. nitem, Phi., NP. —. •y/nwd, display : Av. ni- -\/md, Phi. \/nima, NP. 2. Kj. w = Av. m, Phi. w, NP. ». 2. Kj. w = Av. m, Phi. w, NP. ». han-, verbal prefix: Av. ham, Phi., NP. an-, shin, lamentation : Av. hhshim-, Phi. shin, NP. —. han-, verbal prefix: Av. ham, Phi., NP. an-, shin, lamentation : Av. hhshim-, Phi. shin, NP. —. 3. Kj. n = Av. ?, Phi. n, NP. TO. , roof : Av. ?, Phi. ban, NP. Jam. 3. Kj. n = Av. ?, Phi. n, NP. TO. , roof : Av. ?, Phi. ban, NP. Jam. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the XXVIII. n I have used this symbol to indicate nasalization following a long vowel. The sound is nearly the same as that heard in Urdu, but is less marked. There are but few words in Kj. which possess it. The NP. words tazi greyhound, taj crown, bdzi gaming, have been borrowed and nasalized to lanzhi, tdnj, and banzi. The NP. words tazi greyhound, taj crown, bdzi gaming, have been borrowed and nasalized to lanzhi, tdnj, and banzi. THE PHONOLOGY OF SOUTHERN KUEMANJI 215 Other words in which it occurs are Jconder, where, fo huder; lawenderl, there, for lawederi; lalngir, a partisan, fo lalgir ; spinddr, poplar, for sjnddr ; and possibly Kurmdnj, for Kur(d)-mdd. XXIX. ng g This is the guttural nasal, pronounced as in English "hang", "bang". g This is the guttural nasal, pronounced as in English "hang", "bang". 1. K].ng=Av. n, Phi. —, NP. —. mdng, moon : Av. rnaonha-, mdh, Phi., NP. md 2. Kj. ng = Av. n-g, Phi. n-g, NP. n-g. angust, finger: Av. angushta-, Phi. angust, NP. angusht. , angwen, honey: Av. ?, Phi. angumen, NP. angubin. rang, colour: (Skt. ranga-), Phi. —, NP. rang. 3. Kj. ng Av. ?, Phi. n, NP. ». 5^, anus: Av. ?, Phi., NP. him. Loan-word hdnga, a mine : NP. kdn. Loan-word ddnga, a pimple : NP. ddna. 4. Kj. ng = Av. nt, Phi. w£, NP. nd. 5 , anus: Av. ?, Phi., NP. him. Loan word hdnga, a mine : NP. kdn. Loan-word ddnga, a pimple : NP. ddna. 4. Kj. ng = Av. nt, Phi. w£, NP. nd. j g , , chang, some: Av. chvant, Phi. chvant, NP. chand. fang idea : Av. pantan, Phi., NP. JNMMZ. 5. Kj. mgr = Av. «d, Phi. nd, NP. «d. fowler, bond : Av. -\Zband, Phi., NP. band 6. Kj. ?«7 = nd in loan-word. 6. Kj. ?«7 = nd in loan-word. mangil, kerchief : Ar. J mangil, kerchief : Ar. J XXX. r 1. Kj. r = Av. r, Phi. r, NP. r. asr, tears : Av. asm, Phi. ars, NP. ars. dgir, fire: Av. ator-, Phi. dtdr, NP. ddhar. bar, a reason : Av./ra-, Phl./reA, NP. _/mL hushtir, camel: Av. ushtra, Phi. ushtir, NP. shutur. \fresh, flow: Av. \/rech, Phi., NP. thigh : Av. rawa-, Phi., NP. raw. 2. Kj. r = Av. r, Phi. r, NP. i. geru, gullet: Av. garah-, Phi. garuk, NP. ^MZW 216 THE PHONOLOGY OF SOUTHERN KURMANJI XXXI. r (alveolar, strongly trilled) The use of r, though quite distinct from that of r, does not appear to be the subject of any readily detected rule. In the words par feather, -\/bir cut, the r has its counterpart in NP. In other words, such as pr, full, many : Av. per'na, Phi., NP. pur ; rawa, a drove : NP. rama ; i/rif, snatch away : NP. f/rub; rash, black: NP. rakhsh (Firdausi), the cause of its strengthening is not evident. The sound ia very widely used, and is one of the commonest characteristics of the language. XXXI. r (alveolar, strongly trilled) The use of r, though quite distinct from that of r, does not appear to be the subject of any readily detected rule. In the words par feather, -\/bir cut, the r has its counterpart in NP. In other words, such as pr, full, many : Av. per'na, Phi., NP. pur ; rawa, a drove : NP. rama ; i/rif, snatch away : NP. f/rub; rash, black: NP. rakhsh (Firdausi), the cause of its strengthening is not evident. The sound ia very widely used, and is one of the commonest characteristics of the language. XXXI. r (alveolar, strongly trilled) The use of r, though quite distinct from that of r, does not appear to be the subject of any readily detected rule. In the words par feather, -\/bir cut, the r has its counterpart in NP. In other words, such as pr, full, many : Av. per'na, Phi., NP. pur ; rawa, a drove : NP. rama ; i/rif, snatch away : NP. f/rub; rash, black: NP. rakhsh (Firdausi), the cause of its strengthening is not evident. The sound ia very widely used, and is one of the commonest characteristics of the language. XXXII. I 1. Kj. Z=Av. r, Phi. I, NP. I. gill, complaint: Av. gereza-, Phi. gildk, NP. gileh. 2. Kj. 1 = Av. r, Phi. r, NP. r. gil, detention, holding: Av. \/garw, Phi. -\/graf, NP gir. halata, hill: Av. haraifi-, Phi. har, NP. —. 3. Kj. I = Av. n, Phi. n, NP. n. kul, blunt: Av. ? (Skt. kuntha-), Phi. ?, NP. kund. judl, young, pretty: Av. yuvan, Phi. javdnak, NP. javdn. mdl, house: Av. nmana-, Phi. man, NP. man. ruwdla, rola, beloved, soul: Av. urvan-, Phi. ruvdn, NP. rawdn. zil, large, powerful, heavy: Av. zainti-, Phi. zand, NP. —. la, at upon, from : Av. ana, Phi., NP. —. 4. Kj. I = Av. t, Phi. t, NP. t. kul, short: Av. kutdha-, Phi. kittdk, NP. kutdh. 5. Kj. I = Av. t, Phi. «Z, NP. d. khuld, God : Av. khvadhdta-, Phi. khuddk, NP. khudd. 6. Kj. Z = tZ in loan-word &KZ, key: NP. Mia!. XXXIII. I 1. Kj. _Z = Av. r, Phi. r, NP. r. gulch, kidney : Av. veretka-, Phi. gurtak, NP. gurda. doi gulch, kidney : Av. veretka-, Phi. gurtak, NP. gurda. doi vale: Av. darend, Phi. ?, NP. darah. ^kel, cultivation: vale: Av. darend, Phi. ?, NP. darah. ^kel, cultivation: https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THE PHONOLOGY OF SOUTHERN KURMANJI 217 Av. y/karsh, Phi. \/kdsh, Tear, NP. \/lcdr. sipul, spleen: Av. spereza-, Phi. sparz, NP. sipurz. 2. Kj. Z = Av. r, Phi. I, NP. L sal. year : Av. sareta-, Phi., NP. saL i/mal, rub, sweep : Av. \/marz, Phi., NP. -y/wuiK. \/Ae& allow: Av. -y/harz, Phi., NP. V ^ - 2. Kj. Z = Av. r, Phi. I, NP. L sal. year : Av. sareta-, Phi., NP. saL i/mal, rub, sweep : Av. \/marz, Phi., NP. -y/wuiK. \/Ae& allow: Av. -y/harz, Phi., NP. V ^ - https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the XXXIV. s 1. Kj. s = A v . s, Phi. s, NP. s. asr, tears : Av. asru-, Phi., NP. ows. sipul, spleen : Av. spereza-, Phi. sparz, NP. sipurz. sal, year: Av. sareta-, Phi., NP. saL es£, bone: Av. asta-, Phi. asi, NP. ws£-. hastur, thick, heavy : Av. stawra-, Phi. stapr, NP. sitabr. 2. Kj. s = Av. s, Phi. sA, NP. sft. 2. Kj. s = Av. s, Phi. sA, NP. sft. ispi, louse: Av. spish-, Phi. shupush, NP. shipish. Tees-, tortoise : Av. kasyapa-, Phi. ?, NP. kashaf. 3. Kj. s = Av. sft, Phi. s, sA, NP. sh. anust, finger: Av. angushta-, Phi. angust, NP. angusht. mist, fist: Av. mushti-, Phi., NP. mushl. i. Kj. s = Av. 0 (r), Phi. s, NP. s. awis, pregnant: Av. apudra, Phi. awws, NP. aids, pas, guarding : Av. (padra, Phi. pas, NP. pas. 5. Kj. s = Av. 0, Phi.' z, NP. z, bdsik, arm: Av. bdzu-, Phi. bdzdk, NP. 6a2M. brus-, flashing, sparks : Av. -\/braz, Phi., NP. —. bdsik, arm: Av. bdzu-, Phi. bdzdk, NP. 6a2M. brus-, flashing, sparks : Av. -\/braz, Phi., NP. —. 6. Kj. s = z in loan-word. 6. Kj. s = z in loan-word. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the terras, clean : Ar. ^ J ' . terras, clean : Ar. ^ J ' . 7. Kj. s = Av. s, Phi. A, NP. h. pas, sheep, small cattle : Av. pasu-, Phi. pah, NP. —. dsik, gazelle : Av. dsu-, Phi. dhik, NP. dhu. mast, fish : Av. masya-, Phi. mdhik, NP. waM. 8. Kj. s = Av. s (2), Phi. s, NP. A. mas, great: Av. was-, maz, Phi. mas, NP. wiA. dsin, 8. Kj. s = Av. s (2), Phi. s, NP. A. mas, great: Av. was-, maz, Phi. mas, NP. wiA. dsin, mas, great: Av. was-, maz, Phi. mas, NP. wiA. dsin, mas, great: Av. was-, maz, Phi. mas, NP. wiA. dsin, https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the 218 THE PHONOLOGY OF SOUTHERN KUEMANJI iron : Av. —, Phi. asm, NP. dhan. bendsa, quarrel, offence of violence : Av. —, OP. vi-ndtha, Skt. vi- -y/nac, Phi. vinos NP. gundh. XXXV. sh 1. Kj. sh = Av. sh, Phi. sh, NP. sh. 1. Kj. sh = Av. sh, Phi. sh, NP. sh. j , , drawsh, awl: Av. drafsha-, Phi. drafsh, NP. dirafsh. dush last night: Av. daoshatara-, Phi. dosh, NP. dosh. hishk, dry: Av. hushka-, PH., NP. khushk. hushtir, camel: Av. ushtra-, Phi. ushtir, NP. shutur. nishiw, precipice: Av. nikhshvaepa-, Phi. nishep, NP. nishib. pursha, sleet: Av. parshva-, Phi. parashveh, NP. —. \/^> wash : Av. -\/shu Phi., NP. y/shust. shd, glad, rejoicing: Av. \/shd, Phi. sM<, NP. sMd. 2. Kj. S/J = Av. s, Phi. a, NP. s. asft, millstone: Av. as-man, Phi. as-ydw, NP. ds-ydb. gisht, all: Av. vispo-, Phi. -ws£, -wsp, NP. —. esft, pain, hurt: Av. yaska-, Phi. «/as&, NP. jask. 3. Kj. sft = Av. s, Phi. s, NP. sA. i!as&, dead flesh : Av. nasu-, Phi. nasdy, NP. ZasA. i!as&, dead flesh : Av. nasu-, Phi. nasdy, NP. ZasA. 4. Kj. sh = s in loan-words. yakhshir, prisoner of war: Ar. loan-word through Tk.^~«~J . 4. Kj. sh = s in loan-words. yakhshir, prisoner of war: Ar. loan-word through Tk.^~«~J . 5. Kj. sA = Av. eh, Phi. cA, NP. —, z. ' -sh, enclitic meaning, and, also : Av. -cha, Phi. -ch-, NP. — •\/rish, outpour: Av. \/rech, Phi. \/rech, NP. \/riz. 5. Kj. sA = Av. eh, Phi. cA, NP. —, z. j -sh, enclitic meaning, and, also : Av. -cha, Phi. -ch-, NP. — •\/rish, outpour: Av. \/rech, Phi. \/rech, NP. \/riz. 6. Kj. sh = Av. —, Phi. h, NP. h. kdsh (in kdshkeshan), chaff: Av. ?, Skt. &aca, Phi., NP. hah 7. Kj.sA=Av. z, Phi. z, NP. z. meshik, marrow, brain : Av. mazga-, Phi. mazg, NP. magh 8. Kj. sft = Av. z, Phi. sA, NP. aft. \/dosh, milk : Av. \/^M2;. Phi., NP- ^d 9. Kj. sft = Av. acft, Phi. s, NP. s. jwsft after behind : Ax pascha Phi pas NP pas shuen 9. Kj. sft = Av. acft, Phi. s, NP. s. 9. Kj. sft = Av. acft, Phi. s, NP. s. jwsft, after, behind : Ax.pascha, Phi. pas, NP. pas. shuen, traces, place, consequences: Av. ? -y/scfta, Phi., NP. —. jwsft, after, behind : Ax.pascha, Phi. pas, NP. pas. shuen, traces, place, consequences: Av. ? -y/scfta, Phi., NP. —. THE PHONOLOGY OF SOUTHERN KURMANJI 219 XXXVI. z 1. Kj. z = Av. z, Phi. z, NP. z. . 1. Kj. z = Av. z, Phi. z, NP. z. . y/za, be born.: Av. y/zan, Phi., NP. \/zd. bardz, boar: Av. vardza-, Phi. vardz, NP. gurdz. miz, urine : Av. y/maez, Phi. y/mez, NP. y/mlz. zamdn, tongue: Av. hizu-, Phi. uzvdn, NP. zabdn. 1. Kj. z = Av. z, Phi. z, NP. z. . y/za, be born.: Av. y/zan, Phi., NP. \/zd. bardz, boar: Av. vardza-, Phi. vardz, NP. gurdz. miz, urine : Av. y/maez, Phi. y/mez, NP. y/mlz. zamdn, tongue: Av. hizu-, Phi. uzvdn, NP. zabdn. 2. Kj. z = Av. z, Phi. 5, NP. <Z. y/zdn, know: Av. \Zzdn, OP. y/ddn, Phi., NP. \/ddn. zdicd, bridegroom: Av. zdmdtar, Phi. damddh, NP. ddmdd. az, I : Av. azem, OP. aiawi, Phi., NP. —. zer, heart: Av. -, Phi., NP. dil. 3. Kj. z = Av. 2, Phi. z, zft, NP. z. eKz, thief: Av. daozhda-, Phi. duzhd, NP. (ZMZCZ. muz, wage: Av. mizhda-, Phi., NP. muzd. 3. Kj. z = Av. 2, Phi. z, zft, NP. z. eKz, thief: Av. daozhda-, Phi. duzhd, NP. (ZMZCZ. muz, wage: Av. mizhda-, Phi., NP. muzd. 4. Kj. z = Av. j , Phi. ;, NP. z. ten, goat: Av. buja-, Phi. &w/a, NP. 6MZ. s/zar, bray : Av. V i ^ , PU- ?, NP. v/^a»'- 5. Kj. z = Av. cfc, Phi. i, NP. z. 5. Kj. z = Av. cfc, Phi. i, NP. z. -\/pdrez, abstain, protect, defend : Av. paiti- \Zraech, Phi. •\/pahrej, NP. parhiz. 6. Kj. z = Av. «, Phi. sh, NP. sA. M?z, relatives : Av. khvaetu-, Phi., NP. Jchvesh. 7. Kj. z = Av. s, Phi. s, NP. s. ziw, silver: Av. simd, Phi. asm, NP. slm. nizm, low: Av. nisma, PH., NP. —. gwz, uterus : Av. ?, Phi. ?, NP. &ws. 7. Kj. z = Av. s, Phi. s, NP. s. ziw, silver: Av. simd, Phi. asm, NP. slm. nizm, low: ziw, silver: Av. simd, Phi. asm, NP. slm. nizm, low: Av. nisma, PH., NP. —. gwz, uterus : Av. ?, Phi. ?, NP. &ws. z , , , z , Av. nisma, PH., NP. —. gwz, uterus : Av. ?, Phi. ?, NP. &ws. 8. Kj. z = Av. ?, Phi. sft, NP. sh. zik, belly : Av. ?, Phi. shikambu, NP. shikam. 8. Kj. z = Av. ?, Phi. THE PHONOLOGY OF SOUTHERN KURMANJI THE PHONOLOGY OF SOUTHERN KURMANJI 220 https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the XXXVI. z sft, NP. sh. zik, belly : Av. ?, Phi. shikambu, NP. zik, belly : Av. ?, Phi. shikambu, NP. shikam. 9. Kj. z = Ar. <_/> i«O. 9. Kj. z = Ar. <_/> i«O. azdb, Ar. c_jlAfr. awE/ct, Ar. O 9 W». zaror, Ar. qdzi, Ar. ^_^s»». zdlim, Ar. JU. Aaz, Ar. JO qdzi, Ar. ^_^s»». zdlim, Ar. JU. Aaz, Ar. JO https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to th THE PHONOLOGY OF SOUTHERN KUEMAN.TI 221 https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the ASPIRATE XXXVIII. h XXXVII. zh 1. Kj. zh = Av. zh, Phi. z, j , NP. z. zhnu, azhnii, Jmee : Av. zhnu-, PH., NP. zanu = Av. zdnu~ tuzh, sharp : Av. taezha-, tizhi-, Phi. tej, NP. tez. 2. Kj. zft = Av. z, Phi. 2, NP. z. dzhdr, torment: Av. a- \/zar, Phi., NP. dzdr. -\/dzhut urge, drive: Av. a- i/az, Phi., NP. —. 3. Kj.zh= Av. z, Phi. zh, NP. z. hazhdr, wretched, miserable : Av. ^/zar, Phi. zhdr, NP. zar. 3. Kj.zh= Av. z, Phi. zh, NP. z. hazhdr, wretched, miserable : Av. ^/zar, Phi. zhdr, NP. zar. 4. Kj. zh = Av. y, Phi. i, NP. —. MzA, strength : Av. aoja-, Phi. oj, NP. —. 5. Kj. zh = Av. y, Phi. z, NP. zA. zMr, chamber, cellar : Av. jafra-, Phi. zufar, NP. Z^GM/. 6. Kj. zh = Av.;, Phi. z, NP. z. zyfo'w, woman : Av. jeni-, Phi., NP. zan. \/zhi, live : Av. •\/ji, Phi. •y/ziv, NP. v ^ - -\/;Z^aw; churn, beat: Av. \/jan, Phi., NP. -\Zzan. zhdr, poison ; Av. jadra, Phi., NP. zahr. zyfo'w, woman : Av. jeni-, Phi., NP. zan. \/zhi, live : Av. •\/ji, Phi. •y/ziv, NP. v ^ - -\/;Z^aw; churn, beat: Av. \/jan, Phi., NP. -\Zzan. zhdr, poison ; Av. jadra, Phi., NP. zahr. 7. Kj. zh = Av. cA, Phi. cA, NP. z. •\frezh, pour: Av. \/rech, Phi. •\/rech, NP. vV2- "o^r light, day: Av. raochah-, Phi. rocA, NP. rwz. 8. Kj. zA = ch in loan-word. halozha, plum : NP. dlucheh. 9. Kj. zA = Av. s, Phi. s, NP. s. qizh, locks : Av. gaesa-, Phi., NP. <7& 10. Kj. zh = Av. s, Phi. sA, NP. sA. jezhn, festival: Av. yasna, Phi. yashn, NP. jashn. 11. Kj. zA = Av. sA, Phi. s, sh, NP. s, sh. ^/chezh, taste: Av. Rehash, Phi., NP. Rehash. -\/kuzhr kill: Av. -\/kaosh, Phi., NP. \/Jcush. mizh, midge: Av> makhshi-, Phi., NP. magas. 12. Kj. z& = Av. <Z, Phi. z, NP. zA. zhuzhik, hedgehog: Av. duzhaka-, Phi. zuzak, NP. zhuzhik, hedgehog: Av. duzhaka-, Phi. zuzak, NP. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THE PHONOLOGY OF SOUTHERN KUEMAN.TI XXXVIII. h Occurs only initially, not having survived medially or finally. 1. Kj.»=Av.A,Phl.A, NP.A. hdwan, mortar: Av. hdvana-, Phi., NP. hdvan. hdwin, summer: Av. hdmin, Phi. hdmin, NP. —. hdwan, mortar: Av. hdvana-, Phi., NP. hdvan. hdwin, summer: Av. hdmin, Phi. hdmin, NP. —. 2. Kj. h = Av. h, Phi. —, NP. —. hagar, if: Av. ha-kara-, Phi., NP. agar. han-, verbal prefix: Av. ham-, Phi., NP. an-, hi, genitive particle: Av. he, OP. hya, Phi. i, NP. i. (the izafa). 3. Kj. h = Phi. A, NP. —. harmi, pear : Phi. hormod, NP. armii 4. Kj. A = Av. &, Phi. kh, NP. M. taM, dry: Av. hushka-, Phi., NP. khushk. hu, hog: Av. hu-, Phi., NP. Mu&. 5. Kj. h = Av. —, Phi. kh, NP. M. 5. Kj. h = Av. —, Phi. kh, NP. M. hilka, hek, egg: Av. ?, Phi. khdik, NP. khdya. hirch, bear : Av. aresha-, Phi., NP. fcfors. gg bear : Av. aresha-, Phi., NP. fcfors €. Kj. A = NP. M. €. Kj. A = NP. M. Aewifc, cool: NP. khunuk. j Aewifc, cool: NP. khunuk. 7. Kj. h = kh in loan-word. hawir, dough : Ar. j-2~. 7. Kj. h = kh in loan-word. 7. Kj. h = kh in loan-word. XXXIX. h The guttural aspirate, which is not natural to Kj., being borrowed from Arabic. It is not always preserved in loan- words from Arabic, tending to become a simple aspirate, as in haz, Ar. Ja.>-; hafr, Ar. yi>-; hazir, Ar. ^>\o- in haz, Ar. Ja.>-; hafr, Ar. yi>-; hazir, Ar. ^>\o- In one case only has h become h, namely in hatet, seven : Av. hapta-, which is, however, only thus pronounced in a small area in the south. hawir, dough : Ar. j-2~. 8. Kj. h = y in loan-words. hafiw, orphan : Ar. ^J . helddsh, travelling companion : hafiw, orphan : Ar. ^J . helddsh, travelling companion : Tk yolddsh hewdsh gently : Tk yawdsh hafiw, orphan : Ar. ^J . helddsh, travelling companion : Tk ldd h h d h l Tk d h hafiw, orphan : Ar. ^J . helddsh, travelling companion : Tk. yolddsh. hewdsh, gently : Tk. yawdsh. Tk. yolddsh. hewdsh, gently : Tk. yawdsh. Tk. yolddsh. hewdsh, gently : Tk. yawdsh. 9. Kj. h = Kj. w. hirch = wirch. hishk = wishk. hendsa = wendsa. hurd= wurd. hirch = wirch. hishk = wishk. hendsa = wendsa. hurd= wurd. 10. Kj. h occurs as augment before a or d. (a) In the following original words it is an augment: \Aasa, be easy : Av. s/sl. hasp, horse : Av. aspo-. hawr, (a) In the following original words it is an augment: \Aasa, be easy : Av. s/sl. hasp, horse : Av. aspo-. hawr, https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THE PHONOLOGY OF SOUTHERN KURMANJI 222 cloud: Ay. aivra-. hawrdz, ascent: Av. aiwi-rasta. -\/ha come: Av. -\/ai. hdzh, power: Av. aoja-. \/han, bring: Av. d- i/nay. (b) In the following loan-words: halozha: NP. alucha. handr : NP. andr. hanjvr : NP. anjlr. harzan : NP. arzdn hmr : Ar. ^~wi. hdjiz: Ax. js~\£. halak: Tk. alak ; and many others. y (c) In hastur, Av. stawra-, Phi. stavr, NP. sitabr; and in hawdr (camping ground), Av. vara-, Phi. var, NP. —, it appears with -a, as ha augment. y (c) In hastur, Av. stawra-, Phi. stavr, NP. sitabr; and in hawdr (camping ground), Av. vara-, Phi. var, NP. —, it appears with -a, as ha augment. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the LOSSES OF OEIGINAL LETTERS AND TREATMENT OF SOME CONSONANT GROUPS 1. The commonest loss is that of original d and t, which is supported by hundreds of examples both ancient and modern. The dental is, however, preserved in the initial position. After a long vowel, between vowels, and after n, it invariably disappears. In all the following examples both Phi. and NP. preserve the original t, the former as t or d, the latter as d. ganum, wheat: Av. gantaoma-. wd, wind: Av. vdta-. gan, fetid: Av. gainti-. -\/gar, turn: Av. \/veret. ham, which : Ab. Jcatama-. dzd, brave ; Av. dzdta-. hi, will»w : Av. vaeiti-. spi, good, pretty, white : Av. spaeta-, etc. 2. Original thr preceded by a short vowel becomes dr, while THE PHONOLOGY OP SOUTHERN KURMANJI 223 in Phi. and NP. original short vowel is preserved and thr changed to tr or hr. char, face : Av. chithra-, Phi. chihr, NP. chih char, face : Av. chithra-, Phi. chihr, NP. chihra. zhdr, poison : Av.jathra-, Phi., NP. zahr. shdr, district, township : Av. khshaihra-. Phi. shatr, NP shahr. char, face : Av. chithra-, Phi. chihr, NP. chihra. zhdr, poison : Av.jathra-, Phi., NP. zahr. shdr, district, township : Av. khshaihra-. Phi. shatr, NP. shahr. zhdr, poison : Av.jathra-, Phi., NP. zahr. zhdr, poison : Av.jathra-, Phi., NP. zahr. shdr, district, township : Av. khshaihra-. Phi. shatr, NP shahr. The group era, awra, and ktira, seen in their proper forms in Central and Northern Kurdistan as ider, avder, and lende (Kj. bonder), suggest comparison with Av. ithra, avaihra, and kuthra, with simple loss of ih, as in chwdr, where the w of Av. chathwdr, is preserved and ih lost, as against the reverse process in Phi. and NP., where the ih has been preserved in the h of chahdr, and no trace of w survives. 3. Original medial khr only appears to be represented in the word stir, red ; Av. sukhra. Phi. has preserved the group in sukhr, while NP. has inverted it in surkh. From the presence of suhr in Central and Northern Kj., it would appear the stir is the result of loss of kh before r with the vowel lengthening noticeable with the loss of original ih before r. 3. Original medial khr only appears to be represented in the word stir, red ; Av. sukhra. Phi. has preserved the group in sukhr, while NP. has inverted it in surkh. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the run, light: Av. raokhshna = Phi., NP. roshan. run, light: Av. raokhshna = Phi., NP. roshan. In the second case Kj. loses any consonant preceding n, while Phi. and NP. preserve it. In the second case Kj. loses any consonant preceding n, while Phi. and NP. preserve it. run, oil: Av. raoghna-, Phi., NP. roghan. tin, thirst: Av. tarshna, Phi., NP. tashna. •pani, heel: Av. pashna, PH., NP. pashna. run, oil: Av. raoghna-, Phi., NP. roghan. tin, thirst: Av. tarshna, Phi., NP. tashna. •pani, heel: Av. pashna, PH., NP. pashna. The treatment of original r-n is the same as Phi. and NP. viz. loss of n, as far : Av. parena-, etc. The treatment of original r-n is the same as Phi. and NP. viz. loss of n, as far : Av. parena-, etc. LOSSES OF OEIGINAL LETTERS AND TREATMENT OF SOME CONSONANT GROUPS From the presence of suhr in Central and Northern Kj., it would appear the stir is the result of loss of kh before r with the vowel lengthening noticeable with the loss of original ih before r. Original medial fr is preserved intact in one case, and as ur in another, without inversion as in PhL and NP. wafr, snow : Av. vafra-, Phi. vafr, varf, NP. barf, zhtir, chamber, cellar : Av. jafra-, Phi. zafr, NP. zharf In the second example af appears to have passed through aw to ti. The form zhawr is heard in the extreme south. 4. Original mr only appears in nerm, Av. namra, Phi., NP. narm, which may be a loan-word from Phi. or NP. 5. Original sr appears in asr, tears, Av. asru-, Phi., NP. ars, i.e. not inverted, as in Phi. and NP. 6. The word chawr, fat, grease = Phi. charp, NP. charb, indicates the preservation of a possible hr, fr group. 7. Original consonant(s) plus n. This may be subdivided into khshn, other consonant plus n, and group r-n. In the first case Kj. loses khsh, while Phi. and NP. preserve sh, losing kh. In the first case Kj. loses khsh, while Phi. and NP. preserve sh, losing kh. THE PHONOLOGY OF SOUTHERN KURMANJI 224 8. Treatment of original kh. 8. Treatment of original kh. 8. Treatment of original kh. As an initial, before sh, Kj., like Phi. and NP., drops kh, as in shaw, shir, etc., from Av. khshapa-, khshira-, Phi. shap, shir, NP. shah, shir. As an initial, before sh, Kj., like Phi. and NP., drops kh, as in shaw, shir, etc., from Av. khshapa-, khshira-, Phi. shap, shir, NP. shah, shir. As an initial, before sh, Kj., like Phi. and NP., drops kh, as in shaw, shir, etc., from Av. khshapa-, khshira-, Phi. shap, shir, NP. shah, shir. As an initial, where Phi. and NP. preserve kh, Kj. loses it, as :— As an initial, where Phi. and NP. preserve kh, Kj. loses it, as :— wash, pleasant: Av. ?, Phi. Jchvash, NP. khush. wurd, small: Av. ^/khvar, Phi. khurtak, NP. khurd. shin, blue, green: Av. khshaena-, Phi. khashln, NP khashln. wash, pleasant: Av. ?, Phi. Jchvash, NP. khush. wurd, small: Av. ^/khvar, Phi. khurtak, NP. khurd. shin, blue, green: Av. khshaena-, Phi. khashln, NP. khashln. As a medial, where Phi. and NP. preserve kh, Kj. loses it, as :— As a medial, where Phi. and NP. preserve kh, Kj. loses it, as :— it, as :— bash, part, lot: Av. bakhsh, Phi., NP. bakhsh. mesh, fly : Av. makhshi-, (Phi., NP. magas). turn, seeds : Av. taokhma-, Phi. tokhm, NP. tukhm. wach, twig : Av. vakhsha-, Phi. vakhshj, NP. —. bash, part, lot: Av. bakhsh, Phi., NP. bakhsh. mesh, fly : Av. makhshi-, (Phi., NP. magas). turn, seeds : Av. taokhma-, Phi. tokhm, NP. tukhm. wach, twig : Av. vakhsha-, Phi. vakhshj, NP. —. 9. Kj. loses original sh where it is the final consonant, while Phi. and NP. preserve it. 9. Kj. loses original sh where it is the final consonant, while Phi. and NP. preserve it. ispl, louse : Av. spish-, Phi. shupush, NP. shepish. gije, ear : Av. gaosha-, Phi., NP. gosh, me, ewe : Av. maasha-, Phi., NP. mesh, rewl, fox : Av. revish-, Phi. robds, NP. rubah. re, beard : Av. rmsha-, Phi. resA, NP. rlsh. THE PHONOLOGY OF SOUTHERN KUEMANJI 225 10. In the word sipul, Kj. has lost the z of Av. spereza-, which is preserved in Phi. and NP. in sparz and sipurz. In hnka, " flashing, sparks," a similar loss of z (Av. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the 8. Treatment of original kh. \/braz) is noticed, though it is preserved as s in briska, having the same meaning. g 11. Kj. loses final ch, preserved by Phi. and changing to z in NP. g 11. Kj. loses final ch, preserved by Phi. and changing to z in NP. 11. Kj. loses final ch, preserved by Phi. and changing to z in NP. ro, light, day : Av. raochah-, Phi. roch, NP. ruz ro, light, day : Av. raochah-, Phi. roch, NP. ruz. 's/tw, flow, melt: Av. \/tach, Phi. \/vitach, NP. i/gu ro, light, day : Av. raochah-, Phi. roch, NP. ruz. 's/tw, flow, melt: Av. \/tach, Phi. \/vitach, NP. i/g g y 's/tw, flow, melt: Av. \/tach, Phi. \/vitach, NP. i/guddz. 12. Loss of r. ? initial (unsupported by other examples):— 12. Loss of r. ? initial (unsupported by other examples):— ? initial (unsupported by other examples):— dst, direction, line: Av. rdst-, Phi., NP. rast. Medial:— tin, thirst: Av. tarshna-, Phi. tishn, NP. tishna. ezh, worth : Av. arejah-, Phi. arj, NP. arz. anishk, elbow : Av. areihn-, Phi. aret, ertan, NP. dran baosh, upper part of side : Av. barozhda-, Phi., NP. — \/ba, take, bear : Av. \/bere, Phi., NP. \/bar. Final :— Final :— khasu, mother-in-law : Av. khvasura, Phi. ?, NP. khasur •s/azhi, flood, inundate : Av. d-\/ghzhar, Phi., NP. —. \/pzha, sprinkle continuously: Av. vi-\/ghzhar, Phi., NP. —. 13. Loss of t preserved in Phi. •\/gur, change, return: Av. \/varet, Phi. \/vart, NP. bist, span : Av. vi-tasti-, Phi. vitast, NP. bidhist. 14. Loss of medial b :— tur, radish : NP. turb-lza. turn, wallet, loan-word from Tk. tubra. Loss of b after m :— hdmar, store : Av. ham-\/bere, Phi. ambdr, NP. ambd shamu, Sabbath : NP. shambeh. gumez, dome : NP. gumbad. JBAS. APKIL 1922. 15 15 JBAS. APKIL 1922. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the THE PHONOLOGY OF SOUTHERN KUEMANJI 226 15. Loss of final v. 15. Loss of final v. gd, bull: Av. gdv-, Phi., NP. gdv. 16. Loss of gh preserved in Phi. and NP. dril, a lie : Av. draogha-, Phi. drogh, NP. durogh. run, butter : Av. https://doi.org/10.1017/S0035869X00150257 Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. Downloaded from https:/www.cambridge.org/core. UCL, Institute of Education, on 08 Feb 2017 at 06:50:56, subject to the 8. Treatment of original kh. raoghna-, Phi. rohan, NP. roghan. mir-ishk, hen : Av. meregha-, Phi., NP. murgh. 16. Loss of gh preserved in Phi. and NP. dril, a lie : Av. draogha-, Phi. drogh, NP. durogh. run, butter : Av. raoghna-, Phi. rohan, NP. roghan mir-ishk, hen : Av. meregha-, Phi., NP. murgh. • Authorities consulted : Grundriss der Iranischen Philologie ; Grundriss der Newpersischen Etymologic, Horn; Kurdische Grammatik, Justi; Diclionnaire Kurde Francaise, Justi- Jaba ; An old Zand Pahlavi Glossary, Jamaspji; Avesta- English and English-Avesta Glossary, Bharucha ; An Avesta Grammar, Williams Jackson ; Etudes Iraniennes, Darmesteter. • Authorities consulted : Grundriss der Iranischen Philologie ; Grundriss der Newpersischen Etymologic, Horn; Kurdische Grammatik, Justi; Diclionnaire Kurde Francaise, Justi- Jaba ; An old Zand Pahlavi Glossary, Jamaspji; Avesta- English and English-Avesta Glossary, Bharucha ; An Avesta Grammar, Williams Jackson ; Etudes Iraniennes, Darmesteter. Darmesteter.
https://openalex.org/W3044340027	https://www.nature.com/articles/s41598-020-68911-5.pdf	English	null	Experimental kernel-based quantum machine learning in finite feature space	Scientific reports	2,020	cc-by	7,564	Experimental kernel‑based quantum machine learning in finite feature space Karol Bartkiewicz1,2, Clemens Gneiting3, Antonín Černoch2, Kateřina Jiráková2, Karel Lemr2* & Franco Nori3,4 Karol Bartkiewicz1,2, Clemens Gneiting3, Antonín Černoch2, Kateřina Jiráková2, Karel Lemr2* & Franco Nori3,4 We implement an all-optical setup demonstrating kernel-based quantum machine learning for two- dimensional classification problems. In this hybrid approach, kernel evaluations are outsourced to projective measurements on suitably designed quantum states encoding the training data, while the model training is processed on a classical computer. Our two-photon proposal encodes data points in a discrete, eight-dimensional feature Hilbert space. In order to maximize the application range of the deployable kernels, we optimize feature maps towards the resulting kernels’ ability to separate points, i.e., their “resolution,” under the constraint of finite, fixed Hilbert space dimension. Implementing these kernels, our setup delivers viable decision boundaries for standard nonlinear supervised classification tasks in feature space. We demonstrate such kernel-based quantum machine learning using specialized multiphoton quantum optical circuits. The deployed kernel exhibits exponentially better scaling in the required number of qubits than a direct generalization of kernels described in the literature. Many contemporary computational problems (like drug design, traffic control, logistics, automatic driving, stock market analysis, automatic medical examination, material engineering, and others) routinely require optimiza- tion over huge amounts of data1. While these highly demanding problems can often be approached by suitable machine learning (ML) algorithms, in many relevant cases the underlying calculations would last prohibitively long. Quantum ML (QML) comes with the promise to run these computations more efficiently (in some cases exponentially faster) by complementing ML algorithms with quantum resources. The resulting speed-up can then be associated with the collective processing of quantum information mediated by quantum entanglement.h p g q y q g There are various approaches to QML, including linear algebra solvers, sampling, quantum optimization, or the use of quantum circuits as trainable models for inference (see, e.g., Refs.2–18). A strong focus in QML has been on deep learning and neural networks. Independently, kernel-based approaches to supervised QML, where computational kernel evaluations are replaced by suitable quantum measurements, have recently been proposed10,12 as interesting alternatives. Combining classical and quantum computations, they add to the family of quantum-classical hybrid algorithms. q y g Kernel-based QML (KQML)is particularly attractive to be implemented on linear-optics platforms, as quan- tum memories are not required. Here, we thus investigate the prospect of KQML with multiphoton quantum optical circuits. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Results l Kernel resolution in finite dimensions. An important and widespread kernel class are Gaussian-type kernels, which introduce a flexible notion of proximity among data points. An essential hyperparameter of Gaussian-type kernels is thus their variance (or, more generally, their resolution). The resolution determines a Gaussian kernel’s ability to distinguish data points, which, for given training data, can decide if a model can be trained successfully or not. If kernel resolution is too coarse, resulting decision boundaries miss relevant details in the data; if it is too refined, the model becomes prone to overfitting. Only if the resolution can be chosen suffi- ciently flexibly to be accommodated to the structure of the data, model training can be expected to be successful.if l In the infinite-dimensional feature spaces offered by continuous variable implementations, viable FMs with (in principle) arbitrary resolution can be implemented, e.g., by mapping data into squeezed states12, where the adjustable squeezing factor then determines the resolution of the resulting Gaussian kernel (i.e., its variance). However, within the paradigm of discrete, finite-dimensional quantum information processing, the FHS dimen- sion becomes a scarce resource, resulting in limitations on kernel resolution. As we show now, optimizing the range of kernel resolutions in finite dimensions then forces us to move beyond the scope of Gaussian kernels. gi y p Let us discuss the optimal kernel resolution that can be achieved in N-dimensional FHS, within the class of FMs of the form (1) x →\|ψ(x) = N n=0 √rne2πinx \|n, N n=0 rn = 1, (1) with {\|n} a basis of the Hilbert space and x ∈[−1/2, 1/2) . Any data set can be brought to this form, which is a routine step in data preparation. We stress that the amplitudes rn are independent from the input values x. The resulting kernels then are of the form (2) κ(x, x′) = κ(x −x′) = N n=0 rn e2πin(x′−x) 2 . (2) In this shorthand notation κ(x) ≥0 ∀x and κ(0) = 1 . For the sake of clarity we consider here 1D input data x. For D-dimensional inputs x , each input component xi is encoded separately, requiring an (N · D + D)-dimen- sional FHS. If the FHS is spanned by q qubits, we have N = 2q −1 . www.nature.com/scientificreports/ nonlinear FMs, it is not required to implement nonlinear transformations on the quantum-state encoded data, in contrast to the direct amplitude encoding common in other QML approaches.h nonlinear FMs, it is not required to implement nonlinear transformations on the quantum-state encoded data, in contrast to the direct amplitude encoding common in other QML approaches.h The central idea underlying KQML is that inner products of vectors that are mapped into FHS can be directly accessed by measurements, which then suggests to identify these inner products with kernel functions. By physically measuring the kernel functions κ(x′, x) = \|ϕ(x′)\|ϕ(x)\|2 , it is thus possible to bypass their per pedes computation on a classical machine. Such measurement-based implementation may, in some cases, be signifi- cantly faster than the latter option. y It follows from the representer theorem that a function of the reproducing kernel that minimizes the cost function (a solution to the ML problem) can be written as f ∗(x) = M m=1 amκ(x, xm), where M is the number of training samples, the coefficients am are real parameters subject to the training, and x belongs to feature space. For a given kernel κ , the parameters am can be found efficiently. The objective of ML is to deliver a function f ∗(x) that classifies the non-separable points x1, ..., xM−K and xM−K+1, ..., xM by finding a trade-off between the number of misclassifications and the width of the separating margin. The parameters am can be obtained by solving the following problem: minimize M m=1(\|am\|2 + γ um) such that aiκ(x, xi) ≥1 −ui for i = 1, ..., M −K, and aiκ(x, xi) ≤−(1 −ui) for i = M −K + 1, ..., M, u ≥0, where γ gives the relative weight of the number of misclassified points compared to the width of the margin. In a nutshell, this approach allows to replace the non- linearity of the problem with linear multidimensional quantum computations, which offers a potential speed-up. Experimental kernel‑based quantum machine learning in finite feature space Karol Bartkiewicz1,2, Clemens Gneiting3, Antonín Černoch2, Kateřina Jiráková2, Karel Lemr2* & Franco Nori3,4 To this end, we propose kernels that scale exponentially better in the number of required qubits than a direct generalization of kernels previously discussed in the literature12. We also realize this scheme in a proof-of-principle experiment demonstrating its suitability on the platform of linear optics, thus, proving its practical applicability with current state of quantum technologies.ii Let us explain KQML by first recalling some definitions and theorems, and then we overview the recently proposed method for finding linear boundaries in feature Hilbert space (FHS)12. FHS is defined as a space of complex vectors \|ϕ(x), where ϕ describes a feature map (FM), and x denotes a real vector of dimension D (the input data). FHSs generally have higher dimension than the original data x. This implies that linear decision boundaries in FHS can give rise to nonlinear decision boundaries in the original data space. By virtue of such 1Faculty of Physics, Adam Mickiewicz University, 61‑614 Poznan, Poland. 2RCPTM, Joint Laboratory of Optics of Palacký University and Institute of Physics of Czech Academy of Sciences, 17. listopadu 12, 771 46 Olomouc, Czech Republic. 3Theoretical Quantum Physics Laboratory, RIKEN Cluster for Pioneering Research, Wako‑shi 351‑0198, Japan. 4Department of Physics, The University of Michigan, Ann Arbor, MI 48109‑1040, USA. *email: karol.bartkiewicz@upol.cz; acernoch@fzu.cz; k.lemr@upol.cz Scientific Reports \| (2020) 10:12356 \| https://doi.org/10.1038/s41598-020-68911-5 www.nature.com/scientificreports/ Results l (a) We find that the resolution-optimized kernel Figure 1. Kernel family (2) for different amplitude choices. (a) We find that the resolution-optimized kernel (blue solid) exhibits suppressed side maxima as compared to the MSI kernel (red dashed), while the TSQ kernel (with squeezing factor ζ = 2 , black dotted) maintains a nonvanishing plateau at all x values. For comparison, we also display the respective squeezed-state kernel for N →∞ (gray dotted) and CK (purple dash-dotted). (b) Characteristic amplitude progressions for the example of N = 14 and ζ = 4 . (c) The optimized kernel exhibits a significantly improved resolution progression with N, as compared to the MSI or the TSQ kernel (here with ζ = 3). Figure 1. Kernel family (2) for different amplitude choices. (a) We find that the resolution-optimized kernel (blue solid) exhibits suppressed side maxima as compared to the MSI kernel (red dashed), while the TSQ kernel (with squeezing factor ζ = 2 , black dotted) maintains a nonvanishing plateau at all x values. For comparison, we also display the respective squeezed-state kernel for N →∞ (gray dotted) and CK (purple dash-dotted). (b) Characteristic amplitude progressions for the example of N = 14 and ζ = 4 . (c) The optimized kernel exhibits a significantly improved resolution progression with N, as compared to the MSI or the TSQ kernel (here with ζ = 3). We can use the above compact-space embedding to gain further insight into the nature of our kernel defini- tion (2). If we interpret the states (1) as \|ψ = N n=1 √rn\|p = n ⊗\|n , we can introduce the density operator ρ = \|ψψ\| and trace over the internal spin degree of freedom, We can use the above compact-space embedding to gain further insight into the nature of our kernel defini- tion (2). If we interpret the states (1) as \|ψ = N n=1 √rn\|p = n ⊗\|n , we can introduce the density operator ρ = \|ψψ\| and trace over the internal spin degree of freedom, (5) ρext = Trint(ρ) = N n=1 rn\|p = np = n\|. Results l (5) We then find that the kernel (2) is related to the spatial coherences of the mixed reduced state ρext : κ(x, x′) = \|x\|ρext\|x′\|2.i We define a kernel’s spatial resolution x[κ] by its variance (a hyperparameter typically optimized for Gauss- an kernels) (6) (x[κ])2 ≡ 1/2 −1/2 dx x2 ˜κ(x), (6) where the renormalized kernel ˜κ(x) = κ(x)/R , with R ≡ 1/2 −1/2 dx κ(x) = N n=1 r2 n , describes a valid probability distribution. In the case of the mulit-slit interference states \|ψMSI , one analytically obtains (x[κMSI])2 = 1 12(1 −S1(N)) , with the interferometric “squeezing factor” S1(N) = −12 π2 N−1 j=1 (−1)j N−j Nj2 , and N ≥219. where the renormalized kernel ˜κ(x) = κ(x)/R , with R ≡ 1/2 −1/2 dx κ(x) = N n=1 r2 n , describes a valid probability distribution. In the case of the mulit-slit interference states \|ψMSI , one analytically obtains (x[κMSI])2 = 1 12(1 −S1(N)) , with the interferometric “squeezing factor” S1(N) = −12 π2 N−1 j=1 (−1)j N−j Nj2 , and N ≥219. The kernel (6) minimizing the variance is a solution to the optimization problem: minimize rT·K·r \|r\|2 such that N n=1 rn = 1 , where (7) Knm = 1 12, n = m (−1)\|n−m\| 2(n−m)2π2 , else (7) and r = (r1, . . . , rN)T . In Fig. 1 we compare this optimized kernel with the TSQ and the MSI kernel. The opti- mized kernel comes with strongly suppressed side maxima as compared to the MSI kernel, while the TSQ maintains a nonvanishing plateau for all x values. Consequently, the optimized kernel enables, for a given N, a significantly improved resolution as compared to the other kernel choices. Figure 1b clarifies that amplitudes decaying symmetrically about the “center” state are responsible for improving the kernel resolution. and r = (r1, . . . , rN)T . In Fig. 1 we compare this optimized kernel with the TSQ and the MSI kernel. The opti- mized kernel comes with strongly suppressed side maxima as compared to the MSI kernel, while the TSQ maintains a nonvanishing plateau for all x values. Consequently, the optimized kernel enables, for a given N, a significantly improved resolution as compared to the other kernel choices. Figure 1b clarifies that amplitudes decaying symmetrically about the “center” state are responsible for improving the kernel resolution. Results l In particular, for N = 1 and rn = 1/2 we have κ(x, x′) = cos[π(x′ −x)]2, which realizes a cosine kernel (CK). The class of states (1) comprises also truncated squeezed states \|ψTSQ(x) , with In this shorthand notation κ(x) ≥0 ∀x and κ(0) = 1 . For the sake of clarity we consider here 1D input data x. For D-dimensional inputs x , each input component xi is encoded separately, requiring an (N · D + D)-dimen- sional FHS. If the FHS is spanned by q qubits, we have N = 2q −1 . In particular, for N = 1 and rn = 1/2 we have κ(x, x′) = cos[π(x′ −x)]2, which realizes a cosine kernel (CK). The class of states (1) comprises also truncated squeezed states \|ψTSQ(x) , with (3) √rn = √(2n)!(−tanh ζ)n √ B 2nn!√cosh ζ (3) (ζ denotes the squeezing factor and B renormalizes the state after truncation), and, what we call here, multi-slit interference states \|ψMSI(x) , with constant amplitudes √rn = 1/ √ N . The latter inherit their name from the fact that, by virtue of x\|p = e2πipx (h=1), they are formally equivalent to a balanced superposition of momentum states in a (hypothetical) compact continuous variable Hilbert space (augmented by an internal spin-N degree of freedom), (4) \|ψMSI(x) = 1 √ N N n=1 x\|p = n\|n, (4) giving rise to “N-slit interference” in the position coordinate when projected onto x\| ⊗ 1 √ N N n=1n\|19. Note that polynomial kernels (discussed, e.g., in8,12) fall outside of the state class (1). https://doi.org/10.1038/s41598-020-68911-5 Scientific Reports \| (2020) 10:12356 \| www.nature.com/scientificreports/ Figure 1. Kernel family (2) for different amplitude choices. (a) We find that the resolution-optimized kernel (blue solid) exhibits suppressed side maxima as compared to the MSI kernel (red dashed), while the TSQ kernel (with squeezing factor ζ = 2 , black dotted) maintains a nonvanishing plateau at all x values. For comparison, we also display the respective squeezed-state kernel for N →∞ (gray dotted) and CK (purple dash-dotted). (b) Characteristic amplitude progressions for the example of N = 14 and ζ = 4 . (c) The optimized kernel exhibits a significantly improved resolution progression with N, as compared to the MSI or the TSQ kernel (here with ζ = 3). Figure 1. Kernel family (2) for different amplitude choices. Results l On the other hand, a kernel that maximizes the variance (i.e., κ(x) = 1 ) follows from r1 = 1 and rn = 0 for n = 1 , resulting in the variance (x[κ])2 = 1/12. By a suitable choice of the coefficients rn , we can thus tune the resolution of the kernel between its minimum value obtained for the optimized kernel and its maximum value assumed for a uniform kernel.fi Whereas kernels of the form (2) can also be efficiently computed classically, their quantum evaluation may still deliver a significant speed-up. We illustrate this with an example, the computation of cos2N x. The optimal classical algorithm depends on the properties of N. In the best case scenario, N is a power of 2. Then, in the first step we compute cos2 x. Next, we compute [cos2 x]2, etc. The entire computation then takes log2(N + 1) steps. As we demonstrate below, for the quantum implementation, the required size of the FHS (number of qubits) grows also like log2(N + 1) . However, in contrast, there the associated calculations are replaced by a single measure- ment. We expect similar arguments to hold for more general classes of functions, as well. Beyond the quantum-classical hybrid approach pursued here, the proposed FMs may, if seen as modules to be combined with other quantum computing subroutines, contribute their resource-efficient data point separation ability to an overall setup that comes with an inherently quantum scaling advantage. MSI states, for instance, can be generated in a gate-based quantum computer following the first stage of the phase-estimation algorithm20. Alternative Gaussian‑kernel implementation. Above we have shown that truncated squeezed states and their resulting kernels fall within the state class (1). If we relax the condition that the amplitudes rn be inde- Scientific Reports \| (2020) 10:12356 \| https://doi.org/10.1038/s41598-020-68911-5 www.nature.com/scientificreports/ Figure 2. Training results on a random inseparable data set of 40 samples (up/down-tipped triangles). The performance on a test set (left/right-tipped triangles) of 60 points (the fraction of correctly classified samples that were not used in the QML process) is given in the bottom right corner of each respective subplot. We find that the optimal variance/resolution choice for the Gaussian kernel is s = 2 . For s = 3 we deal with overfitting. Results l Shown are the simulation results both for an exact Gaussian kernel and for the truncated FM (8) comprising 4 terms ( q = 2 ). The learned classification boundaries are given as contour plots. The slight difference in performance compared to the theoretical prediction is due to statistical fluctuations in the experimental data and the relatively small test set (misclassification of a single near-boundary point results in a 0.02 performance drop). Figure 2. Training results on a random inseparable data set of 40 samples (up/down-tipped triangles). The performance on a test set (left/right-tipped triangles) of 60 points (the fraction of correctly classified samples that were not used in the QML process) is given in the bottom right corner of each respective subplot. We find that the optimal variance/resolution choice for the Gaussian kernel is s = 2 . For s = 3 we deal with overfitting. Shown are the simulation results both for an exact Gaussian kernel and for the truncated FM (8) comprising 4 terms ( q = 2 ). The learned classification boundaries are given as contour plots. The slight difference in performance compared to the theoretical prediction is due to statistical fluctuations in the experimental data and the relatively small test set (misclassification of a single near-boundary point results in a 0.02 performance drop). pendent from the input values x, we can formulate an alternative data encoding into truncated squeezed states according to (8) \|φ(x) = Z N n=0 (sx)n √ n! \|n, (8) where N = 2q −1 , x = x1 + ix2, and Z−1 = N n=0 (s\|x\|)2n n! . Note that this feature map is defined for 2D inputs x = (x1, x2)T . For large N this kernel again reproduces to good approximation a Gaussian kernel, as (9) κ(x′, x) = \|ϕ(x′)\|ϕ(x)\|2 ≈exp [−s2(x1 −x′ 1)2 −s2(x2 −x′ 2)2], (9) where the variance is set by the hyperparameter s. In particular, as shown in Fig. 2, this approximation is valid or q = 2 and relatively small values of s.it We find that this kernel performs, for the number of qubits q = 2 and after numerically optimizing the hyperparameter to s = 2 , on average as well as the cosine kernel for the same total number of qubits equal to N = 1 (see Fig. 4). Figure 3. Optical circuit implementing both the FM and the model circuits. The performance of the setup in QML is shown in Fig. 4 for N = 1 and D = 2. The experimental setup consists of polarizing beam splitters (PBSs), beam dividers (BDs), quarter-wave and half-wave plates (QWPs and HWPs, respectively), and single photon detectors Dn for n = 1a, 1b, 2a, 2b, 3, 4 . D3 and D4 are H/V polarization resolving (implemented as a PBS and two standard detectors). The kernel κ(x′, x)exp = [ p,s=H,V CC(D2s, D3p)−CC(D2V, D3H)+CC(D2H, D3V)]/ m>n 6 n=1 CC(Dm, Dn) is given as a ratio of coincidences CC(Dm, Dn) registered by photon detectors Dn and Dm to the total number of photons. Figure 3. Optical circuit implementing both the FM and the model circuits. The performance of the setup in QML is shown in Fig. 4 for N = 1 and D = 2. The experimental setup consists of polarizing beam splitters (PBSs), beam dividers (BDs), quarter-wave and half-wave plates (QWPs and HWPs, respectively), and single photon detectors Dn for n = 1a, 1b, 2a, 2b, 3, 4 . D3 and D4 are H/V polarization resolving (implemented as a PBS and two standard detectors). The kernel κ(x′, x)exp = [ p,s=H,V CC(D2s, D3p)−CC(D2V, D3H)+CC(D2H, D3V)]/ m>n 6 n=1 CC(Dm, a ratio of coincidences CC(Dm, Dn) registered by photon detectors Dn and Dm to the total number of p gh (x′, x)exp = [ p,s=H,V CC(D2s, D3p)−CC(D2V, D3H)+CC(D2H, D3V)]/ m>n 6 n=1 CC(Dm, Dn) is given as ratio of coincidences CC(Dm, Dn) registered by photon detectors Dn and Dm to the total number of photons. (10) κ(x′, x) = \|ϕ(x′)\|ϕ(x)\|2 = D n=1 cos2N(x′ n −xn), (10) w h e r e t h e F M t a k i n g a n o r m a l i z e d f e at u r e xn ∈[−π/2, π/2) t o F H S i s \|ϕ(x) = D n=1 N k=0 N k sink(xn) cosN−k(xn)\|kn. Note that N is related to the number of qubits q per dimen- sion as q = ⌈log2(N + 1)⌉. This FM can also be considered a constant-phase representation of constant-amplitude states. This is the same as representing states either in a basis of eigenstates of x or z components of a collective spin operator. Results l Moreover, by appropriate parameter reconfiguration, it would be possible to realize this type of kernel using the same experimental setup. From an experimental perspective, however, it is more conveni- ent (and thus scalable) to implement the feature map associated with the powers of the cosine kernel, which is exclusively implemented by setting phases and polarization angles. Cosine kernels. The kernel selected for our proof-of-principle demonstration of KQML is Scientific Reports \| (2020) 10:12356 \| https://doi.org/10.1038/s41598-020-68911-5 www.nature.com/scientificreports/ Figure 3. Optical circuit implementing both the FM and the model circuits. The performance of the setup in QML is shown in Fig. 4 for N = 1 and D = 2. The experimental setup consists of polarizing beam splitters (PBSs), beam dividers (BDs), quarter-wave and half-wave plates (QWPs and HWPs, respectively), and single photon detectors Dn for n = 1a, 1b, 2a, 2b, 3, 4 . D3 and D4 are H/V polarization resolving (implemented as a PBS and two standard detectors). The kernel κ(x′, x)exp = [ p,s=H,V CC(D2s, D3p)−CC(D2V, D3H)+CC(D2H, D3V)]/ m>n 6 n=1 CC(Dm, Dn) is given as a ratio of coincidences CC(Dm, Dn) registered by photon detectors Dn and Dm to the total number of photons. In particular, (cos(x)\|0 + sin(x)\|1)/ √ 2 ⇔(\|0′ + e2ix\|1′)/ √ 2, where \|0 = (\|0′ + \|1′)/ √ 2 and \|1 = (\|0′ −\|1′)/ √ 2.h sion as q = ⌈log2(N + 1)⌉. This FM can also be considered a constant-phase representation of constant-amplitude states. This is the same as representing states either in a basis of eigenstates of x or z components of a collective spin operator. In particular, (cos(x)\|0 + sin(x)\|1)/ √ 2 ⇔(\|0′ + e2ix\|1′)/ √ 2, where \|0 = (\|0′ + \|1′)/ √ 2 and \|1 = (\|0′ −\|1′)/ √ 2.h This mapping uses exponentially less resources (qubits) than the direct product of the map from Ref.12, i.e., \|ϕ(x) = D n=1 N m=1 1 k=0 sink(xn) cos1−k(xn)\|kn,m, where the number of qubits per dimension is q = N. Using the powers of CKs allows us to adjust the kernel resolution by choosing the proper value of N. Thus, the number of used qubits can be related directly to the variance of the kernel. The number of qubits here plays the same role as the squeezing parameter in the experimental proposal given in Ref.12. The CK can also include additional (D −1) degrees of freedom by virtue of a FM defined as (11) \|ϕ(x) = D n=1 N k=0 ei2yn−1 N k sink(xn) cosN−k(xn)\|kn, (11) https://doi.org/10.1038/s41598-020-68911-5 Scientific Reports \| (2020) 10:12356 \| www.nature.com/scientificreports/ Figure 4. Training results on a random inseparable data set of 40 samples (up/down-tipped triangles). The performance on a test set (left/right-tipped triangles) of 60 points (the fraction of correctly classified samples that were not used in the QML process) is given in the bottom right corner of each respective subplot. We see that the best choice of CK is N = 1 . For N = 2 we deal with overfitting and for N = 1/2 the kernel is too coarse to give as good results as for N = 1 . The learned classification boundaries are given as contour plots. The slight difference in performance of KQML in relation to the theoretical prediction is due to statistical fluctuations of the experimental data and relatively small test set (misclassification of a single near-boundary point results in 0.02 performance drop). Figure 4. Training results on a random inseparable data set of 40 samples (up/down-tipped triangles). The performance on a test set (left/right-tipped triangles) of 60 points (the fraction of correctly classified samples that were not used in the QML process) is given in the bottom right corner of each respective subplot. We see that the best choice of CK is N = 1 . For N = 2 we deal with overfitting and for N = 1/2 the kernel is too coarse to give as good results as for N = 1 . The learned classification boundaries are given as contour plots. The slight difference in performance of KQML in relation to the theoretical prediction is due to statistical fluctuations of the experimental data and relatively small test set (misclassification of a single near-boundary point results in 0.02 performance drop). where y0 = 0, the number of terms here is (N + 1)D, and the associated kernel measured by postselection is κ(x′, x) = D n=1 cos2N(x′ n −xn) cos2(y′ n−1 −yn−1). Conclusions We report on the first experimental implementation of supervised QML for solving a nonlinear multidimensional classification problem with clusters of points which are not trivially separated in the feature space. We hope that our research on QML will help to improve ML technologies, which are a major power-horse of many industries, a vivid field of research in computer science, and an important technique for solving real-world problems. We believe that both the theoretical and the experimental investigation of FM circuits and their constraints regarding kernel resolution and compression for a limited FHS (i.e., FHS size dependent FMs) constitutes a crucial step in the development of practical KQML for support-vector-machine QML8–10,12,13. We demonstrate that a linear-optical setup with discrete photon encoding is a reliable instrument for this class of quantum machine learning tasks. We also report obtaining exponentially better scaling of FHS in the case of CK than in the case of taking direct products of qubits12. The same can hold for other more complex kernels implemented in finite FHS, which could appear unfeasible, but in fact require nontrivial FMs (e.g., the resolution-optimized kernels shown in Fig. 1). Thus, KQML can provide a promising perspective for utilizing noisy intermediate-scale quantum systems21–24, complementing artificial quantum neural networks25–29 and other hybrid quantum-classical algorithms30–32.h y q g The classical computational cost of the power kernel computation is O[log(N)] and the quantum cost is a constant value depending on the precision of the computation. In the classical case, one needs to perform O[log(N)] computation steps that can not run in parallel due to the recursive nature of the classical algorithm. In the quantum case, one needs to run 1 computation step but on log(N) qubits. As in any quantum computation, the precision of the calculation depends on the number of measurements and it can be considered constant for a given computational problem. This observation itself is a valuable result and a quantum advantage. The quantum advantage of the presented approach is apparent in terms of the complexity of calculations, i.e., O[log(N)] versus O(1) . Consider the number of samples needed for quantum calculations. It depends on the confidence level (z) and admissible error: ǫ . For a given pair of z and error ǫ , one needs O(1/ǫ2) repetitions of the experiment. This is just a constant overhead. Discussion The data accumulation time can be shortened by orders of magnitudes by fine tuning the parameters of the setup and by using brighter photon sources. using linear interpolation. The data accumulation time can be shortened by orders of magnitudes by fine tuning the parameters of the setup and by using brighter photon sources. Conclusions In the classical case, this constant overhead can be smaller, but the complexity of calculations can be larger as the it is N-dependent. Only if we face significantly lower than unity qubit-number- dependent efficiency η (i.e., circuit-size dependent losses), for a given z-value the complexity of quantum com- putations should be considered as being O(η( −log(N))/ǫ2) . However, the power scaling also applies to the total error probability of classical computations of log(N) steps. Note, however, that both η and single-step error probability of classical computing are not fundamentally limited and can be arbitrary close to 1 or 0, respectively.i p y p g y y p y Our quantum kernels can be used for solving high-dimensional classification problems and could poten- tially be computed faster than their classical counterparts. Popular problems solved by classification algorithms include image recognition (e.g. face detection or character recognition), speech recognition (e.g. voice user interfaces), medical diagnoses (e.g. associating results of medical tests with a class of diseases), real-time specific data extraction from vast amounts of unstructured data (e.g. classification of patterns in unstructured data) and many more. Classification can also be used as an initial phase for predictive computations that help to make the best decision based on the available data (e.g., managing risk, security, traffic, procurement etc.). We believe that this quantum-enhanced approach is useful especially in cases where it is difficult or impossible to achieve the result on time with classical computing. Discussion We have experimentally implemented KQML to solve three classification problems on a two-photon optical quantum computer. In our experiment we implemented a D = 2, N = 1 kernel (using all the modes from Fig. 3, we can set at most D = 5 with q = 1 ). We used two photons, but only the top mode of the dual-rail encoding. Including more modes would lead to kernels causing overfitting (see Fig. 4). i We have performed measurements for M = 40 two-dimensional samples ( D = 2 ), drawn from two classes (see horizontally/vertically-tipped triangles in Fig. 4). This procedure was repeated for three benchmark classifi- cation problems. For each benchmark 40 × 39/2 = 780 measurements were performed to create a corresponding Gram matrix (GM), which was subsequently used to find the best linear classification boundary as given by the representer theorem. In other words, a custom kernel κ(xm, xn) = κ(xn, xm) for m, n = 1, 2, ..., M was measured. This kernel was used as a custom precomputed kernel for the scikit-learn SVC classifier in python. h p pi py Pairs of H-polarized photons were prepared in a type-I spontaneous parametric down-conversion process in a β–BaB2O4 crystal. The crystal was pumped by a 200 mW laser beam at 355 nm (repetition rate of 120 MHz). The coincidence rate, including all possible detection events from Fig. 3, was approximately 250 counts per second. The setup operates with high fidelity (98%) and the dominant source of errors can be attributed the Poissonian photon count statistics. The design of this setup is modular and its easy to incorporate more qubits by simply adding additional blocks. We measured each point for a time necessary to collect about 2,500 detection events. Thus, excluding the time needed to switch the setup parameters, the whole measurement for a single benchmark problem takes about two hours. p To prepare the contour plot of the decision function based on the experimental data shown in Fig. 4 and to quantify the performance of the trained model on the relevant test sets, we have also measured the GM for 1,225 points and used its symmetries to fill in the unmeasured values. The values for points in between have been found Scientific Reports \| (2020) 10:12356 \| https://doi.org/10.1038/s41598-020-68911-5 www.nature.com/scientificreports/ using linear interpolation. References I. Computational speedups using small quantum devices. Phys. Rev. Lett. 121, 250501 (2018). . et al. Error mitigation extends the computational reach of a nois g p y q p 25. Shen, Y. et al. Deep learning with coherent nanophotonic circuits. Nat. Photonics 11, 441 (2017). 25. Shen, Y. et al. Deep learning with coherent nanophotonic circuits. Nat. Photonics 11, 441 (2017). 26. Bueno, J. et al. Reinforcement learning in a large-scale photonic recurrent neural network. Optica 5, 756 (2018).i 27. Tacchino, F., Macchiavello, C., Gerace, D. & Bajoni, D. An artificial neuron implemented on an actual quantum processo Quantum Inf. 5, 26 (2019). Quantum nf. 5, 6 ( 0 9). 28. Kak, S. C. Quantum neural computing. In Advances in Imaging and Electron Physics, Vol. 94, 259 (Elsevier, Amsterdam, 1995). f 28. Kak, S. C. Quantum neural computing. In Advances in Imaging and Electron Physics, Vol. 94, 259 (Elsevier, Amsterdam, 199 29 Farhi E & Neven H Classification with quantum neural networks on near term processors (2018) arXiv:1802 06002 28. Kak, S. C. Quantum neural computing. In Advances in Imaging and Electron Physics, Vol. 94, 259 (Elsevier, Amsteri i q p ( ) 30. Li, Y. & Benjamin, S. C. Efficient variational quantum simulator incorporating active error minimization. Phys. Rev. X 7, 021050 (2017).fi i 30. Li, Y. & Benjamin, S. C. Efficient variational quantum simulator incorporating active error minimization. Phys. Rev. X 7, 021050 (2017).fi 31. Kandala, A. et al. Hardware-efficient variational quantum eigensolver for small molecules and quantum magnets. Nature 549 (2017). ( ) 32. Mitarai, K., Negoro, M., Kitagawa, M. & Fujii, K. Quantum circuit learning. Phys. Rev. A 98, 032309 (2018). ( ) 32. Mitarai, K., Negoro, M., Kitagawa, M. & Fujii, K. Quantum circuit learning. Phys. Rev. A 98, 032309 (2018). Acknowledgementsi g Authors acknowledge financial support by the Czech Science Foundation under the project No. 19-19002S. The authors also acknowledge the projects No. CZ.1.05/2.1.00/19.0377 of the Ministry of Education, Youth and Sports of the Czech Republic financing the infrastructure of their workplace. K.J. is supported in part by the Palacky University internal grant No. IGA-PrF-2020-007. F.N. is supported in part by: NTT Research, Army Research Office (ARO) (Grant No. W911NF-18-1-0358), Japan Science and Technology Agency (JST) (via the CREST Grant No. JPMJCR1676), Japan Society for the Promotion of Science (JSPS) (via the KAKENHI Grant No. JP20H00134, and the JSPS-RFBR Grant No. JPJSBP120194828), and the Foundational Questions Institute Fund (FQXi) (Grant No. FQXi-IAF19-06), a donor advised fund of the Silicon Valley Community Foundation. References Supervised learning with quantum-enhanced feature spaces. 4. Wang, W. & Lo, H.-K. Machine learning for optimal parameter prediction in quantum key distribution. Phys. Rev. A 100, 062334 (2019). 15. Hendry, D. & Feiguin, A. E. Machine learning approach to dynamical properties of quantum many-body systems. Phys. R 100, 245123 (2019). 16. Zhang, Y. et al. Machine learning in electronic-quantum-matter imaging experiments. Nature 570, 484–490 (2019). Č . Zhang, Y. et al. Machine learning in electronic-quantum-matter Č 17. 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Harnessing disordered-ensemble quantum dynamics for machine learning. Phys. Rev. Appl. 8, 02 (2017). 23. Dunjko, V., Ge, Y. & Cirac, J. www.nature.com/scientificreports/ the last pair of waveplates is used both to flip the polarization and to perform the Hadamard transformation. Finally, the PBS transmits only H-polarized photons for further processing.h the last pair of waveplates is used both to flip the polarization and to perform the Hadamard transformation. Finally, the PBS transmits only H-polarized photons for further processing.h y y g The procedure of measuring the kernel κ(x′, x) can be extended to include additional dimensions, resulting in measuring the kernel ¯κ(x′, x) = κ(x′, x) cos2(y −y′) following from FM (11). Instead of the transformation U†(x′)U(x), we consider R†(y′)U†(x′)U(x)R(y), where R(y) = e2iy\|HH\| is a phase shift applied to a prese- lected H-polarized photon in the bottom part of the setup, and R†(y′) = e−2iy′\|HH\| is a phase shift to the postselected H-polarized photon in the same part of the setup. The phase difference between the postselected upper and lower H-polarized photons can be measured as cos2(y −y′). This is done with PBS′ which transmits diagonally-polarized photons \|D = (\|H + \|V)/ √ 2 and reflects antidiagonal photons \|A = (\|H −\|V)/ √ 2, and polarization-resolving single-photon detectors (see caption of Fig. 3). Received: 30 March 2020; Accepted: 24 June 2020 References 1. Mohri, M., Rostamizadeh, A. & Talwalkar, A. Foundations of Machine Learning (MIT Press, Cambridge, 2012). 2. Schuld, M., Sinayskiy, I. & Petruccione, F. An introduction to quantum machine learning. Contemp. Phys. 56, 172 (2015). y y q g p y 3. Cai, X.-D. et al. 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Quantum machine learning in feature M. & Killoran, N. Quantum machine learning in feature Hilbert sp g p 13. Havlíček, V. et al. Methods O ti l i Optical circuit for KQML. States given by Eq. (11) can be prepared in a quantum optical setup. In the reported proof of principle experiment, we can set N = 3 and D = 2 . This means that, effectively, the experiment deploys q = 2 qubits per dimension. The FM is defined via single-photon polarization states (H/V polarization) as well as dual-rail encoding (T/B for top/bottom rail, respectively) (12) \|ϕ(x) = 2 n=1 c3(xn)\|HTn + √ 3s(xn)c2(xn)\|HBn + √ 3c(xn)s2(xn)\|VBn + s3(xn)\|VTn , (12) where c(xn) ≡cos(xn) and s(xn) ≡sin(xn). This approach is resource-efficient as it only requires two photons to encode x into the FHS state of N = 3 and D = 2.h where c(xn) ≡cos(xn) and s(xn) ≡sin(xn). This approach is resource-efficient as it only requires two photons to encode x into the FHS state of N = 3 and D = 2. l l h d d Th f h k f ll An optical circuit implementing this FM is depicted in Fig. 3. The top part of the FM circuit works as follows: first, it transforms the standard input \|HB using wave plates resulting in \|HB →(\|HB + \|VB)/ √ 2. Next, a beam divider separates polarization modes in space, i.e., we have (\|HB + \|VT). Now, the effective operation of wave plates in the top and bottom modes can be described as first transforming \|VT →µT\|HT + νT\|VT and \|HB →µB\|HB + νB\|VB. The parameters are set as µT = √ 2c3(xn), νT = √ 2s3(xn), µB = √ 6c2(xn)s(xn), νB = √ 6c(xn)s2(xn).hh This whole operation is unitary and can be described as U(x)\|HH = \|ϕ(x). The complex conjugate of opera- tion U(x) is U†(x′) and it can be used to express the kernel as κ(x′, x) = \|HH\|U†(x′)U(x)\|HH\|2. Thus, the circuit U†(x′) for projecting the state \|ϕ(x) to \|ϕ(x′) can be constructed as the inverse of the feature embedding U(x) circuit, but for setup parameters set for x′ . The next action of the plates in the top and bottom rails is to perform a reverse transformation, but for xn = x′ n. Next, the plates flip the polarizations in the respective rails. Now, the interesting part of the engineered state is in the top rail with flipped polarization. To implement U(x′)†, Scientific Reports \| (2020) 10:12356 \| https://doi.org/10.1038/s41598-020-68911-5 www.nature.com/scientificreports/ Additional information Correspondence and requests for materials should be addressed to K.B., A.Č. or K.L. Correspondence and requests for materials should be addressed to K.B., A.Č. or K.L Reprints and permissions information is available at www.nature.com/reprints. Author contributions K.B., C.G., and F.N. developed the theoretical framework, and wrote the paper. K.B. planned the experiment, processed the experimental data. A.Č., K.J. and K.L. designed and built the experimental setup and performed the measurements. All authors discussed the results and participated in the manuscript preparation. https://doi.org/10.1038/s41598-020-68911-5 Scientific Reports \| (2020) 10:12356 \| www.nature.com/scientificreports/ 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 license, and indicate if changes were made. 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W4394603965.txt	https://www.biorxiv.org/content/biorxiv/early/2024/04/09/2024.04.05.588226.full.pdf	en		Turn `noise' to signal: accurately rectify millions of erroneous short reads through graph learning on edit distances	bioRxiv (Cold Spring Harbor Laboratory)	2,024	cc-by	18,452	bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Turn ‘noise’ to signal: accurately rectify millions of erroneous short reads through graph learning on edit distances Pengyao Ping 1,2 , Shuquan Su1,2 , Xinhui Cai1,2 , Tian Lan1,2 , Xuan Zhang Peng4,5 , Yi Pan 3 , Wei Liu 1,2 , Jinyan Li 3 1,2 , Hui 1 Data Science Institute, University of Technology Sydney, PO Box 123, Broadway, Ultimo, 2007, NSW, Australia 2 School of Computer Science, Faculty of Engineering & Information Technology, University of Technology Sydney, PO Box 123, Broadway, Ultimo, 2007, NSW, Australia 3 School of Computer Science and Control Engineering, Shenzhen Institute of Advanced Technology (SIAT), Chinese Academy of Sciences, 1068 Xueyuan Avenue, Shenzhen University Town, Shenzhen, 518055, Guangdong, China 4 Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, 636921, Singapore 5 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore Corresponding author: jinyan.li@siat.ac.cn Abstract Although the per-base erring rate of NGS is very low at 0.1% to 0.5%, the percentage/probability of erroneous reads in a short-read sequencing dataset can be as high as 10% to 15% or in the number of millions. Correction of these wrongly sequenced reads to retrieve their huge missing value will improve many downstream applications. As current methods correct only some of the errors at the cost of introducing many new errors, we solve this problem by turning erroneous reads into their original states, without bringing up any non-existing reads to keep the data integrity. The novelty of our method is originated in a computable rule translated from PCR erring mechanism that: a rare read is erroneous if it has a neighbouring read of high abundance. With this principle, we construct a graph to link every pair of reads of tiny edit distances to detect a solid part of erroneous reads; then we consider them as training data to learn the erring mechanisms to identify possibly remaining hard-case errors between pairs of high-abundance reads. Compared with state-of-the-art methods on tens of datasets of UMI-based ground truth, our method has made a remarkably better performance under 19 metrics including two entropy metrics that measure noise levels in a dataset. Case studies found that our method can make substantial impact on genome abundance quantification, isoform identification, SNP profiling, and genome editing efficiency estimation. For example, the abundance level of the reference genome of SARS-CoV-2 can be increased by 12% and that of Monkeypox can be boosted by 52.12% after error correction. Moreover, the number of distinct isomiRs is decreased by 31.56%, unveiling there are so many previously identified isomiRs that are actually sequencing errors. April 5, 2024 1/37 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Author summary Detecting short-read sequencing errors and correcting the related erroneous reads is a long-standing problem in bioinformatics. Current error correction algorithms correct only small parts of the errors but simultaneously introduce thousands of non-existing sequences. We present a new method to rectify erroneous reads under 300 bp produced by PCR-involved miRNA-sequencing, small RNA sequencing, or paired-end RNA sequencing, regardless of platform or sample type. Our method is the first kind considering the PCR erring mechanism and machine learning technique to improve sequencing data quality by turning millions of erroneous short reads into their original state without bringing up any non-existing sequences into the read set. Our error correction method can make a significant impact on a wide range of cutting-edge downstream applications. The observations and advantages in the case studies lay down strong evidence to question the accuracies of current downstream research outcomes and open new avenues to conduct downstream analysis whenever short-read data are adopted. Introduction 1 Next-generation sequencing (NGS) techniques and platforms have dramatically changed the world of genomics and computational biology [1–3]. High throughput DNA-sequencing has enabled large scale whole-genome sequencing and gene-targeted sequencing; NGS-based RNA-seq has provided ever higher coverage and sharper resolution of dynamic transcriptome for a wide range of applications such as isoform discovery, differential gene expression analysis, alternative gene splicing and allele-specific expression profiling [2]. However, NGS inevitably self-made sequencing errors including base deletions, insertions and substitutions at various steps like sample handling, library preparation, polymerase chain reaction (PCR), and/or at the base calling step [4, 5]. Although the erring rate is estimated very low at 0.1% - 0.5% per base in Illumina short-read sequencing, huge numbers of erroneous bases have been generated and stored at every sequencing dataset (e.g., about 197,402 base errors in a miRNA-sequencing dataset ERR187525, and about 997,020 base errors in a paired-end whole-genome sequencing dataset SRR22085311 which have been found through this study). As these mistaken bases are randomly distributed across all the reads in a dataset, the percentage/probability of erroneous reads in a dataset can be very high (e.g., as high as 10-15%). Suppose the per-base erring probability is estimated as p at a sequencing platform, and assume these erring events are independent at all the base positions in a read, then the probability perror (r) of a read r containing one or multiple base errors is given by perror (r) = L X L i=1 i pi (1 − p)(L−i) = 1 − (1 − p)L 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 (1) where, L = ∥r∥, the length of read r. If p = 0.1% and L = 100, then perror (r) = 9.52%. In other words, the percentage of erroneous reads in a dataset is about 9.52% when the per-base erring rate is estimated as 0.1% and the length of reads L = 100bp. If the per-base erring rate p is estimated as 0.15%, then there are about 13.94% of erroneous reads in the dataset. This is a fundamental issue previously unrecognized concerning the high percentages of erroneous reads in NGS datasets. These erroneous reads are usually treated as data noise implicitly or explicitly excluded for downstream data analysis such as de novo genome/transcriptome assembly and differential gene expression profiling [6, 7]. Or, April 5, 2024 2 2/37 22 23 24 25 26 27 28 29 30 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. these erroneous reads are un-purposely considered as genuine true reads in the data analysis which may have led to inaccurate or wrong conclusions. To restore the huge missing value of these high percentages of erroneous reads in each sequencing dataset, it is highly demanded to do accurate rectification of all these errors, as opposed to treating them as noise removal, to boost the data quality and integrity so as to improve the downstream applications. One of the main sources of the sequencing errors is from PCR (polymerase chain reaction), a technique that makes fast duplications of small segments of DNA which has been used by NGS to amplify the fragmented DNA/RNA molecules for effective sequencing. Most of the time, PCR makes perfect copies of the fragmented segments of DNA/RNA, but occasionally it introduces base-pair substitutions, deletions, insertions, or even yields new hybrid sequences during template switching [8]. Thus, after the PCR amplification, one or two copies in the duplications of a DNA segment may show inconsistent bases. Fig. 1a illustrates how base errors arise when amplifying one DNA template during PCR amplification. PCR errors not only occur in the library preparation but also during sequencing processes such as clonal molecules [5]; Fig. 1b is an example that depicts how errors are introduced in the process of bridge amplification during Illumina sequencing. Such PCR erring incidents are then inherited by NGS’s base calling step that converts the nucleotide sequence into a digital string named a read. The conversion is not 100% accurate as well, similar to PCR introducing minor mistakes (Fig. 1c) [4, 9]. So, there exist various erring incidents in the sequencing. However, one thing nearly certain is that: an erroneous read must be a low-frequency rare read if the error occurred at the late cycles of PCR. This is because the probability of the same mistakes happening at the same positions is extremely low, especially in 200-300bp reads. Efficient detection of these erroneous reads from a dataset of hundreds of millions of reads is challenging. First, some low-frequency rare reads are genuine reads not containing any sequencing errors. This is attributed to the uneven PCR amplification rates at different segments of the DNA—poorly amplified molecules will be sequenced to a lesser extent than the highly amplified molecules and vice versa [10, 11]. Second, an amplified segment after PCR erring may become identical to a high-frequency molecule. As a result, for a pair of high-frequency reads A and B that are very similar, it is impossible to judge, without machine learning of the PCR erring mechanisms, whether A’s copies contain B’s wrongly amplified copy, or whether B’s copies contain A’s wrongly amplified copy. We construct a graph rG(R) using the unique reads r1 , r2 , . . . , rn along with their respective frequencies from a reads dataset R (a multiset of reads) to detect erroneous reads under the sophisticated help of graph-based machine learning. Let f req(r) represent the abundance level or the frequency of a read r, or the number of copies of r in the sequencing data. For each of the unique reads in R, we represent it as a node in the graph and label the node with the read’s frequency. There is an edge e(i,j) between node ri and node rj if the edit distance between read ri and read rj is 1 or 2. Specifically, when searching for edges with an edit distance of 2, only substitutions are taken into account. A read u is a neighbouring read of read v if there is an edge between them. As understood from the PCR erring mechanism in NGS, the pairing of two neighbouring reads u and v implies that a copy of u is a wrongly amplified/sequenced copy of the v molecule, or a copy of v is a wrongly amplified/sequenced copy of the u molecule, or both. When f req(v) is low while f req(u) is high, we rectify the erroneous read v by removing this node from the graph, while increase f req(u) by f req(v). That is, we turn the ‘noise’ read v (low-frequency rare read) into its normal state u. We denote such a set of erroneous reads in the graph as edit-erring-READS and the isolated nodes with high frequencies as error-free-READS. April 5, 2024 3/37 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. a A ... G C-A error T-G error G ... ... G C C G ... C ... G C G C G C G C ... (0) DNA Template (1) 1st Cycle (2) 2nd Cycle (3) 3rd Cycle (4) 4th Cycle (5) 5th Cycle (6) 6th Cycle b Flow cell errors ... A-G error G T (1) Flow cell hybridization c ① (3) Strands denature (2) Bridge amplification ② ③ ① ② (4) Ready for sequencing ② ① ③ ... ③ T A T G C T ... ... ③ T T C C A G ... ... ① G-T error ② ① (1) Repeating N cycles to image a read with N bases ① ② ③ C A G A A T ... ② . ③ ... Clusters ... Imaging Dye-labeled dNTPs G A A G AG (2) Captured fluorescent singnals (3) Sequencing Reads Fig 1. Schematic diagrams to illustrate how base errors are generated during library preparation and sequencing process. (a) Schematic illustration of base error generation when amplifying one DNA template during conventional PCR amplification. Base ‘T’ mutated to ‘G’ in the 3rd cycle, the ‘C’-to-‘A’ error occurs in the 4th cycle and this error is inherited by the subsequent cycles. (b) Schematic graph depicting PCR errors generated in the process of bridge amplification during Illumina sequencing. An example of ‘A’-to-‘G’ is inherited. (c) An overview of base calling during Illumina sequencing. Using a small edit distance of 1 or 2 to define the edges of the graph is because those erroneous reads containing one mistaken base or two constitute the majority of the total erroneous reads in the dataset. The majority percentage is given by PEmax L i (L−i) i p (1 − p) error%(p, 1, Emax) = i=1 (2) 1 − (1 − p)L April 5, 2024 4/37 83 84 85 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. where p is the base erring probability, Emax is a maximum edit distance allowable to define an edge. If L = 100, p = 0.1% and Emax = 2, then error%(p, 1, Emax) = 99.84%. This indicates that 99.84% of all the possible erroneous reads in the dataset are those reads containing one base error or two (Emax). The second challenge in the correction of erroneous reads in the graph rG(R) is to deal with the situation when a low-frequency read is linked to multiple high-frequency reads, or two (or more) high-frequency reads are linked each other in the graph (denoted as ambiguous errors). Hence, we model the situations as a classification problem and use machine learning techniques to predict whether the duplications of a high-frequency read contain or not contain wrongly sequenced copies of its neighbouring high-frequency reads. This is a novel classification problem not formulated in any literature. In this work, we use edit-erring-READS and error-free-READS as training data and extract multiple features of different dimensions from the data, and then utilize an optimized gradient boosting classifier of the eXtreme Gradient Boosting (XGBoost) [12] to make the prediction under a supervised learning framework. As the training data is rG(R)-specific, the prediction model is able to learn the inherent erring patterns of each specific sequencing platform that conducts the specific biomolecular samples’ sequencing. So, our machine learning approach is competent to handle the rectification of erroneous reads that have a length less than 300 bp produced by any PCR-involved miRNA-sequencing, small RNA-sequencing, or paired-end RNA-sequencing regardless of the difference in the platforms or in the biomolecular samples. We name our method ‘noise2read’. Results 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 Comparing with state-of-the-art methods including k-mer-methods [13–19], multiple sequence alignment based methods [20–24], and other methods [25–27], our noise2read consistently outperforms under 19 metrics on eight UMI-based wet-lab datasets and five simulated single-end and paired-end datasets constructed in this study. It also has superior performance on eight UMI-based wet-lab datasets and four simulated miRNA datasets established previously in published literature. Case studies on twelve single-end and two paired-end sequencing datasets (about abundance change of viral reference genomes, isoform identification, SNP profiling, genome base editing efficiency estimation) reveal that noise2read can make substantial impacts on downstream applications. We first present real distributions of the erroneous reads containing various numbers of base errors in a dataset through UMI-based cluster analysis. High prevalence of erroneous reads containing one or two base errors from UMI-based cluster and distribution analysis We utilized the sequence information of UMI tags to investigate the distributions of erroneous reads that contain different numbers of base editing errors. Specifically, we divided the reads in a UMI group into high-frequency reads and low-frequency reads. Then, we calculated the edit distance between each unique low-frequency read and each unique high-frequency read. Then, each of the unique low-frequency read has the smallest edit distance with the set of unique high-frequency reads. Given each of these smallest distances, we record the number of low-frequency sequences that have this edit distance with the set of high-frequency reads. We applied the above process to the datasets of SRR1543964-SRR1543971 by defining a high-frequency read as a read with a copy count no less than five (note: clusters with ambiguous base ‘N’ in the UMI sequence are not included in this analysis). April 5, 2024 86 5/37 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. We observed that there exist two different high-frequency sequences that have been tagged with the same UMI, as similarly reported in the literature [28]. For instance, as seen in S1 Fig, each of these UMI clusters has two high-frequency reads, between which the edit distance is bigger than 100 (111 or 129 respectively), demonstrating that such two high-frequency reads within the same UMI cluster should be originated from two different molecules although they were tagged with the same UMI. Moreover, there exist big editing distances (e.g., 116) between high and low-frequency reads within the same UMI cluster, it would be unreasonable to assume only base-editing-error-relationship between all the low and high frequency reads. In fact, a low-frequency sequence with a small edit distance to a high-frequency read is more likely caused by PCR or sequencing errors. Here, we assume that those low-frequency reads with an edit distance equal to or less than 4 may be erroneous reads caused by PCR or sequencing errors. In this context, among these eight data sets, at least 60% of the erroneous reads in 95.21%-96.70% of the UMI clusters are caused by 1 or 2 base errors, as depicted in the stacked bar chart in S2 Fig. Five more UMI clusters are shown in S3 Fig: 81.25%, 95.24%, 100%, 94.73% and 100% of low-frequency reads have the 1 and 2 base difference with the set of high-frequency reads in the same UMI cluster. These findings indicate that those erroneous reads containing one mistaken base or two constitute a more significant proportion of the total erroneous reads in the data set. Based on our theoretical analysis and UMI cluster analysis, the proposed algorithm, noise2read, is set to correct erroneous reads containing base errors less than 3. Constructed training data for noise2read 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 Noise2read is a progressive three-stage error correction method. As introduced above, its novelty sits in the computable rule translated from PCR erring mechanism: a rare read is erroneous if it has a neighboring read of high abundance. With this principle, we construct a graph to link every pair of reads of a small edit distance to detect a substantial part of erroneous reads in the graph. Then we take them as training data to learn the platform-specific erring mechanism to identify possibly remaining hard-case errors between pairs of frequent reads in the graph, namely specific training data is used at different platforms. An Auto Machine Learning (AutoML) module is centered in the process of noise2read, which is used multiple times in the different stages for the prediction of ambiguous or amplicon errors. AutoML has a component for the preparation of training and objective data and has a component for the parameter optimization of the gradient boosting-based classifiers (Fig. 2). The first stage (shaded in blue) rectifies low-frequency leaf nodes (genuine errors) and ambiguous errors by a traversal on the 1nt-edit-distance read graph 1nt-rG(R0 ) constructed from the original reads dataset R0 . Here, every edge in the 1nt-edit-distance read graph means the edit distance between the two nodes is one nucleotide (i.e., 1nt). The second stage (shaded in pink) conducts correction of genuine and ambiguous errors at the 2nt-edit-distance read graph 2nt-rG(R1 ) constructed from the first-stage corrected dataset R1 . Here, every edge in the 2nt-edit-distance read graph means the edit distance between the two nodes is two nucleotides (i.e., 2nt). Particularly, we consider only substitution relationships for constructing 2nt-edit-distance edges since the majority of NGS data conforms to a consistent read length. The third stage (shaded in yellow) is designed to eliminate specific errors at an updated 1nt-edit-distance graph 1nt-rG(R2 ) only for the amplicon sequencing data but using the same AutoML module for prediction. Graph rG(R) is often a disconnected graph. Fig. 3 shows nine sub-graphs of rG(D1) constructed at the first stage, where D1 is a simplified version of SRR1543964. It is interesting to see that there are many clustered low-frequency leaf reads linked to one high-frequency read, while there also exist edges that link pairs of high-frequency reads. April 5, 2024 134 6/37 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Input R0 Output R I I I I I I 1nt-rG(R0) 1a 1nt-rG(R2) 3f Amplicon Correction edit-erring3e 3b & error-freeREADS 3d 1b edit-erring& error-freeREADS 2d AutoML 1nt-error Correction 2nt-error Correction 2e 2f R2 1d 2b I R1 No edit-erring2c & error-freeREADS 1f I Amplicon? 3c 1c 1e 3a Yes Read Length > L? No 2a Yes 2nt-rG(R1) 1a - ○) 1f Fig 2. Overview of the workflow of noise2read. The first stage ( ○ 2a - ○) 2f rectify 1nt- and 2nt-based-errors to their and the second stage (○ 3a - ○) 3f is optional only for normal states, respectively. The third stage (○ further correction specified to the amplicon sequencing data. The integrative Auto Machine Learning (AutoML) module is used multiple times by feeding different edit-erring-READS and error-free-READS in each stage. Fig. 4 is a zoomed version with more details about a subgraph in Fig. 3a, where the high-frequency nodes are highlighted in orange and the low-frequency nodes are highlighted in pink. By definition, every edge in this graph implies that the linked reads have only one base difference. With these sub-graphs, noise2read • directly turns those leaf nodes of low-frequency into their high-frequency parent nodes (their normal states r1 , r2 , ..., or r7 ); • uses the AutoML module to identify the parent node of two low-frequency nodes r8 and r9 , as these two low-frequency nodes are each linked to more than one high-frequency read (r8 is linked to r2 , r3 and r4 ; r9 is linked to both of r1 and r3 ); and • uses the AutoML module to judge whether there are erroneous reads between the linked high-frequency nodes (e.g., between r2 and r3 , between r5 and r7 ). Although noise2read is a three-stage progressive error correction method, we usually take the first two stages because they are sufficient to eliminate the majority of the errors in many typical NGS datasets. Only in the cases where the data has extensive coverage, such as amplicon sequencing, the option to use the third step is chosen for additional error correction. April 5, 2024 7/37 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. a c b d e f g h i Fig 3. Nine subgraphs, denoted as (a-i), from 1nt-rG(R0 ) in the first correction stage for the dataset D1 derived from SRR1543964. Each node in the subgraphs represents a read and is labelled with its frequency inside the circle. The edge connecting two nodes indicates that their edit distance equals 1. The number of nodes in the subgraphs (a, b, c, d, e, f, g, h) or (i) is 79, 69, 10, 621, 190, 111, 2788, 83 or 445, respectively. Entropy reduction and information gain after error correction The error correction effect or the noise/uncertainty reduction by an error correction method in a dataset can be measured by Shannon’s entropy and information gain. For a read dataset R, its Shannon entropy H is given by X H=− pr ∗ log2 pr (3) 202 203 204 205 r∈R where, pr is the percentage frequency of r in R. An ideal correction should eliminate all the errors/noises in the dataset while not introducing any new errors, or new sequences. Therefore, the entropy H ′ (R′ ) of a corrected read dataset R′ should consist of two parts: one is about the original reads, the other is about the wrongly introduced reads. We define H ′ (R′ ) as April 5, 2024 8/37 206 207 208 209 210 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. r1 r6 r9 r5 r3 r8 r7 r2 r4 Fig 4. This subgraph is a zoomed-in version of Figure 3a; it contains six high-frequency reads labelled as r1 to r7 and highlighted in orange, as well as contains 72 low-frequency reads shaded in pink. H ′ (R′ ) = H ′ (R′ ∩ R) + H ′ (R′ − R) X H ′ (R′ ∩ R) = − pr ∗ log2 pr (4) r∈{R′ ∩R} ′ ′ H (R − R) = − X pr′ ∗ log2 pr′ r ′ ∈{R′ −R} Information gain reflects the amount of information gained from the original state of the reads after the error correction, defined as △I(R′ ; R) = H(R) − H ′ (R′ ) = H(R) − H ′ (R′ ∩ R) − H ′ (R′ − R) 212 (5) The fewer unique reads is in a dataset, the less uncertainty, and the entropy will decrease after mistaken bases are corrected. To see the error correction effect on the data quality improvement, we propose to visualise the information change via a heatmap of taking items of △I as minor rectangular points and marking the wrongly April 5, 2024 211 9/37 213 214 215 216 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. introduced sequences as noises red dots. A visualization of △I for D1 is shown in Fig. 5 and those for D2 - D8 are presented at S4-S10 Figs. (details of these datasets are described at the section of Evaluation Criteria of Methods). The primary colour of the heatmaps close to zero strongly suggests that the correction conserves the original high-frequency information by all the methods. The negative and positive scores on the colour bar describe the information gain and loss, respectively. Seen from Fig. 5, noise2read is better than the other methods to reduce noise level as there is nearly no score bigger than zero. Those red points depict information loss brought by wrongly introduced new sequences, leading to new errors to increase false positives and negatives. noise2read does not yield any non-existing reads, and the colour in Fig. 5b darker than that in Fig. 5a implies that noise2read has more information gained from the additional amplicon sequencing correction. Moreover, to intuitively quantify the information gain or loss, we considered the changes only in low-frequency sequences before and after error correction. We denote the frequent reads as a subset F Rτ of R, and we calculate the entropy by removing F Rτ from R or R′ . Then, we focus on the entropy change given by △H = H(R − F Rτ ) − H ′ (R′ − F Rτ ) 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 (6) where τ is a threshold for defining high-frequency reads, H(R − F Rτ ) and H ′ (R′ − F Rτ ) represent the non-frequent reads’ entropy before and after correction, respectively. We calculated the entropy change △H for the sequencing datasets D1 - D8 and five simulated datasets D9 - D13 after error correction (details shown in Table 1). noise2read achieves the most considerable information gain on all these datasets. Specifically, the increased information by noise2read on the simulation datasets outperforms the other methods. The extensive information gain is because noise2read can rectify almost all the errors in the simulated datasets. 233 234 235 236 237 238 239 240 Table 1. Non-frequent reads’ information gain (△H with τ = 4) on the UMI-based wet-lab datasets D1-D8 and simulated datasets D9-D13. Best scores are highlighted in bold. Methods Datasets Coral RACER Fiona Lighter Pollux Bcool Care noise2read D1 0.588 6.023 4.077 4.686 2.508 0.916 1.776 6.409 D2 0.770 6.116 4.393 4.842 2.534 0.023 1.557 6.339 D3 0.921 6.369 4.891 5.505 3.044 2.333 0.469 6.239 D4 0.590 6.215 4.638 5.083 3.011 1.702 0.907 6.202 D5 0.752 6.360 4.589 5.127 2.821 0.046 1.302 6.297 D6 0.970 6.316 4.800 5.026 2.930 1.986 1.357 6.567 D7 0.898 6.306 4.883 5.422 3.096 0.100 0.446 6.194 D8 1.156 5.974 4.344 4.604 3.014 1.824 1.598 6.187 Average 0.831 6.210 4.577 5.037 2.870 1.116 1.176 6.304 D9 0.621 -1.349 -0.209 1.362 0.462 1.874 2.941 10.484 D10 2.072 -0.673 3.318 3.040 3.152 0.960 1.721 12.733 D11 2.849 0.273 2.129 3.910 3.459 0.723 1.483 12.380 D12 1.250 0.039 2.476 4.535 2.784 1.103 0.881 12.249 D13 1.159 0.811 2.991 5.193 3.159 1.725 0.909 13.161 Average 1.590 -0.180 2.141 3.608 2.603 1.277 1.587 12.201 Performance comparison on UMI-contained sequencing datasets The above eight datasets D1-D8 are subsets of wet-lab data sequenced using a UMI-contained high-fidelity sequencing technique (a.k.a. safe-SeqS) [29, 30]. Detailed April 5, 2024 10/37 241 242 243 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. a c b noise2read e d RACER g f Bcool h Lighter Care noise2read Pollux i Fiona Coral Fig 5. Visualisation of information gain for noise2read, noise2read, Care [23, 24], RACER [14], Bcool [26], Pollux [17], Lighter [15], Fiona [21], and Coral [20] on dataset D1. Here, noise2read denotes the result without further amplicon correction. Heatmaps (c, d, e, f, g, h) and (i) display the incorrectly introduced reads as red points. The number of red points shown on each heatmap corresponds to 502, 2310, 7808, 2935, 8523, 13899 and 722, respectively. generation of these datasets can be found in the first subsection of Evaluation criteria at the section Methods. We evaluated the performance of noise2read in comparison with seven other computational error correction methods at both the base-level and read-level under various metrics, including recall (TPR), TNR, fall-out (FPR), FNR, Area Difference (AD), Precision, Positive Gain, Accuracy, and Purity Entropy (E). Detailed definitions of these metrics can be found in Metrics section of Methods. The comparative performance with the seven methods Coral [20], RACER [14], Fiona [21], Lighter [15], Pollux [17], Bcool [26], and Care [23, 24] are summarized in Fig. 6a for D1 and those in S11 Fig and S12 Fig for D2-D8. S2-S9 Tables. are provided to further supplement the results. Our method noise2read has achieved the best performance on all the datasets under all the metrics. The high-TPR and low-FNR performance indicate that noise2read can turn most noise while leaving the lowest number of actual noise as signals; The high TNR illustrates that noise2read can introduce fewer new errors by preserving most signals unchanged, while the low FPR suggests that noise2read introduces none or few new noises without bringing up any non-existing sequences after the correction process. In detail, noise2read surpassed all the other methods on D1, achieving a score 0.924 higher than the second-best method RACER which has a score of 0.859. Notably, noise2read exhibited exceptional performance in Recall, Precision, Positive Gain, April 5, 2024 11/37 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Accuracy, and Purity Entropy, as evidenced by the values in Table 2. The positive gain percentage of noise2read is 7.26% and 48.15% higher than RACER and Care. noise2read and its amplicon mode achieved the finest purity entropy of 0.05 and 0.077, sounder than the second-best method RACER which has a score of 0.110. The progressive process gradually converts noise into signals; for example in Table 2, the 1st, 2nd and 3rd stages convert 72.9% (81,630), 93.2% (104,373), and 96.2% (107,717) of the errors into signals on D1, respectively. noise2read is mainly designed for any short-read sequencing data whenever PCR is involved. Without the 3rd step, it also achieves sound performance (refer to S2-S9 Tables.) by restoring most erroneous reads into their normal states and not introducing false positive reads. The 3rd step for further correction on amplicon sequencing data maintains fewer original error-free reads than the second stage and correspondingly introduces more noise but not new sequences. RACER can rectify 93.4% of the noise but newly introduces almost 22 times the number of new errors compared to our method. Care newly introduces 52 false positives but can only correct 64.9% of the erroneous reads. The other methods can only correct less than 65% of the errors but simultaneously give rise to thousands of new mistakes. 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 Table 2. TP, FP, FN, TN, Recall, Precision, Positive Gain, Accuracy and purity Entropy(E) at the read level for comparing our method noise2read (decomposed at different stages) with seven existing methods on the UMI-based wet-lab dataset D1. Best scores are highlighted in bold. Metrics Methods noise2read stage 1 stage 2 stage 3 Coral RACER Fiona Lighter Pollux Bcool Care TP FP FN TN Recall Precision Positive Gain Accuracy E 81630 104373 107717 22677 104589 72792 61330 20537 40970 72673 0 0 201 4249 4347 136472 145999 73710 3889 52 30391 7648 4304 89344 7432 39229 50691 91484 71051 39348 693059 693059 692858 688810 688712 556587 547060 619349 689170 693007 0.729 0.932 0.962 0.202 0.934 0.650 0.547 0.183 0.366 0.649 1.000 1.000 0.998 0.842 0.960 0.348 0.296 0.218 0.913 0.999 0.729 0.932 0.960 0.165 0.895 -0.568 -0.756 -0.475 0.331 0.648 0.962 0.991 0.994 0.884 0.985 0.782 0.756 0.795 0.907 0.951 0.232 0.077 0.050 0.518 0.110 0.757 0.802 0.732 0.447 0.3 We conducted additional performance comparisons between our noise2read and ten state-of-the-art approaches on eight UMI-based ground-truth datasets all established previously by the literature [30]. We refer these datasets as D34 to D41. Detailed information about these datasets can be found at Supplemental Table 1. To conduct a fair comparison, we employed the evaluation procedures presented in the literature [30] to compute the confusion matrix at the read level. By this definition, a read is deemed erroneous if even a single base is incorrect, namely, a read is error-free only when all its bases are correct. Table 3 shows the comparative performance of noise2read on D41 in comparison with Bless [18], Coral [20], Lighter [15], Reckoner [19], Sga [25], BFC [16], Pollux [17], Fiona [21], RACER [14], and Care [23, 24]. For comparative performance on D34-40, please see S17 Table. and S18 Table.. These comparison results highlight that noise2read consistently achieved the highest number of true positives on all of D34-D41, except for Fiona’s TP on D41, which is slightly bigger than noise2read. Importantly, noise2read demonstrates the lowest count of false positives among all these datasets. noise2read performs exceptionally good in Precision, Accuracy, AD and Positive Gain on all of the eight benchmark datasets. April 5, 2024 12/37 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Table 3. The comparative performance between noise2read and methods Bless [18], Coral [20], Lighter [15], Reckoner [19], Sga [25], BFC [16], Pollux [17], Fiona [21], RACER [14], and Care [23, 24] on the UMI-based benchmark dataset D41 previously established in the literature [30]. Metrics Kmer Positive Methods TP TN FN FP TPR FNR TNR FPR Precision Accuracy AD Size Gain Bless 30 39345 509513 23498 1751 0.63 0.37 1.00 0.00 0.957 0.96 0.39 0.60 Coral 30 23172 497906 48255 4774 0.32 0.68 0.99 0.01 0.829 0.91 0.09 0.26 Lighter 30 51934 497165 19336 5672 0.73 0.27 0.99 0.01 0.902 0.96 0.51 0.65 Reckoner 30 24143 501767 47233 964 0.34 0.66 1.00 0.00 0.962 0.92 0.11 0.32 Sga 26 13129 501767 58582 629 0.18 0.82 1.00 0.00 0.954 0.90 0.03 0.17 BFC 30 18415 500964 53345 1383 0.26 0.74 1.00 0.00 0.930 0.90 0.06 0.24 Pollux 30 26308 430041 33643 83210 0.44 0.56 0.84 0.16 0.240 0.80 -0.11 -0.95 Fiona 54983 483470 13675 21979 0.80 0.20 0.96 0.04 0.714 0.94 0.56 0.48 RACER 50106 444352 9857 69792 0.84 0.16 0.86 0.14 0.418 0.86 0.45 -0.33 NA Care 43213 501631 28896 367 0.60 0.40 1.00 0.00 0.992 0.95 0.36 0.59 noise2read 54316 501759 18011 21 0.75 0.25 1.00 0.00 0.9996 0.97 0.56 0.75 Performance on simulated short-read datasets and those with artificially modified bases Based on actual sequencing datasets of various read lengths of 75bp, 101bp and 150bp, we simulated datasets D9-D13 with mimic UMIs. D9 was simulated from a single-end dataset with SRA accession SRR12060401, while D10 and D11 were simulated using the forward sequencing data (R1) and the reverse complementing sequence data (R2) from a paired-end data with SRA accession SRR9077111. Similarly, D12 and D13 were simulated using R1 and R2 from a paired-end data with SRA accession SRR11092062. The simulation procedure is presented in Evaluation Criteria of Methods. Error correction performance are shown in Fig. 6b-l, S13 Fig and S10 Table. for dataset D9 and S14-S21 Figs. and S11-S14 Tables. for datasets D10-D13. noise2read super outperforms the other methods under all the metrics on all the simulated datasets. Specifically for the AD performance, noise2read (0.989) has 39.3%, 61.9% and 123.3% higher performance than that of Lighter(0.71), Care(0.611) and Fiona (0.443) (Fig. 6b). noise2read reaches the best precision, gain and accuracy and achieves a substantial positive gain (Fig. 6c).As shown in Fig. 6d, noise2read is the only method significantly decreasing the purity Entropy after correction. Information gain visualisations in Fig. 6e-l indicate the information is still dominated by most of the original signal after correction. All the other methods wrongly introduced new sequences (in a number of 164 to 9698) after correction. The other methods’ performance fluctuates widely. For instance, at the read level, the performance ranking of the top three methods in terms of AD on dataset D9 (Fig. 6b) is Lighter, Care and Fiona. However, the performance ranking is Fiona, Care, and Coral on D10 (S14(b) Fig), and Lighter, Fiona and Care (S16(b)Fig) on D12. April 5, 2024 13/37 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. b a c d g e f h i Care j RACER k Pollux Bcool l Fiona Coral Fig 6. Comparison of True Positive Rate (TPR), True Negative Rate (TNR), False Positive Rate (FPR), False Negative Rate (FNR) and Area Difference (AD) at the read-level for noise2read and seven other methods on the UMI-based wet-lab dataset D1 is shown in (a). noise2read* denotes the result without amplicon correction. Performance comparisons at the read-level on simulated dataset D9 are shown in (b-d). Information gain visualisations for D9 are presented in (e-l). Heatmaps in (f, g, h, i, j, k, l) display the red noise, with 1223, 164, 9698, 377, 2378, 3651 and 1255 points, respectively. April 5, 2024 14/37 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Moreover, we simulated four single-end miRNA datasets (denoted by D14-D17) using the simulation procedure proposed in miREC [27] plus an additional step of mimicking UMIs to these datasets. Of them, D14-D15 contain substitution and indel errors, while D16-D17 contain only substitution errors. Comparison results between noise2read (with or without high-frequency ambiguous error prediction) and miREC are shown in Table 4 and S15 Table. noise2read can rectify more errors than miREC, achieving more TP and less FN after correction. The miREC method and noise2read can achieve similar, reasonably good results in accuracy, precision and fall-out (S15 Table). However, from the recall, Positive Gain, purity Entropy E and information gain △H performance on all four datasets, noise2read is better than miREC. 320 321 322 323 324 325 326 327 328 329 Table 4. Performance comparison between noise2read (with and without enabling the high-frequency ambiguous error prediction) and miREC on miRNA simulation datasets D14-D17 at the read level. Metrics Datasets Methods TP FP FN TN Recall Positive Gain △E △H miREC 4224 0 267 218980 0.941 0.941 0.129 4.755 D14 noise2read 4411 11 80 218969 0.982 0.980 0.137 9.053 noise2reada 4409 3 82 218977 0.982 0.981 0.137 9.053 miREC 4135 5 271 219060 0.938 0.937 0.126 4.821 D15 noise2read 4305 7 101 219058 0.977 0.975 0.134 8.485 noise2reada 4308 4 98 219061 0.978 0.977 0.134 8.485 miREC 6418 16 309 216728 0.954 0.952 0.179 5.301 D16 noise2read 6577 17 150 216727 0.978 0.975 0.186 8.184 noise2reada 6579 16 148 216728 0.978 0.976 0.186 8.184 miREC 6398 0 306 216767 0.954 0.954 0.179 5.337 D17 noise2read 6577 1 127 216766 0.981 0.981 0.187 8.769 noise2reada 6574 0 130 216767 0.981 0.981 0.187 8.769 a Performance by noise2read without prediction of errors between high-frequency reads. b High-frequency threshold τ = 4. Error correction changes the abundance level by 12% for the reference genome of SAS-Cov-2 and by 52.12% for Monkeypox The SARS-CoV-2 and Monkeypox viruses have severely affected the health of human beings. The reference genome sequences have been extensively utilized to understand the origin and phylogeny, and also as a fundamental framework for the design of mRNA vaccines. We investigate how much abundance is changed for the reference genomes of these two viruses after our algorithm noise2read rectifies the base errors contained in the short-read sequencing data of the reference genomes. The study will help understand the within-host viral mutants of the reference genome and their abundance compositions. We removed human-related reads from paired-end sequencing dataset SRR11092062 [31] using Bowtie2 [32] and the human genome reference GRCh38.p13. The paired R1 and R2 of SRR11092062 after the filtering process were denoted as D18 and D19, respectively. Then, we performed read alignments for all the reads in D18 and D19 using Bowtie2, and extracted only those reads that have perfect matches with the viral reference genome MN996528.1 [31] to calculate the base coverage (abundance level) of the viral reference genome. We note that the reference genome MN996528.1 of SARS-CoV-2 was de novo assembled from paired-end short-read sequencing dataset SRR11092062 after human-related reads were filtered. Our method noise2read corrected 181,360 erroneous reads in D18 and corrected 138,575 erroneous reads in D19. In particular, 2328 corrected reads can be perfectly April 5, 2024 15/37 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. aligned to the genome. This leads to the base coverage of the reference genome increased by 12% on average, namely an elevation of the sequencing coverage from 96.07 to 107.75 (or from the 19,144 perfectly matched reads to 21472) (Figure 7a, b). Figure 7c depicts a frequency distribution under a scaled density curve for the coverage difference after error correction. Some regions of the genome maintain the same low level of base coverage without change after the reads correction. Interestingly, some base coverage becomes lower after the error correction (e.g., position 13541) suggesting there exists a within-host mutant that gains new support of perfectly matched reads turned from the reference genome. In the abundance change analysis for the Monkeypox virus, we used the paired-end whole-genome sequencing dataset SRR22085311 ( its paired R1 and R2 denoted as D20 and D21 here) and the reference genome GCA 025947495.1 [33]. Our noise2read rectified a huge number of erroneous reads in D20 (400,622 out of 3,599,812 reads, i.e., 11.13%), and another huge number of erroneous reads in D21 (456,242 out of 3,599,812 reads, i.e., 12.67%). Figure 7e presents a coverage comparison chart before and after the error correction, alongside a coverage difference chart (Figure 7f), where it can be seen that the average base coverage of the reference genome is increased by 52.12% from depth 1216.75 to 1850.95 after a huge number of 651,410 reads were retrieved to perfectly align with the genome.The frequency distribution of the base coverage differences as another angle viewing the abundance change for the Monkeypox virus is presented in (Figure 7d), where the abundant and perfectly matched reads aligned to the genome are highlighted again. Especially, those positive shifts towards a higher coverage (Figure 7e,f) confirm much more about the ground truth of the known reference genome and the detection of possible new variants of the genome. April 5, 2024 16/37 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. a b c d e Fig 7. Comparison of base coverage before and after correction for SARS-Cov-2 and Monkeypox virus genomes using perfectly matched reads. (a) and (b) show the base coverage and its difference for the SARS-Cov-2 data, while (e) and (f) present the base coverage and coverage difference for the Monkeypox virus data. Frequency distributions of the coverage difference are shown in (c) and (d) with scaled density curves for the SARS-Cov-2 and Monkeypox virus data, respectively. April 5, 2024 17/37 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Accurate error correction improves detection of isomiRs and refines SNPs profiling MicroRNAs (miRNAs), non-coding RNA molecules approximately 22 nucleotides long, can modulate gene expression post-transcriptionally through the silencing and decay of target mRNAs [34]. Dysregulation of miRNAs plays crucial roles in many biological mechanisms, and it is also a main reason in cancer and autoimmune disorders [35, 36]. By miRNA sequencing, various types of isoforms (i.e., isomiRs) have been detected [37]. However, whether the base differences found in the isomiRs are actual biological variations or synthetic artefacts due to the PCR or sequencing errors or both is difficult to judge. Here, we study how our error correction changes the identification and quantification of isomiRs from short RNA-seq datasets and how it refines the profiling of known single-nucleotide polymorphisms (SNPs) in isomiRs. We downloaded ten single-end small RNA-sequencing datasets of lymphoblastoid cell lines from five population groups in the 1000 Genomes Project [38]. These datasets (denoted as D22-D31 here) were cleaned by removing the adapter sequences via cutadapt [39]. We used IsoMiRmap [40] under the setting of pre-defined miRNA reference sets from the database miRBase(v22) [41] as a “miR-space” to quantify known isomiRs and SNPs for D22-D31 before and after our sequencing error correction. IsoMiRmap tags an identified isomiR as an exclusive isomiR if it only exists in the miR-space with one or more occurrences but not elsewhere in the human reference genome, otherwise recognized as an ambiguous isomiR. These quantification results are summarised in Table 5. The number of unique ambiguous isomiRs is decreased by 24.12%-31.75% or in numbers from 151 to 245, but their total counts are increased by a number between 160 and 640 among the ten datasets after the error correction; the number of exclusive isomiRs is decreased by 34.46%-37.48% but their total counts are increased by a number between 5095 and 14,441. These results suggest that some previously identified isomiRs are artefacts containing sequencing errors rather than natural isoforms. On the other hand for the profiling of the known SNPs, the number of unique SNPs is decreased by 34.13% 59.09%, and their counts are also decreased by 4.40% - 35.56% except for two increased by 1.41% and 1.61% respectively. This observation unveils that some of the previously annotated SNPs are actually sequencing errors. Similar quantitative and qualitative changes observed in the profiling of these known SNPs in the isomiRs distinguishing true SNPs from sequencing errors enables more accurate annotation of SNPs. The significant change of the isomiRs quantification after correction is because an average of 235,146 (2.62%) sequences were corrected by noise2read in the ten datasets (Table 5). To understand more about the frequency change of isomiRs and SNPs, we categorised the isomiRs according to their original miRNAs, then we utilised scatter graphs with Kepler plots to understand the associations between the number of identical isomiRs and total isomiRs’ count (log10 transformation) before and after the error correction of the sequencing reads. The leftward shift on the x-axis (Fig. 8a and 8b for exclusive isomiRs of D22 and D23,respectively, Fig. 8c, d for ambiguous isomiRs and known SNPs of D22, and S22-S24 Figs for the other miRNA datasets) indicates a reduction of the count of unique isomiRs, while the upward change on the y-axis indicates an increase in authentic isomiRs. These significant changes in isomiRs and SNPs highlight the importance of correction for accurately characterizing isomiR and SNP profiles, making contributions to the annotation of isomiRnome. April 5, 2024 18/37 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 April 5, 2024 272107 207804 235146 D30 Average 144252 D29 D31 264327 288649 D27 D28 147763 268616 D25 324175 D24 D26 236699 2.62% 1.66% 3.40% 2.45% 4.23% 2.24% 1.43% 2.17% 1.91% 4.02% 2.70% 197071 D22 D23 Correction Percentage Read Corrected Number Datasets 25.15% 31.75% 25.73% 619 632 851 871 827 27.21% 26.16% 26.78% 31.18% 27.65% 634 670 309 594 915 449 24.12% 25.59% 27.13% 475 407 518 626 547 708 821 688 508 485 331 Decrease Corrected Original Unique Reads Number 18546 10752 14092 32558 8735 29849 19761 24767 17653 10867 16425 Original 18964 10912 14517 33198 9124 30344 20147 25191 17978 11365 16859 Corrected 2.53% 1.49% 3.02% 1.97% 4.45% 1.66% 1.95% 1.71% 1.84% 4.58% 2.64% Increase Total Reads Count Ambiguous isomiRs 35.88% 36.69% 34.46% 4590 3427 5229 8134 7158 36.07% 36.75% 35.52% 37.48% 34.95% 5200 4377 2959 4855 6788 4733 36.59% 35.44% 35.98% 3403 3347 3916 5367 5184 6112 7464 5553 3512 5511 3489 Decrease Corrected Original Unique Reads Number 1165945 685935 938218 2598394 741500 1215062 779496 1763443 732858 928151 1276397 Original 1175539.2 691030 947607 2612835 751526 1223420 786162 1776084 739190 940139 1287399 Corrected 0.89% 0.74% 1.00% 0.56% 1.35% 0.69% 0.86% 0.72% 0.86% 1.29% 0.86% Increase Total Reads Count Exclusive isomiRs 52.98% 53.45% 49.31% 79 73 144 154 168 59.09% 44.55% 34.13% 57.14% 54.59% 59.02% 41.38% 50.56% 63 83 51 84 50 51 64 126 119 122 87 132 185 101 56 116 54 Decrease Corrected 704 1540 184 284 2639 3909 362 253 396 450 6220 1491 142 183 2523 3964 318 192 266 392 6320 606 Corrected 15.57% 22.83% 35.56% 4.40% -1.41% 12.15% 24.11% 32.83% 12.89% -1.61% 13.92% Decrease Total Reads Count Original SNPs Original Unique Reads Number Table 5. Known isomiRs and SNPs profiling change from miRNA sequencing data before and after correction. bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 19/37 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. a b c d Fig 8. Scatter graphs compare the numbers of isomiRs and known SNPs before and after correction, while the Kepler plots provide additional visualizations of the data distribution. a and b compare the numbers of exclusive isomiRs from D22 and D23, respectively, and c and d compare the numbers of ambiguous isomiRs and known SNPs from D22. Accurate error correction significantly improves the quality of outcome sequences edited from a disease gene by ABEs and CBEs Base editing is a new genome editing technique that uses CRISPR systems and enzymes to introduce point mutations into cellular DNA or RNA for modeling and understanding genetic diseases [42, 43]. However, deciding whether a nucleotide position is exactly editable in a genomic context is inefficient by wet-lab experiments, and the base editors may yield many unexpected genotypic output sequences when the editable window covers multiple target nucleotides. Deep-learning-based prediction tools have been developed to predict the base-editing efficiency and outcome-sequence copy numbers from Adenine and cytosine base editors (ABEs and CBEs) [44]. The training data used by these prediction tools are extracted from short-read DNA/RNA sequencing data. Here, we investigate how much the number of unique reads (unique outcome sequences) changes after our sequencing error correction. We removed those records in which the target sequence has only one outcome sequence from the training data of HT ABE Train and HT CBE Train used in the literature [44]. Then, we cleaned them to form two datasets (denoted by D32 for ABEs April 5, 2024 20/37 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. and D33 for CBEs here), and applied noise2read to D32 and D33 separately. As a result, the number of unique outcome sequences in D32 is reduced by 2309 from 28892 to 26583 (7.99%), and the number of unique outcome sequences in D33 is reduced by 5042 from 27312 to 22270 (18.46%). The number reduction of unique outcome sequences is because some low-frequency reads are not a result of base editing but due to sequencing errors. In total, noise2read recognised 5109 erroneous reads in the ABE dataset and 10271 erroneous reads in the CBE dataset and turned all of them into normal states. This error correction has significantly improved the quality of the training data that would be very helpful for enhancing the prediction of base editing efficiencies. Runtime and memory consumption 438 439 440 441 442 443 444 445 446 447 448 We compared CPU runtime and peak memory used by noise2read with those by Care [23, 24], RACER [14], Bcool [26], Pollux [17], Lighter [15], Fiona [21], and Coral [20] on data sets D1-D8. We executed all the programs on an Intel(R) Xeon(R) Gold 6238R CPU clocked at 2.20GHz, leveraging 56 CPU cores for parallel computing. For the model training of noise2read, a single Tesla V100S-PCIE-32GB GPU was employed. To gauge memory usage across all the programs, we used the library psutil [59]. These runtime and memory consumption comparisons are presented in Table 6. 449 450 451 452 453 454 455 456 Table 6. Runtime(T) and Memory(M) usage by different methods on the datasets D1-D8. CPU D1 D2 D3 D4 D5 D6 D7 D8 Methods cores T M T M T M T M T M T M T M T M Coral 3.3 65528 3.4 91045 3.0 54182 3.8 99376 3.9 99673 3.3 68805 3.7 61512 2.4 49311 Fiona 56 36.4 1736 20.2 1702 22.5 1811 25.2 2111 29.1 1794 33.4 1755 19.9 1846 15.2 1353 Lighter 1 568 1 564 1 568 1 568 1 568 1 564 1 566 1 566 RACER 64 3.1 111 2.7 100 3.7 130 4.1 146 3.4 123 3.4 125 3.7 135 2.2 110 Pollux 1 1277.4 219 1072.2 197 1445.3 204 1722.2 217 1425.1 209.07 1455.3 219 1480.1 217 844.9 198 Bcool 15.9 12 4.6 12 8.1 12 11.7 12 3.9 12 11.6 12 5.5 12 5.3 12 Care 1 788 1 793 1.0 813 1 737 1 812 1 823 1 822 1 589 56 noise2read 137.3 4405 121.5 4012 126.8 6629 143.1 5723 125.8 4755 136.3 6557 123.0 5393 110.2 3472 noise2reada 171.0 4373 161.0 5350 146.0 4824 199.0 7449 173.0 4699 178.0 5189 160.0 5014 198.0 3767 a. The performance of noise2read is enhanced through additional amplicon correction. The CPU model of Intel(R) Xeon(R) Gold 6238R CPU @ 2.20GHz was used by all the methods. 1 GPU of Tesla V100S-PCIE-32GB was used for the model training of noise2read. The runtime is given in minutes; Memory consumption is given in MB. Lighter and Care exhibited fast speeds, completing corrections within a minute by taking a small amount of memory consumption for each of the 8 data sets. On the other hand, Pollux had the slowest speed due to its inability to run in parallel. noise2read spent the second highest memory consumption and made the second slowest speed. (We note that we do not suggest using a large number of multiprocessing processes for noise2read to run, as we have observed that those situations could suddenly consume a significant amount of memory, and the program ran out of memory and terminated.) The separate time consumption by noise2read at its different stages as recorded in the built-in log files across all datasets D1-D41 are presented in S19 Table. It can be observed that a significant amount of time was spent on tasks such as constructing “2nt-edit-distance read graphs”, “performing feature extraction”, and “model training”. To shorten the running time on large data sets, it is suggested to choose a smaller number of negative samples and set a smaller number of trials (e.g., 20) for the construction of sub-optimal models. Additionally, opting not to predict errors within April 5, 2024 21/37 457 458 459 460 461 462 463 464 465 466 467 468 469 470 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. high-frequency reads will also save noise2read a substantial amount of time and memory usage but fortunately without much performance sacrifice on error correction. We note that although noise2read is slow, it never introduces any non-existing reads into the datasets. This is a unique merit in all of current sequencing error correction methods. While the speed of error correction is undeniably a crucial factor in evaluating the performance of a correction method, an even more critical consideration is whether the method introduces new errors. A fast error-introducing method damages the quality of the whole dataset and may become unexpectedly harmful to downstream data analysis, although its fast speed is advantageous in the preprocessing error correction stage. Our method does not have this speed advantage so far, but it never introduces new errors, guaranteeing the integrity of the datasets. In future work, we consider efficiency tricks to improve the speed of feature extraction and machine learning. Discussion 472 473 474 475 476 477 478 479 480 481 482 483 A long-standing problem in sequencing data analysis is how to reduce sequencing base errors and erroneous reads as much as possible before any downstream applications. Existing short reads correction methods utilize biochemical-based experimental designs such as unique molecular identifiers (UMIs) to count and track molecules [10], or take computational methods including k-mer-methods [13–19], multiple sequence alignment based methods [20–24], and the other methods [25, 26]. One limit of the UMI-based strategies is that errors/mutations can also happen at UMIs. Serious concern about the computational methods is that they have significantly overcorrected reads by introducing pseudo new sequences or shifting one type of error into another, often leaving numerous reads uncorrected. Some of these methods only focus on restoring substitution mistakes but do not support indels’ correction. Besides, instance-based methods such as miREC [27] were designed to handle specific sequencing data type miRNA sequencing reads. And it assumes that frequent sequences contain no mistakes, thus it cannot be used to correct potential errors between high-frequency reads or cannot deal with those singletons with no relationships to the high-frequency reads. Following the principle of the PCR erring incidents and sequencing process, we constructed special graphs of short reads to capture the relationships between edit-erring and error-free reads. Through novel modelling of the errors between high-frequency reads and their high- or low-frequency neighbours as a classification problem, we have successfully predicted almost all the errors using machine learning techniques. Validation experiments on the UMI-based wet lab and simulated datasets of known ground truth have demonstrated that the proposed noise2read algorithm can eliminate most of the PCR and sequencing errors without introducing any non-existing sequences into the read set. Moreover, we investigated the impact of error-corrected data on downstream data applications. We have found that: (1) The abundance level change of the reference genome of SARS-CoV-2 and Monkeypox after the sequencing error correction is remarkable, which may allow us to rethink how to get a precise genome sequence for these viruses; (2) For the isomiRs and SNPs profiling, the counts of some isomiRs and SNPs are decreased while some others are increased, which is of great significance to identifying actual isomiRs and SNPs and re-annotating the isomiRnome. (3) Both ABE and CBE should have higher base editing efficiency than currently estimated. The accurate and higher base editing efficiency with correct preprocessing may improve the original deep-learning prediction accuracy. Altogether, these observations and advantages lay down strong evidence to question the accuracies of current downstream research outcomes and open new avenues to conduct downstream analysis whenever short-read data are adopted. April 5, 2024 471 22/37 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. A small edit distance such as 1 or 2 is currently used to define the edges of rG(R). When the edit distance threshold Emax is enlarged, more edges will be created for rG(R) and possibly more erroneous reads will be identified. The tradeoff is that the computational complexity of constructing these new edges is exponential while newly identified erroneous reads become less and less when Emax increase. In fact, these erroneous reads constitute an extremely small percentage (< 0.16%) of the total erroneous reads in theory. In future work, we will test the computational complexity when Emax is set as 3, and explore how to change the correction steps. The speed and memory usage of noise2read still needs improvement, especially the parts for building the 1nt- and 2nt-edit-distance read graphs and AutoML training for prediction. The easy-usable and automatic tuning of the classifiers’ parameters facilitates wide-range explorations, but we note that noise2read may yield a slightly different result at different trials, even setting the same seeds. We also note that noise2read will derive more false positives when dealing with errors between high-frequency reads of extremely short length (e.g. < 30bp). This limit may be overcome by extracting more or fewer features from the reads. Furthermore, we already attempted using deep learning architecture (e.g., CNN, LSTM) to detect the errors, but a better performance was not achieved than by current noise2read. To make noise2read from outstanding to an exceptional tool, novel conception of new features for the short reads and attention-based deep learning models are potential solutions as our future work. 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 Methods 542 Implementation of our noise2read algorithm 543 The proposed algorithm noise2read was implemented using the python language and packaged as an easily-usable prototype publicly available. It mainly consists of modules for the construction of 1nt- and 2nt-edit-distance read graphs, for the creation of the training sets edit-erring-READS and error-free-READS for machine learning, for Auto Machine Learning (AutoML) prediction, and for the error correction steps. Special considerations in the construction of edit-distance graph of short reads By setting a high-frequency threshold τ , noise2read finds the 1nt- or 2nt-edit-distance edges between unique high-frequency reads (with frequency > τ ) and all the other unique reads in a read dataset, and then it takes all of these unique reads as nodes, their counts as attributes and the detected associations to build a graph. The rationale for not detecting the 1nt- or 2nt-edit-distance read pairs in the low-frequency reads is that it is computationally challenging and meaningless to distinguish whether one read of low abundance is mutated from the other low-frequency read (e.g., it is hard to determine if there are mutations or sequencing errors between two reads each with a frequency of one and with one base or two-base difference). The rationale for 2nt-edit-distance error correction is that some NGS data contains two base errors in some long read (e.g., 150 bp), and we set a threshold l (e.g., 30) of the sequence’s minimum length to determine whether to perform 2nt-edit-distance error correction. Noise2read does not perform a pairwise alignment for searching the 1nt- or 2nt-edit-distance edges between the high-frequency reads and all the other reads in the read set. Instead, it enumerates all the possible 1nt- or 2nt-edit-distance (substitutions only for the 2nt) reads for all of the high-frequency reads and stores them in the Python Set. Then, it invokes the Python built-in function intersection to obtain the edges. It April 5, 2024 521 23/37 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. may not be the best way to find all the edges using hash tables in this manner. However, such a strategy can find all required edges instead of finding an approximate number of edges. We constructed the 2nt-edit-distance-based graph by searching only substitution relations as edges. This idea is based on the observation that substitutions are the most prevalent type of sequencing error [45], and on that ambiguous nucleotides are often denoted by the symbol ‘N’ [46, 47] during sequencing. Moreover, NGS read lengths are usually consistent and fixed in a single sequencing run, owing to the fixed number of sequencing cycles in technologies like Illumina sequencing. This uniform read length is achieved since the read size is directly tied to the number of sequencing cycles performed, and each cycle corresponds to the sequencing of a single base. On the other hand, if a deletion or insertion exists in the read, the sequence length will change, and such a sequence will not appear in a uniform-length sequencing dataset. Noteworthy, noise2read can handle indel errors when insertion or deletions are represented by the symbol ‘N’. Construction of edit-erring-READS and error-free-READs as training data By defining a maximum frequency threshold τerr (τerr ≤ τ ), we considered two kinds of erroneous reads: genuine errors and ambiguous errors. Genuine errors are referred to those leaf nodes whose frequency τ ′ is less than or equal to τerr (τ ′ ≤ τerr ) and which have a neighbouring node with a higher frequency than τ . This set of erroneous reads is denoted as edit-erring-READS. Genuine errors can be directly rectified to their correct states. While we define two kinds of ambiguous errors: (a) those nodes (reads) r with a low-frequency τ ′ that are each connected to multiple (≥ 2) high-frequency nodes; (b) wrongly sequenced reads existing between a pair of similar high-frequency reads as the second kind of ambiguous error instances. In other words, in the constructed 1nt-edit-distance-based read graph, if there are edges between two similar high-frequency sequences, there may be sequencing errors between them. Moreover, amplicon sequencing utilises ultra-deep PCR amplifications for a specific gene target and supports hundreds to thousands of amplicons multiplexed sequencing in one assay to achieve high coverage, but ultra-deep PCR simultaneously amplifies PCR errors. To this end, we further construct a 1nt-edit-distance-based read graph for amplicon min sequencing data and consider those reads of frequencies less than τamp (e.g., 50) as potential amplicon errors mutated from its neighbouring reads of extremely max high-frequency larger than τamp (e.g., 1500). We consider isolated nodes of high frequencies bigger than τ as error-free reads. We take those isolated nodes of high frequencies in the 1nt- or 2nt-edit-distance graphs to build the training set error-free-READS. Auto Machine Learning (AutoML) prediction 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 Unlike the direct rectification of genuine errors into their original state, we model whether a high-frequency read contains true mutations or sequencing errors from its high- or low-frequency neighbours as a classification problem. We created an auto Machine Learning (AutoML) module for its end-to-end prediction. The flowchart illustrated in Fig. 9 outlines the steps involved in the AutoML module. April 5, 2024 568 24/37 607 608 609 610 611 Non-erring Samples Genuine errors Transformed Data 14 15 Predictions Best Model 8 5 Training Data 3 Objective Data 9 Fit Transform 6 16 12 13 10 Scaled Data 11 XGBoost Classifier Pruning Strategy 2 4 7 Sampling Strategy edit-erring& error-freeREADS 1 Ambiguous errors Feature Construction bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Optuna Fig 9. An overview workflow of the Auto Machine Learning (AutoML) module for end-to-end prediction. Formulation of the classification problem 612 We consider edit-erring-READS as positive training instances, while error-free-READS as negatives. For a low-frequency node with a degree greater than two, we calculate its probability of mutation from all its high-frequency neighbouring nodes and take the node with the highest probability as its correct sequence. For the second type of ambiguous error prediction, we integrate the predicted results of the first kind into the training data. In the current version, we only use the predicted ambiguous samples as negative samples for high-ambiguous error prediction to reduce training time and complexity. The mutation observed in high-frequency reads exhibits a bidirectional nature. Therefore, we only consider the prediction result with a higher probability when the bidirectional predictions match. In other words, if the absolute difference between the probabilities of the two-way predictions is less than a specific value, we discard the prediction; otherwise, we choose the prediction having a higher probability. Feature representation for the training and objective data 614 615 616 617 618 619 620 621 622 623 624 625 A short DNA or RNA sequence can be represented as r = b1 b2 ...bl , where bi ∈ {A, C, G, T, N } or bi ∈ {A, C, G, U, N }. Here, A, G, C, T and U represent the nitrogenous bases Adenine, Guanine, Cytosine, Thymine and Uracil, respectively. The letter N denotes an uncertain nucleotide, and l ∈ N represents the total number of bases in r. We extract features from r by considering its substrings of length k (where 1 ≤ k ≤ l), also known as k-mers. Each training instance consists of two reads in the edit-erring-READS or error-free-READS, examples of the training instances can be found in S1 Text. The features in reads are extracted using descriptors: (i) Fourier Transformation [48], (ii) Shannon’s and Tsallis’s entropy [48, 49], and (iii) Fickett’s score [50, 51]. Specifically, the features for a pair of reads with one or two base differences in a training instance may be identical. Therefore, features are only extracted from the absolute correct read (i.e., the first read in training instances) in order to avoid redundancy. These feature extraction methods are briefly depicted as follows: (i) Fourier Transformation. Sequences are converted into numeric representations by mapping each base to its atomic number (A:70, G: 78, C: 58, T or U: 66 and N: -1) [52]. Fourier Transformation converts the numerical sequence into a frequency domain representation using Discrete Fourier Transform (DFT). Here, Fast Fourier Transform April 5, 2024 613 25/37 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. (FFT) and a power spectrum analysis were conducted to extract features. This approach has been applied in several biological sequence analysis [53, 54], which can reveal hidden periodicity and repetitive elements in gene sequence. Mathematical exploration and examples of this approach can be found in [48]. (ii) Shannon’s and Tsallis’s entropy. We first transformed each read sequence into its frequency representation by selecting a k-mer length of k = 1, 2, 3 and then computed both the absolute and relative frequencies of all possible k-mers. We then separately applied Shannon’s and Tsallis’s entropy to each k. These approaches capture the degree of disorder and uncertainty present in the sequence. Mathematical definitions and examples of these entropy approaches can be seen in [48]. (iii) Fickett’s score involves the computation of four position-based values and four nucleotide-based content values from the sequence. The position indicator represents the degree of preference for each codon at one codon position over another, while the nucleotide-based content indicator measures the percentage of sequence per base. Then each indicator can be converted into a probability using the previous research output. The Fickett score is obtained by calculating the sum of probability multiplied by weight for each indicator. Detailed definitions and examples of this method refer to literature [51]. Additionally, we used the (iv) read counts and (v) characterised the error types and respective motifs as features. For instance, consider the two reads ACATG and ACGTG, the error is a substitution of C with G. Here, C-G is the error type, and CAT and CGT are the corresponding motifs. Similarly, for the two reads CGTG and ACGTG, the error is an insertion of A, the error type is represented as X-A, and the motifs are XA and AC. We define and normalise the feature vector V of error types or motifs as follows V = (v1 , v2 , ..., vi , ..., vn ) (7) counti + priori vi = total count Here, i represents an error type, where counti refers to the total number of occurrences of error type i and total count refers to the total number of all error types present in the data; priori is a small pre-defined value (e.g., 0.01) assigned to each item to avoid dividing by zero in cases where a certain error type or motif is not present in the data. Before training, each type of feature is standardised separately by removing the mean and scaling to the unit variance using the pre-processing method “StandardScaler” of Scikit-learn [55]. To address class imbalance issue in the training data, we used the Synthetic Minority Over-sampling Technique (SMOTE) [56] with sampling performed by Imbalanced-learn [57]. Model optimisation and prediction 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 The eXtreme Gradient Boosting (XGBoost) [12] is a well-established and efficient machine learning algorithm for classification. Optuna [58] is a framework that employs sampling and pruning heuristics to automatically discover optimal hyperparameter settings by conducting multiple trials. We chose XGBoost as our classifier and utilized Optuna to optimize the hyperparameters to achieve fast and accurate predictions. We have pre-set some parameters for the classifier, including the tree method, regularisation term, number of estimators, and learning rate. A logistic regression was used to produce the probability for binary classification, and we aimed to maximize the test accuracy as the objective for selecting the best model via multiple trials (e.g., 20). For each task, noise2read utilized AutoML to create a new Optuna study object for training and selecting the best prediction model. For example, we trained and selected the four best models for predicting 1nt- and 2nt-based ambiguous, 1nt-based high ambiguous, and amplicon errors. April 5, 2024 644 26/37 678 679 680 681 682 683 684 685 686 687 688 689 690 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Error correction for isolated nodes 691 After prediction, we restore all the edit-erring-READS to their normal state. We then adopted a third-party method Bcool [26] to deal with the errors contained in the isolated nodes, including many singletons, in the 1nt-edit-distance read graph. However, we keep only those corrected sequences by Bcool, which present in the original or in the first round of the corrected dataset without any genuine and ambiguous errors, to prevent generating non-existing new sequences. Evaluation criteria 693 694 695 696 697 698 Generation of the gold-standard wet-lab datasets with UMI-based ground truth We have developed a novel approach for generating ground truth datasets, motivated by Mitchel’s method presented in literature [30]. Our method involves first correcting the Unique Molecular Identifier (UMI) errors using noise2read before clustering sequences with identical UMI tags. We did not attempt to correct those UMI sequences (if exist) which contain 2 base errors or more. The reason is that a UMI sequence is short with an extremely low probability containing two or more errors. Our detailed correction step for UMI sequences is just the construction of a 1nt-edit-distance read graph from the UMI sequences, and then the identification of those leaf nodes from the graph whose frequency is less than or equal to the maximum frequency threshold for the error reads and which also have a neighboring node with a higher frequency. Such a strategy might under-correct the errors in UMIs but will also avoid the over-correction issue for the UMIs. Additionally, within each UMI group, we retain one high-frequency read accounting for more than half of the group, which will avoid the impact when two or more sequences are bound to the same UMI tag. Moreover, we observed that many raw reads contain a difference of more than three bases compared to their ground truth counterparts (see S16 Table) in [30]. For instance, in the dataset derived from SRR1543964, there are over 10,853 reads with a length of 238, each containing at least three base errors. Under the assumption in the introduction of this study, the likelihood of a read with a length of 238 having more than three base errors is relatively low, at only 0.00186, given a per-base error rate of 0.1%. It is impossible to construct a dataset of absolute ground truth from a real sequencing dataset, even with the help of UMI tagging. Using UMI to create a ground truth from real sequencing data is equivalent to using UMI for error correction. To our knowledge, no substantiated evidence supports the rationality of altering three or more bases within a sequence to establish a ground truth. Through our above theoretical and edit distance analysis, we chose to drop those reads in each UMI cluster that have >2 errors to construct ground truth datasets. While this approach is not an ideal method in generating ground truth datasets, it can accurately capture a substantial portion of sequences in their actual states and those sequencing errors. We calculated and summarized the total numbers and the proportions of the sampled reads in the construction of UMI-based ground truth datasets used in this study and those in the benchmark study (see S20 Table). noise2read obtained a similar percentage of sampled reads by using the strict sampling strategy (only choosing one high frequency read in each UMI cluster and dropping all those reads in each UMI group that have >2 errors) in comparison with Mitchell’s method. Generation of UMI-based simulated datasets from wet-lab datasets 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 We employed two different processes for generating UMI-based simulation datasets to evaluate the proposed method’s performance. The first process was designed to simulate April 5, 2024 692 27/37 737 738 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. single-end miRNA datasets similarly as worked in the literature [27]. Ideally, each unique read corresponds to a UMI. Therefore, we generate one unique UMI (numbers are used here instead of base sequences) for each unique sequence in the generated error-free read set. We then write these mimicked UMIs to each record in the edit-erring read set by mapping the sequence ID. Taking motivations from the previous simulation approach [27], we introduced an innovative method to generate UMI-based simulation datasets that can be applied to a broader range of NGS datasets, extending beyond just miRNA sequencing data. The new approach consists of four steps: (1) Correcting a real sequencing dataset using a simplified version of noise2read which excludes complex feature extraction and machine learning components, opting instead for basic feature of frequency levels. This simplified approach is intended to prevent any potential bias in favour of noise2read when evaluating it with these simulated datasets. (2) Reads with counts below a predetermined threshold (e.g., 5) are filtered out to eliminate noise. This process generates a subset denoted as S0. Subsequently, those reads whose sequence counts surpass a predefined threshold (e.g., 30) within S0 are extracted, constituting an error-prone subset named S1, while the remaining reads form an error-free subset referred to as S2. (3) In this stage, reads are randomly sampled from S1 at predetermined error rates per read. These error-prone reads encompass instances with either 1 or 2 base errors, without overlaps. Subsequently, 1 or 2 base errors are randomly induced within these error-prone reads, resulting in simulated raw datasets termed S3. These settings in steps (2) and (3) will further avoid any potential bias favouring noise2read that might inadvertently arise during the first step of the simulation. (4) Combine S2 and S3 to create simulated datasets with errors, combine S1 and S2 to create simulated datasets of ground truth, and then create mimic UMIs for the simulated datasets using the same methodology above. Metrics 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 To accurately evaluate the performance of error correction methods, we propose using confusion matrices at the read-level and base-level to measure changes in reads within the same UMI cluster rather than relying on the sequencing IDs generated by the instruments. The rationale is that for the constructed UMI-based datasets in this study, there is only one unique error-free sequence (of multiple occurrences) in each UMI cluster. Therefore, in a UMI cluster, we only need to compare the edit distance between every other unique read and this error-free read before and after error correction. Then, we are able to compute the confusion matrix using the relevant read count information before and after correction. More than half of the calculation time was saved this way. Otherwise, if we use the sequencing ID as the index, we must compare the edit distances twice for each group (same sequencing ID) of the raw, error-free, and corrected reads, even if they are the same. The absolute values of the true positives in each dataset are associated with the number of reads rather than the number of UMIs. The total number of positives of the actual condition equals the sum of the True positive and False negative. At the read level, a read is deemed erroneous if even a single base is incorrect. Conversely, a read is considered error-free only when all its bases are correct. TP defines the number of edit-erring reads perfectly modified after correction, and TN is the amounts of error-free reads without any changes after modification. While FP denotes the counts of error-free reads that are incorrectly adjusted by introducing new errors, FN represents the number of unchanged or wrongly fixed edit-erring reads. Similarly, at the base level, TP, TN, FP and FN concerned about the mistaken or accurate bases changing before and after correction. Additionally, we employ the edit distance changes instead of the multiple sequence alignment (MLA) strategies used in [30] among raw, authentic and modified reads to April 5, 2024 739 28/37 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. get the confusion conditions as the MLA is highly time-consuming. Another reason is that MLA has more alternative alignment results since it compares three reads. In contrast, the edit-distance-based strategy only compares the ground truth read to its raw or corrected one, respectively. When counting the FN on the base level, we measure the absolute edit distance difference with the accurate read before and after correction. Then, we derive the True Positive Ratio (TPR, a.k.a. recall or sensitivity), False Negative Ratio (FNR), True Negative Ratio (TNR), False Positive Ratio (FPR, a.k.a. fall-out), precision, gain and accuracy from the confusion matrix. TPR and FNR are the ratio of the number of edit-erring reads or bases correctly rectified and wrongly kept as negatives to the total number of actual edit-erring reads or bases, respectively. TNR and FPR are defined as the ratio of the number of error-free reads or bases correctly kept as negatives and wrongly rectified to the total number of actual error-free reads or bases, respectively. From the information theory perspective, TPR is the ratio of noise turning to signal; in contrast, FNR is the unconverted ratio of noise to signal. While FPR is the percentage of new noise introduced, TNR is the ratio of the original signal preserved. TP FN , FNR = TP + FN TP + FN (8) TN FP TNR = , FPR = FP + TN FP + TN An ideal performance should achieve high TPR and TNR while keeping FNR and FPR low. Therefore, we can construct a cross-coordinate system where derived scores from the confusion matrix are assigned to each of the four directions. The four index values of TPR in the upper axis, FPR in the lower axis, TNR in the right axis and FNR in the left axis form a rectangle. The larger the overlapping area between the rectangle and the upper right quadrant, the better the performance. Therefore, we define a quantitative metric of the overlapping Area Difference (AD) to assess the performance as follows, 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 TPR = AD = T P R ∗ T N R − T P R ∗ F N R − F N R ∗ F P R − F P R ∗ T N R TP , TP + FP P ositive Gain = 806 807 808 809 810 811 (9) Moreover, we also calculate the Precision, Positive Gain and Accuracy denoted as follows to evaluate the correction performance at read-level or base-level. Precision evaluates the ratio of precise modifications among all the completed corrections and all the errors, while Positive Gain indicates the positive effect among all the real errors. The accuracy is the proportion of accurate modifications, including true positives and negatives, to the total number of reads or bases concerned. P recision = 805 812 813 814 815 816 817 TP − FP TP + FN (10) TP + TN TP + TN + FP + FN Furthermore, based on the read-level definition for the dataset of known ground truth, we classify reads into two categories: edit-erring and error-free. Then we measure the purity of the dataset using entropy defined as Accuracy = E = −p ∗ log2 p − (1 − p) ∗ log2 (1 − p) 819 820 (11) where p is the probability of randomly selecting one error-prone or error-free read from all sequences. The lower the dataset entropy, the fewer edit-erring reads exist in the dataset. April 5, 2024 818 29/37 821 822 823 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Data availability 824 Generated datasets of UMI-based ground truth are available at D1-D8, their original sequencing data was obtained from SRR1543964-SRR1543971. Simulated ground truth datasets with mimic UMIs are available at D9-D13 with their original sequencing data downloaded from SRR12060401, SRR9077111 and SRR11092062. Simulated miRNA sequencing datasets with mimic UMIs are available at D14-D17. Processed SARS-Cov-2 data are available at D18-D19 with the corresponding source data available at SRR11092062. Monkeypox sequencing data D20-D21 were obtained from SRR22085311. Datasets of D22-D31 for the case study of isomiRs identification and SNPs profiling are available at ERR187525, ERR187844, ERR187542, ERR187668, ERR187759, ERR187669, ERR187622, ERR187665, ERR187892, ERR187500. Datasets for Adenine and cytosine base editor analysis are available at D32-D33 which are generated from Supplementary Table2 of the literature [44]. UMI-based ground-truth datasets (D34-D41 in this study) previously established in the literature [30] can be found at https://doi.org/10.6084/m9.figshare.11776413. S1 Table summarizes the information of datasets D1-D41 used in this study. Code availability 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 The algorithm, noise2read, developed in this study is packaged and released on the Python Package Index (PyPI) at https://pypi.org/project/noise2read/ and Bioconda at https://anaconda.org/bioconda/noise2read with source code publicly available at https://github.com/Jappy0/noise2read and documentation publicly available at https://noise2read.readthedocs.io/en/latest/. Supporting information 841 842 843 844 845 846 Supporting information, including Supplementary Figures 1-24 (presented in a single PDF), Supplementary Tables 1-20 (presented in a single PDF) and Supplementary Notes, are extended data supporting the results of this study. S1 Fig. The example of two UMI clusters shows the number of low-frequency sequences with the corresponding smallest edit distance compared to the high-frequency sequences in the data set SRR1543971. S2 Fig. The proportion of the UMI clusters with at least 60% of the erroneous reads caused by 1 or 2 base errors under the assumption that those low-frequency reads with an edit distance equal to or less than 4 may be erroneous reads caused by sequencing errors S3 Fig. The example of five UMI clusters shows low-frequency sequences with the corresponding smallest edit distance compared to the high-frequency sequences in the data set SRR1543971. S4-S10 Figs. Information gain visualisation for noise2read, Care, RACER, Bcool, Pollux, Lighter, Fiona and Coral on datasets D2-D10. April 5, 2024 825 30/37 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. S11 Fig. The comparison results regarding Positive Rate (TPR), True Negative Rate (TNR), False Positive Rate (FPR), False Negative Rate (FNR), Area Difference (AD) at base-level between noise2read with seven other computational methods on the UMI-based wet-lab datasets D1-D8. S12 Fig. The comparison results regarding Positive Rate (TPR), True Negative Rate (TNR), False Positive Rate (FPR), False Negative Rate (FNR), Area Difference (AD) at read-level between noise2read with seven other computational methods on the UMI-based wet-lab datasets D2-D8. S13-S17 Figs. Comparison of True Positive Rate (TPR), True Negative Rate (TNR), False Positive Rate (FPR), False Negative Rate (FNR), Area Difference (AD), Precision, Gain and Accuracy at the base- and read-level, purity Entropy (E) and Non-frequent reads Entropy at the read-level between noise2read and Care, RACER, Bcool, Pollux, Lighter, Fiona and Coral on simulated datasets D9-D13. S18-S21 Figs. Information gain visualisation for noise2read, Care, RACER, Bcool, Pollux, Lighter, Fiona and Coral on datasets D10-D13. S22-S24 Figs. Scatter graphs compare the numbers of ambiguous isomiRs, exclusive isomiRs and known SNPs from miRNA sequencing datasets D23-D31 after error correction of the reads, while the Kepler plots provide additional visualisations of the data distribution. S1 Table. The information of datasets D1-D33 used in this study. 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 S2-S9 Tables. The comparison results regarding TP, FP, FN, TN, Accuracy, Precision, Recall, Gain and Fall-out on the base- and read-level and the purity Entropy change (△E) and non-frequent reads’ information gain (△H) on the read-level between noise2read and other seven methods including Coral, RACER, Fiona, Lighter, Pollux, Bcool and Care on the UMI-based real sequencing datasets D1-D8. S10-S14 Tables. The comparison results regarding TP, FP, FN, TN, Accuracy, Precision, Recall, Gain and Fall-out on the base- and read-level and the purity Entropy change (△E) and non-frequent reads’ information gain (△H) on the read-level between noise2read and other seven methods including Coral, RACER, Fiona, Lighter, Pollux, Bcool and Care on the simulated datasets D9-D13. S15 Table. Evaluation comparison regarding TP, FP, FN, TN, Accuracy, Precision, Recall, Gain and Fall-out on the base- and read-level and the purity Entropy change (△E) and non-frequent reads’ information gain (△H) on the read-level between noise2read with and without enabling high ambiguous error prediction and miREC on miRNA simulation datasets D14-D17. S16 Table. The number of reads pairs regarding edit distance between raw and ground truth datasets generated from SRR1543964 to SRR1543971 in literature [30]. S17 Table. The comparative performance between noise2read and the methods of Bless, Coral, Lighter, Reckoner, Sga, BFC, Pollux, Fiona, RACER, and Care on the UMI-based benchmark data sets D34-D37 established in the literature [30]. April 5, 2024 862 31/37 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 bioRxiv preprint doi: https://doi.org/10.1101/2024.04.05.588226; this version posted April 9, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. S18 Table. The comparative performance between noise2read and the methods of Bless, Coral, Lighter, Reckoner, Sga, BFC, Pollux, Fiona, RACER, and Care on the UMI-based benchmark data sets D38-D41 established in the literature [30]. S19 Table. The runtime in different stages and computational resources provided during error correction by noise2read on the data sets of D1-D41. S20 Table. The total number of the sequences after sampling from the original data sets, as well as their percentage, when constructing UMI-based ground truth data sets in this study and the benchmark study, respectively. S1 Text. Supplementary Notes for software commands used and examples of the training and predicting instances. (PDF) Acknowledgments 903 904 905 906 907 908 909 910 911 912 We would like to thank the computational resources provided by the UTS (University of Technology Sydney) eResearch High-Performance Computer Facilities. 913 914 Author Contributions 915 Conceptualization: Jinyan Li, Pengyao Ping, Xuan Zhang. 916 Data Curation: 917 Pengyao Ping, Xuan Zhang, Xinhui Cai, Hui Peng. Formal Analysis: Pengyao Ping, Jinyan Li, Xuan Zhang, Tian Lan, Shuquan Su, Xinhui Cai, Hui Peng. 918 919 Funding Acquisition: Jinyan Li. 920 Investigation: 921 Pengyao Ping, Xuan Zhang. Methodology: Pengyao Ping, Jinyan Li, Xuan Zhang, Tian Lan, Shuquan Su. 922 Project Administration: Jinyan Li. 923 Resources: Pengyao Ping, Jinyan Li. 924 Software: Pengyao Ping. 925 Supervision: Jinyan Li, Wei Liu. 926 Validation: 927 Pengyao Ping. 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https://openalex.org/W2983506500	https://europepmc.org/articles/pmc6874468?pdf=render	English	null	Not afraid of low diastolic blood pressure anymore?	Aging	2,019	cc-by	1,666	Maciej Siński, Piotr Sobieraj, Jacek Lewandowski From a pathophysiologic perspective, a low DBP may adversely affect myocardial perfusion because, unlike in other vascular beds, blood flow in the epicardial coronary arteries is maintained during dias- tole [2]. It is therefore reasonable to postulate that the positive effect on cardiovascular risk of achieving a low SBP target may be blunted by the increased hazard associated with a low DBP. Such concept was proven right in various studies however analysis of large population-based trials of individuals with high cardio- vascular risk have been ambiguous. Epidemiologic studies have demonstrated that systolic blood pressure (SBP) is the most important factor influencing cardiovascular risk. The results of clinical studies including the Systolic Blood Pressure Intervention Trial (SPRINT) [1] preceded the changes in blood pressure goals proposed by hypertension societies. SPRINT showed that intensive lowering of the SBP to a goal of <120 mmHg could be beneficial and safe in contrast to the standard goal up to that point of <140 mmHg. In practice, it is unlikely that the SBP can be lowered without simultaneously reducing diastolic blood pressure (DBP). This may then limit how tightly the SBP can be controlled. For more than 30 years, investigators have argued as to whether lowering DBP increases the risk of myocardial infarction and coronary artery disease in patients with hypertension. From a pathophysiologic perspective, a low DBP may adversely affect myocardial perfusion because, unlike in other vascular beds, blood flow in the epicardial coronary arteries is maintained during dias- tole [2]. It is therefore reasonable to postulate that the positive effect on cardiovascular risk of achieving a low SBP target may be blunted by the increased hazard associated with a low DBP. Such concept was proven right in various studies however analysis of large population-based trials of individuals with high cardio- vascular risk have been ambiguous. Ongoing analysis of SPRINT findings has indicated that the increased cardiovascular risk in for participants with a low DBP may be explained by factors other than simply the DBP, such as higher age and higher pre- valence of cardiovascular or chronic kidney disease. This suggests that a low DBP is not an independent risk factor for cardiovascular events [3, 4]. This also seems to be the case in other studies of subjects with high cardiovascular risk [5]. Maciej Siński, Piotr Sobieraj, Jacek Lewandowski groups. Only 28.2% of the patients in SPRINT were ≥75 years, and the mean age of participants in ONTARGET and TRANSCEND with a low DBP (<70 mmHg) did not exceed 70 years [6]. Elderly patients often have a high SBP but a low DBP. It is possible that cardiovascular risk in older subjects could thus be more impacted by a low DBP than in younger individuals. However, when stroke incidence in SPRINT was taken into account, an outcome measure which is highly age dependent, low DBP was not independently associated with a higher incidence of stroke [8]. We performed an additional analysis of the SPRINT study population focused on 1079 subjects >80 years old (mean age 83.4 ± 3 years). The SPRINT data were obtained from the National Heart, Lung and Blood Institute (NHLBI) Biologic Specimen and Data Repository Information Coordinating Center, but our findings are our own and do not necessarily reflect the opinions or views of the SPRINT Research Group or the NHLBI. The population we analyzed was at high risk of a clinical composite endpoint of myocardial infarction, acute coronary syndrome without infarction, stroke, acute decompen- sated heart failure, or cardiovascular death, of which there were 103 events (9.5%) during the trial. The risk of the composite endpoint was increased with a DBP <64 mmHg (Figure 1). However, analysis using a Cox proportional hazard risk model revealed that, after adjustment for age, sex, history of cardiovascular disease, SBP, current smoking status, and body mass index, a low DBP was not independently associated with an increased risk for the endpoint (Figure 1). Epidemiologic studies have demonstrated that systolic blood pressure (SBP) is the most important factor influencing cardiovascular risk. The results of clinical studies including the Systolic Blood Pressure Intervention Trial (SPRINT) [1] preceded the changes in blood pressure goals proposed by hypertension societies. SPRINT showed that intensive lowering of the SBP to a goal of <120 mmHg could be beneficial and safe in contrast to the standard goal up to that point of <140 mmHg. In practice, it is unlikely that the SBP can be lowered without simultaneously reducing diastolic blood pressure (DBP). This may then limit how tightly the SBP can be controlled. For more than 30 years, investigators have argued as to whether lowering DBP increases the risk of myocardial infarction and coronary artery disease in patients with hypertension. Editorial Editorial AGING 2019, Vol. 11, No. 21 AGING 2019, Vol. 11, No. 21 AGING 2019, Vol. 11, No. 21 www.aging-us.com Not afraid of low diastolic blood pressure anymore? Not afraid of low diastolic blood pressure anymore? Maciej Siński, Piotr Sobieraj, Jacek Lewandowski In contrast to this view, how- ever, analysis of the ONTARGET and TRANSCEND findings indicate that the benefit of intensive lowering of SBP may be limited because of concomitant DBP reduction [6]. Similar conclusions have been drawn by other investigators analyzing the SPRINT results showing that a DBP <55 mmHg recorded at a single visit was responsible for attenuation of the benefits of SBP reduction in both of the trial’s treatment arms [7]. Thus, the discussion regarding the influence of lowering the DBP continues. It is tempting to answer the question posed in the title positively. We have convincing evidence that a low DBP should not stand in the way of intensive blood pressure reduction. On the other hand, as clinicians, we are aware that some of our older patients faint while on hypertensive treatment, so we cannot stop asking question, “Aren’t we going too low?” Thus, there is a need for more studies specifically among elderly patients focusing on the effect of a low DBP. In a superb review concerning the J curve, Messerli and Panjrath present a figure showing that subjects with coronary artery disease who have undergone re- vascularization tolerate a lower DBP better than those who have not been revascularized [2]. Keeping that in mind, we should still consider low DBP as a marker of increased risk and focus on risk reduction. The results being highlighted here do not apply to all age AGING 9229 www.aging-us.com gure 1. Left panel - multivariable Cox proportional hazard risk model evaluating association of on-treatment DBP with composite nical endpoint. Right panel - hazard ratio for composite clinical endpoint according to achieved on-treatment DBP in SPRINT articipants over 80 years old. DBP, diastolic blood pressure; SBP, systolic blood pressure; CI, confidence interval; composite clinical ndpoint: myocardial infarction, acute coronary syndrome without infarction, stroke, acute decompensated heart failure, or cardiovascular eath. DBP was included in the model at discrete variable thresholds of 60 mmHg and 70 mmHg and was not an independent risk factor. Figure 1. Left panel - multivariable Cox proportional hazard risk model evaluating association of on-treatment DBP with composite clinical endpoint. Right panel - hazard ratio for composite clinical endpoint according to achieved on-treatment DBP in SPRINT participants over 80 years old. Maciej Siński: Department of Internal Medicine, Hypertension and Vascular Diseases, Medical University of Warsaw, Warsaw 02-097, Poland 1. Sprint Research Group et al. N Engl J Med. 2015; 373:2103–16. https://doi.org/10.1056/NEJMoa1511939 PMID:26551272 Maciej Siński, Piotr Sobieraj, Jacek Lewandowski DBP, diastolic blood pressure; SBP, systolic blood pressure; CI, confidence interval; composite clinical endpoint: myocardial infarction, acute coronary syndrome without infarction, stroke, acute decompensated heart failure, or cardiovascular death. DBP was included in the model at discrete variable thresholds of 60 mmHg and 70 mmHg and was not an independent risk factor. Figure 1. Left panel - multivariable Cox proportional hazard risk model evaluating association of on-treatment DBP with composite clinical endpoint. Right panel - hazard ratio for composite clinical endpoint according to achieved on-treatment DBP in SPRINT participants over 80 years old. DBP, diastolic blood pressure; SBP, systolic blood pressure; CI, confidence interval; composite clinical endpoint: myocardial infarction, acute coronary syndrome without infarction, stroke, acute decompensated heart failure, or cardiovascular death. DBP was included in the model at discrete variable thresholds of 60 mmHg and 70 mmHg and was not an independent risk factor. Figure 1. Left panel - multivariable Cox proportional hazard risk model evaluating association of on-treatment DBP with composite clinical endpoint. Right panel - hazard ratio for composite clinical endpoint according to achieved on-treatment DBP in SPRINT participants over 80 years old. DBP, diastolic blood pressure; SBP, systolic blood pressure; CI, confidence interval; composite clinical endpoint: myocardial infarction, acute coronary syndrome without infarction, stroke, acute decompensated heart failure, or cardiovascular death. DBP was included in the model at discrete variable thresholds of 60 mmHg and 70 mmHg and was not an independent risk factor. 1. Sprint Research Group et al. N Engl J Med. 2015; 373:2103–16. https://doi.org/10.1056/NEJMoa1511939 PMID:26551272 Correspondence: Maciej Siński Email: msinski@wum.edu.pl Keywords: low diastolic blood pressure, cardiovascular risk, SPRINT trial Copyright: Siński 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 2. Messerli FH, Panjrath GS. J Am Coll Cardiol. 2009; 54:1827–34. https://doi.org/10.1016/j.jacc.2009.05.073 PMID:19892233 2. Messerli FH, Panjrath GS. J Am Coll Cardiol. 2009; 54:1827–34. https://doi.org/10.1016/j.jacc.2009.05.073 PMID:19892233 2. Messerli FH, Panjrath GS. J Am Coll Cardiol. 2009; 54:1827–34. https://doi.org/10.1016/j.jacc.2009.05.073 PMID:19892233 3. Sobieraj P, et al. Eur Heart J. 2019; 40:2094–95. https://doi.org/10.1093/eurheartj/ehz225 PMID:31006014 4. Sobieraj P, et al. Sci Rep. 2019; 9:13070. https://doi.org/10.1038/s41598-019-49557-4 PMID:31506550 Received: October 9, 2019 Published: November 7, 2019 5. Kjeldsen SE, et al. Blood Press. 2016; 25:83–92. https://doi.org/10.3109/08037051.2015.1106750 PMID:26511535 6. Böhm M, et al. Eur Heart J. 2018; 39:3105–14. https://doi.org/10.1093/eurheartj/ehy287 PMID:29873709 7. Lee TC, et al. Am J Med. 2018; 131:1228–1233.e1. https://doi.org/10.1016/j.amjmed.2018.05.022 PMID:29906425 8. Sobieraj P, et al. J Am Heart Assoc. 2019; 8:e010811. https://doi.org/10.1161/JAHA.118.010811 PMID:30744452 AGING 9230 www.aging-us.com
https://openalex.org/W2560507862	https://link.springer.com/content/pdf/10.1007/s11427-016-0351-y.pdf	English	null	Regional immunity in tissue homeostasis and diseases	Science China. Life Sciences/Science China. Life sciences	2,016	cc-by	4,605	Zhigang Tian1, Xuetao Cao2, Yongyan Chen1 & Qunyan Lyu3 Zhigang Tian1, Xuetao Cao2*, Yongyan Chen1 & Qunyan Lyu3 1Institute of Immunology and The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science & Technology of China, Hefei 230027, China; 2National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai 200433, China; 3Department of Health Sciences, National Natural Science Foundation of China, Beijing 100085, China Received November 17, 2016; accepted November 22, 2016; published online December 3, 2016 Citation: Tian, Z., Chen, X., Chen, Y., and Lyu, Q. (2016). Regional immunity in tissue homeostasis and diseases. Sci China Life Sci 59, 1205–1209. doi: 10.1007/s11427-016-0351-y The immune system functions in the organ/tissue of the body. The immune cell differentiation and function are influenced by the organ/tissue microenvironments in which they reside, and the interaction of immune cells with the organ/tissue microenvironments may affect and even determine the outcome of the immune responses (Hu and Pasare, 2013; Zajac and Harrington, 2014). It has been increasingly appreciated that the immunologic characterization from professional immune organs including thymus, bone mar- row, spleen, and lymph node, is greatly distinguished from that in mucosal immune organs including skin, intestine, lung, pancreas, uterus and kidney, and other organs such as liver, bone, brain, maternal-fetal interface, and so on. Each organ/tissue is characterized with its own anatomy and microenvironment, in which immune cell populations and subsets may have unique functional molecules, forming “unique regional immune features”. The regional immune responses play an important role in tissue homeostasis and are extensively involved in the pathogenesis of the major diseases related to each tissue/organ. novel tissue-resident immune cell subsets, organ-special- ized immune recognition, organ-specific factors instructing regional immune responses in liver, lung, intestine, bone, brain, uterus, adipose, kidney, tumor, etc. For example, specialized tissue-resident macrophages, including Kupffer cells in the liver, alveolar macrophages in the lung, microglia in the brain, osteoclasts in the bone, and histiocytes in the intestinal connective tissue, exhibit distinct functions due to their resident tissue (Murray and Wynn, 2011). Specialized tissue-resident regulatory T cells in the visceral adipose tis- sue (VAT Tregs), muscle Tregs, bone Tregs and skin memory Tregs, have also been reported with their unique functions in local tissues (Zhou et al., 2015). Innate immune cells continuously receive signals from the resident environment, such as commensal microbiota signals in mucosal organs, which will influence the outcome of immune responses. SCIENCE CHINA Life Sciences SCIENCE CHINA Life Sciences Special Topic: Organ/Tissue-specific immunity • EDITORIAL • Special Topic: Organ/Tissue-specific immunity December 2016 Vol.59 No.12:1205–1209 doi: 10.1007/s11427-016-0351-y • EDITORIAL • Corresponding author (email: tzg@ustc.edu.cn) *Corresponding author (email: caoxt@immunol.org) The Author(s) 2016. This article is published with open access at link.springer.com Zhigang Tian1, Xuetao Cao2*, Yongyan Chen1 & Qunyan Lyu3 Human hepatic CD56bright NK cells express trNK cell markers such as CD69 and CXCR6, and exhibit unique transcriptiona profiles compared to cNK cells (Hudspeth et al., 2016). Similar to murine liver trNK cells, a proportion of human hepatic CD56bright NK cells with the ex- pression of CD49a are T-bet+Eomes−(Marquardt et al., 2015). Additionally, in this issue Han et al. identified a new subset of inflammation-induced CD69+ Kupffer cells during Liste- ria monocytogenes infection (Zhang et al., 2016). Hepatic CD69+ Kupffer cells expressed higher levels of CD11b, but produced lower levels of F4/80, which could not be depleted by clodronate liposome administration. Hepatic CD69+ Kupffer cells suppressed Ag-nonspecific and OVA-specific CD4+ T cell proliferation through membrane-bound TGF-β1 (mTGF-β1). CD69+ Kupffer cells may contribute to pro- tect host from pathological injure by preventing immune over-activation. and Tian summarized the phenotypic and functional features of liver trNK cells (Peng and Tian, 2016). In mice, liver NK cells can be divided into two distinct subsets: CD49a−DX5+ cNK and CD49a+DX5−trNK cells which reside in the liver sinusoid blood and confer memory-like responses in contact hypersensitivity (CHS) models (Peng et al., 2013). Liver trNK cells and cNK cells require different transcription factors for their development, thus representing sepa- rate developmental lineages (Constantinides et al., 2014; Daussy et al., 2014; Klose et al., 2014; Mackay et al., 2016). They also overviewed recent advances in human liver trNK cells and discussed the striking shared hallmarks of trNK cells in different tissues. Human hepatic CD56bright NK cells express trNK cell markers such as CD69 and CXCR6, and exhibit unique transcriptiona profiles compared to cNK cells (Hudspeth et al., 2016). Similar to murine liver trNK cells, a proportion of human hepatic CD56bright NK cells with the ex- pression of CD49a are T-bet+Eomes−(Marquardt et al., 2015). Additionally, in this issue Han et al. identified a new subset of inflammation-induced CD69+ Kupffer cells during Liste- ria monocytogenes infection (Zhang et al., 2016). Hepatic CD69+ Kupffer cells expressed higher levels of CD11b, but produced lower levels of F4/80, which could not be depleted by clodronate liposome administration. Hepatic CD69+ Kupffer cells suppressed Ag-nonspecific and OVA-specific CD4+ T cell proliferation through membrane-bound TGF-β1 (mTGF-β1). CD69+ Kupffer cells may contribute to pro- tect host from pathological injure by preventing immune over-activation. Zhigang Tian1, Xuetao Cao2*, Yongyan Chen1 & Qunyan Lyu3 Osteoimmunology was firstly introduced in 2000, in- dicating the bone and immune system have an intense interaction and should be considered as a functional unit (Arron and Choi, 2000). Osteoblasts (OBs) contribute to hematopoietic progenitor growth and HSC pool maintenance through Notch signal and osteopontin (OPN) production (Calvi et al., 2003; Nilsson et al., 2005). The development and response of immune cells associate to bone remodeling; on the other hand, activated immune cells are involved in bone metabolism regulation, and mediate osteoporosis and bone erosion under pathological conditions such as rheuma- toid arthritis (RA), spondyloarthropathy (SPA), osteoporosis (OP), and periodontal disease (PD). In this issue, Zhao et al. presented current understanding of osteoimmunology and emphasized on the RANK/RANKL/OPG signaling in bone remodeling and immune responses (Zhao et al., 2016). Recent research progress in the interactions between T cells, B cells, DCs and bone cells, in the role of several kinds of cytokines including M-CSF, TNF-α, IL-1, IFN-γ, IL-4, IL-10, IL-6, TGF-β, OPN and complement system in bone homeostasis were also summarized in this review, particu- larly their pathogenesis in major diseases. During human pregnancy, an orchestrated evolutionary ma- ternal adaption toward tolerance of the semiallogeneic fetus is required. In the decidua, remodeling of the immune system involves NK cells, macrophages, T cells and DCs to alter the local microenvironment (Fu et al., 2014; Lima et al., 2014). NK cells as the largest population of immune cells during the first trimester in human decidua, accounting for 50%–70% of the total number of immune cells after 9–12 weeks of pregnancy, showed a close reaction to invasive fetal tro- phoblasts and stromal cells (Colucci and Kieckbusch, 2015; Tao et al., 2015). Evidence shows decidual NK cells act as sentinel cells to control local inflammation and tolerance, and mediate the regulation of fetal growth. In this issue, Fu et al. summarized the concepts and rationale for the remodeled immune system during pregnancy, and the formation and education of decidual NK cells (Fu and Wei, 2016). Most decidual NK cells are CD56brightCD16−NK cells, which express CXCR4, and can be selectively recruited into the decidua by interaction with chemokine CXCL12 secreted by DSCs and EVTs (Hanna et al., 2003; Wu et al., 2005). The central nervous system (CNS) as an immune privi- leged organ, owns its local tissue barrier and immunosup- pressive microenvironment. Zhigang Tian1, Xuetao Cao2*, Yongyan Chen1 & Qunyan Lyu3 In this issue, Zhang et al. reviewed the innate recognition of microbial-derived signals in immunity and inflammation (Zhang and Liang, 2016), which will help us understand the local immune responses. Liver is an unique organ with predominant innate immu- nity, enriched with Kupffer cells, NK cells, NKT cells and γδ T cells (Gao et al., 2008). Current studies have revealed that NK cells are not a homogeneous population, but instead consist of distinct subsets including conventional NK (cNK) cells and tissue-resident NK (trNK) cells. In this issue, Peng With the research progressed, a more clear description of the regional immune features has been presented, such as life.scichina.com link.springer.com The Author(s) 2016. This article is published with open access at link.springer.com Tian, Z., et al. Sci China Life Sci December (2016) Vol. 59 No. 12 1206 B cells have all been found in adipose tissue and were involved in obesity-related metabolic dysfunc- tions (Cipolletta et al., 2012; Feuerer et al., 2009; Liu et al., 2009; Lynch et al., 2012; Molofsky et al., 2013; Talukdar et al., 2012; Winer et al., 2009; Winer et al., 2011; Wu et al., 2011). Particularly, macrophages as the critical effector cells in orchestrating inflammation have been most extensively investigated to explore their roles in obesity-as- sociated metabolic inflammation and insulin resistance (McNelis and Olefsky, 2014). In this issue, Qiu et al. reviewed the metabolic activation and functional properties of adipose tissue macrophage (ATM) including M1 and M2 macrophages (Qiu et al., 2016), which would help us better understand the pathogenic and/or protective roles of ATM and the translational opportunities for developing novel therapeutics against obesity, type 2 diabetes and other related metabolic diseases. and Tian summarized the phenotypic and functional features of liver trNK cells (Peng and Tian, 2016). In mice, liver NK cells can be divided into two distinct subsets: CD49a−DX5+ cNK and CD49a+DX5−trNK cells which reside in the liver sinusoid blood and confer memory-like responses in contact hypersensitivity (CHS) models (Peng et al., 2013). Liver trNK cells and cNK cells require different transcription factors for their development, thus representing sepa- rate developmental lineages (Constantinides et al., 2014; Daussy et al., 2014; Klose et al., 2014; Mackay et al., 2016). They also overviewed recent advances in human liver trNK cells and discussed the striking shared hallmarks of trNK cells in different tissues. Zhigang Tian1, Xuetao Cao2*, Yongyan Chen1 & Qunyan Lyu3 Interestingly, Glial cells includ- ing activated microglia and astrocytes, participate in local immune regulation within the CNS. Further, a specialized CNS-cytokine network was constituted and regulated the de- velopment and recovery from autoimmune diseases within the CNS (Xiao and Link, 1998). Additionally, numerous myeloid cell subsets were found in CNS-adjoining tissues, namely the meninges, the perivascular space, and the choroid plexus (Brendecke and Prinz, 2015). These immune cells have been demonstrated to be involved in the initiation, pro- gression and resolution of multiple sclerosis (MS). In this is- sue, Yan et al. reported a unique group of patients with neu- Adipose tissues, including white, brown and beige fat, play pivotal roles in metabolic homeostasis (Rosen and Spiegelman, 2014). Macrophages, mast cells, neutrophils, eosinophils, ILC2, T cells and Tian, Z., et al. Sci China Life Sci December (2016) Vol. 59 No. 12 1207 romyelitis optica spectrum disorder (NMOSD) who carried autoantibodies of aquaporin-4 (AQP4) and myelin-oligoden- drocyte glycoprotein (MOG) (Yan et al., 2016). They found NMOSD patients carrying both anti-AQP4 and anti-MOG antibodies exhibited combined features of prototypic NMO and relapsing-remitting form of MS, whereas NMOSD with only anti- MOG antibody exhibited an “intermediate” phe- notype between NMOSD and MS, indicating that anti-MOG antibody might be pathogenic in NMOSD patients and that determination of anti-MOG antibody may be instructive for management of NMOSD patients. the tissue-resident immune cell subsets and their interac- tions with tissue/organ microenvironments, clarifying their immunological mechanisms in organ-related major diseases, explaining the local environmental factors involved in the tissue residency, which will provide a theoretical basis for the prevention and control of these major diseases and reveal the possible immune intervention targets and immune treatment. Compliance and ethics The author(s) declare that they have no conflict of interest. Arron, J.R., and Choi, Y. (2000). Bone versus immune system. 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Zhigang Tian1, Xuetao Cao2*, Yongyan Chen1 & Qunyan Lyu3 reported lactic acid in tumor microen- vironments inhibited the production of IFN-γ and IL-4 in NKT cells, and these dysfunctions of NKT cells could sim- ply be induced by low extracellular pH through inhibiting mTOR signaling (Xie et al., 2016). It is interesting that tu- mor acidic microenvironments could regulate immune cell re- sponse through modulating cellular metabolisms. Addition- ally, in the tumor microenvironment a variety of immune fac- tors will alter and aggravate the disease process. Also, Lu et al. reported IL-17 could inhibit the formation of malig- nant pleural effusion (MPE) via promoting the differentia- tion of Th9 and Th2 cells in the tumor microenvironments (Lu et al., 2016). Fu, B., and Wei, H. (2016). Decidual natural killer cells and the immune microenvironment at the maternal-fetal interface. Sci China Life Sci 59, 1224–1231. Fu, B., Tian, Z., and Wei, H. (2014). TH17 cells in human recurrent preg- nancy loss and pre-eclampsia. Cell Mol Immunol 11, 564–570. Gao, B., Jeong, W.I., and Tian, Z. (2008). Liver: an organ with predominant innate immunity. Hepatology 47, 729–736. Hanna, J., Wald, O., Goldman-Wohl, D., Prus, D., Markel, G., Gazit, R., Katz, G., Haimov-Kochman, R., Fujii, N., Yagel, S., Peled, A., and Mandelboim, O. (2003). CXCL12 expression by invasive trophoblasts induces the specific migration of CD16−human natural killer cells. Blood 102, 1569–1577. Hu, W., and Pasare, C. (2013). Location, location, location: tissue-specific regulation of immune responses. J Leukocyte Biol 94, 409–421. Hudspeth, K., Donadon, M., Cimino, M., Pontarini, E., Tentorio, P., Preti, M., Hong, M., Bertoletti, A., Bicciato, S., Invernizzi, P., Lugli, E., Tor- zilli, G., Gershwin, M.E., and Mavilio, D. (2016). Human liver-resident CD56bright/CD16neg NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways. J Autoimmun 66, 40–50. Hu, S., Jia, X., Li, J., Zheng, X., Ao, J., Liu, G., Cui, Z., and Zhao, M. (2016). T cell infiltration is associated with kidney injury in patients with anti-glomerular basement membrane disease. Sci China Life Sci 59, 1282–1289. Biographical Sketch Zhigang Tian is the current President of Chinese Society of Immunology (CSI) and Council member of International Union of Immunological Societies (IUIS) and Federation of Immunological Societies of Asia-Oceania (FIMSA), a professor at University of Science and Technology of China (USTC) in Hefei, China, where also works as a Director of Institute of Immunology, Director of The Key Lab of Innate Immunity and Chronic Diseases of Chinese Academy of Science, and President of Medical Center, and the former dean of School of Life Sciences of USTC. Zhigang used to work as a visiting scientist in NCI/NIH, USA during 1994–1996 and in Kanazawa University, Japan during 2002–2003. Currently, Tian’s laboratory is credited with seminal discoveries regarding basic knowledge and clinical study of NK cells, particularly liver-resident NK cells, regulatory NK cell subsets, and NK cell-based immunotherapy. Zhigang is a co-Editor-in-Chief of Cellular and Molecular Immunology, and editorial board members of up to ten journals including Hepatology, Journal of Autoimmunity, European Journal of Immunology and The Journal of Biological Chemistry, and has published more than 250 papers in peer-reviewed journals including Cell, Nature Immunology, Immunity, The Journal of Experimental Medicine, The Journal of Clinical Investigation, Nature Communications, Proceedings of the National Academy of Sciences of the United States of America, etc. Xuetao Cao is professor and President of Chinese Academy of Medical Sciences & Peking Union Medical College. He received Ph.D. from Second Military Medical University (Shanghai, China) in 1990, became Professor in Immunology in 1992 and Director of the Institute of Immunology in 2001 at the same University. He is Professor and Director of National Key Laboratory of Medical Immunology (2006.5–). He was elected as member of Chinese Academy of Engineering in 2005, Foreign Associate of German Academy of Sciences in 2013, and Foreign Member of EMBO in 2015. His major interests are innate response, inflammation and tumor immunotherapy. As corresponding author, he publishedmore than 230 original papers in peer-reviewed journals including Cell, Nature, Science. He is Co-Editor-in-Chief of Cellular and Molecular Immunology, editorial board member of Cell, Annual Reviews of Immunology, Science Translational Medicine, eLife, Cell Research, etc. Yongyan Chen is an associate Professor of University of Science & Technology of China (USTC). Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Zhigang Tian1, Xuetao Cao2**, Yongyan Chen1 & Qunyan Lyu3 Cell Mol Immunol 12, 543–552. Qiu, Y., Shan, B., Yang, L., and Liu, Y. (2016). Adipose tissue macrophage Tian, Z., et al. Sci China Life Sci December (2016) Vol. 59 No. 12 1209 Biographical Sketch Biographical Sketch As a member of Zhigang Tian’s lab in USTC, she has been attempting to study the innate immunity of liver, including the biology of the overwhelming innate immune cells NK cells and NKT cells in the liver, and their roles during HBV infection and HBV-associated liver diseases; and the mechanisms of HBV-induced liver immunotolerance. Qunyan Lyu, professor and Division Director, Division IV of Department of Health Sciences, National Natural Science Foundation of China. She received her Ph.D. in Mircobiology in 1993, and has been responsible for the management of natural science fund in the field of immunology or medical immunolgy for about 20 years. She has engaged herself in impletmenting the funding plans to support basic research, identifying and forstering scientifc talents, accepting project applications, organizing peer review process, administrating funded projects etc, in the field of immunology or medical immunolgy. As a professional scientific manager, she is also playing active roles in formulation of the priority funding areas, funding schemes, major research plans, annual funding arrangements as well as other disciplinary development strategies for the field of immunology or medical immunology.
https://openalex.org/W4361264859	https://figshare.com/articles/journal_contribution/Supplementary_Figure_1_Legend_from_Pediatric_i_KIT_i_Wild-Type_and_Platelet-Derived_Growth_Factor_Receptor_Wild-Type_Gastrointestinal_Stromal_Tumors_Share_KIT_Activation_but_not_Mechanisms_of_Genetic_Progression_with_Adult_Gastrointestinal_/22367649/1/files/39812718.pdf	English	null	Supplementary Figure 1 Legend from Pediatric <i>KIT</i>–Wild-Type and Platelet-Derived Growth Factor Receptor α–Wild-Type Gastrointestinal Stromal Tumors Share KIT Activation but not Mechanisms of Genetic Progression with Adult Gastrointestinal Stromal Tumors	null	2,023	cc-by	188	Supplemental Figure 1. To determine the amount of viable tumor tissue and morphologic appearance, frozen sections prepared from three cryopreserved samples with sufficient material for analysis. Cryosections of the same tumor specimen used in protein and SNP analyses were evaluated (LEICA 1800 CRYOCUT CRYOSTAT, Wetzlar, Germany). Supplemental Figure 1. To determine the amount of viable tumor tissue and morphologic appearance, frozen sections prepared from three cryopreserved samples with sufficient material for analysis. Cryosections of the same tumor specimen used in protein and SNP analyses were evaluated (LEICA 1800 CRYOCUT CRYOSTAT, Wetzlar, Germany). Supplemental Figure 1. To determine the amount of viable tumor tissue and morphologic appearance, frozen sections prepared from three cryopreserved samples with sufficient material for analysis. Cryosections of the same tumor specimen used in protein and SNP analyses were evaluated (LEICA 1800 CRYOCUT CRYOSTAT, Wetzlar, Germany). Five μm thick sections were mounted on precoated glass slides (Plus cold, Erie Scientific Company, Portsmouth, NH) and stained with H&E frozen section kit (Newcomer Supply, Middleton, WI, Cat. number 9128A). These validation studies showed that each specimen had epitheliod cell morphology, and contained 10% or less nonneoplastic stromal elements.
https://openalex.org/W2010075654	https://europepmc.org/articles/pmc2808239?pdf=render	English	null	Multiorgan Detection and Characterization of Protease-Resistant Prion Protein in a Case of Variant CJD Examined in the United States	PloS one	2,010	cc-by	8,710	Abstract Britton Fund to PG; grant NIH R01 NS062787 and the Creutzfeldt-Jakob Disease Foundation to WQZ, as for cost of supplies and salaries. 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: pxg13@case.edu * E-mail: pxg13@case.edu . These authors contributed equally to this work. . These authors contributed equally to this work. ‘‘prion’’. However, occasionally not direct correlation has been reported between PrPres and infectivity [6]. Multiorgan Detection and Characterization of Protease- Resistant Prion Protein in a Case of Variant CJD Examined in the United States Silvio Notari1., Francisco J. Moleres1., Stephen B. Hunter2, Ermias D. Belay3, Lawrence B. Schonberger3, Ignazio Cali1, Piero Parchi4, Wun-Ju Shieh3, Paul Brown5, Sherif Zaki3, Wen-Quan Zou1, Pierluigi Gambetti1* Silvio Notari1., Francisco J. Moleres1., Stephen B. Hunter2, Ermias D. Belay3, Lawrence B. Schonberger3, Ignazio Cali1, Piero Parchi4, Wun-Ju Shieh3, Paul Brown5, Sherif Zaki3, Wen-Quan Zou1, Pierluigi Gambetti1* Silvio Notari1., Francisco J. Moleres1., Stephen B. Hunter2, Ermias D. Belay3, Lawrence B. Schonberger3, Ignazio Cali1, Piero Parchi4, Wun-Ju Shieh3, Paul Brown5, Sherif Zaki3, Wen-Quan Zou1, Pierluigi 1 1 Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, United States of America, 2 Department of Pathology and Laboratory Medicine, Emory University Hospital, Atlanta, Georgia, United States of America, 3 National Center for Zoonotic, Vector-Borne, and Enteric Diseases, Center for Disease Control and Prevention, Atlanta, Georgia, United States of America, 4 Dipartimento di Scienze Neurologiche, Universita’ di Bologna, Italy, 5 CEA/DSV/iMETI/SEPIA, France Abstract Background: Variant Creutzfeldt–Jakob disease (vCJD) is a prion disease thought to be acquired by the consumption of prion-contaminated beef products. To date, over 200 cases have been identified around the world, but mainly in the United Kingdom. Three cases have been identified in the United States; however, these subjects were likely exposed to prion infection elsewhere. Here we report on the first of these subjects. Methodology/Principal Findings: Neuropathological and genetic examinations were carried out using standard procedures. We assessed the presence and characteristics of protease-resistant prion protein (PrPres) in brain and 23 other organs and tissues using immunoblots performed directly on total homogenate or following sodium phosphotungstate precipitation to increase PrPres detectability. The brain showed a lack of typical spongiform degeneration and had large plaques, likely stemming from the extensive neuronal loss caused by the long duration (32 months) of the disease. The PrPres found in the brain had the typical characteristics of the PrPres present in vCJD. In addition to the brain and other organs known to be prion positive in vCJD, such as the lymphoreticular system, pituitary and adrenal glands, and gastrointestinal tract, PrPres was also detected for the first time in the dura mater, liver, pancreas, kidney, ovary, uterus, and skin. Conclusions/Significance: Our results indicate that the number of organs affected in vCJD is greater than previously realized and further underscore the risk of iatrogenic transmission in vCJD. Citation: Notari S, Moleres FJ, Hunter SB, Belay ED, Schonberger LB, et al. (2010) Multiorgan Detection and Characterization of Protease-Resistant Prion Protein in a Case of Variant CJD Examined in the United States. PLoS ONE 5(1): e8765. doi:10.1371/journal.pone.0008765 ation: Notari S, Moleres FJ, Hunter SB, Belay ED, Schonberger LB, et al. (2010) Multiorgan Detection and Characterization of Protease-Re Case of Variant CJD Examined in the United States. PLoS ONE 5(1): e8765. doi:10.1371/journal.pone.0008765 Editor: Delia Goletti, National Institute for Infectious Diseases L. Spallanzani, Italy Received October 19, 2009; Accepted December 18, 2009; Published January 19, 2010 Copyright: 2010 Notari et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was supported by National Institutes of Health grant AG14359, Centers for Disease Control and Prevention (CCU515004), and the Charles S. PLoS ONE \| www.plosone.org Sodium Phosphotungstate Precipitation (NaPTA) p g p Precipitation with NaPTA was carried out according to Wads- worth et al [14] with minor modifications. Briefly, 100 mg of wet tissue were homogenized (10% wt/vol) with PBS lacking Ca2+ and Mg2+, 2% Sarkosyl (pH 7.4), followed by centrifugation at 10006g for 5 minutes to remove cellular debris. A fraction of the supernatant was collected and frozen for immunoblot analysis, whereas a second fraction of 500 ml was mixed with an equal volume of PBS prepared as above. Samples were adjusted to a final concentration of 50 units/ml of Benzonase and 1 mM of MgCl2 and incubated at 37uC for 30 minutes, followed by the addition of 81.3 ml of a pre-warmed solution containing 4% NaPTA and 170 mM MgCl2. After incubation for another 30 minutes at 37uC and constant agitation, samples were centrifuged at 16,0006g for 30 minutes. The supernatant was discarded whereas the pellet was re- suspended in 200 ml of PBS containing 0.1% Sarkosyl (pH 7.4) with the addition of 50 ml EDTA 250 mM (pH 8), in order to remove the white precipitate present in solution. After an additional centrifu- gation at 16,0006g for 30 minute, supernatants were discarded and the pellets re-suspended in 30 ml of PBS containing 0.1% Sarkosyl. Precipitation with NaPTA was carried out according to Wads- worth et al [14] with minor modifications. Briefly, 100 mg of wet tissue were homogenized (10% wt/vol) with PBS lacking Ca2+ and Mg2+, 2% Sarkosyl (pH 7.4), followed by centrifugation at 10006g for 5 minutes to remove cellular debris. A fraction of the supernatant was collected and frozen for immunoblot analysis, whereas a second fraction of 500 ml was mixed with an equal volume of PBS prepared as above. Samples were adjusted to a final concentration of 50 units/ml of Benzonase and 1 mM of MgCl2 and incubated at 37uC for 30 minutes, followed by the addition of 81.3 ml of a pre-warmed solution containing 4% NaPTA and 170 mM MgCl2. After incubation for another 30 minutes at 37uC and constant agitation, samples were centrifuged at 16,0006g for 30 minutes. The supernatant was discarded whereas the pellet was re- suspended in 200 ml of PBS containing 0.1% Sarkosyl (pH 7.4) with the addition of 50 ml EDTA 250 mM (pH 8), in order to remove the white precipitate present in solution. Sodium Phosphotungstate Precipitation (NaPTA) After an additional centrifu- gation at 16,0006g for 30 minute, supernatants were discarded and the pellets re-suspended in 30 ml of PBS containing 0.1% Sarkosyl. Genetic Analysis Genotyping was performed on genomic DNA extracted from blood as previously described [25]. Introduction Variant Creutzfeldt-Jakob disease (vCJD) was first reported in 1996 as a novel non-inherited form of prion disease [1]. Although the disease bears some of the classical features of the sporadic form of the human transmissible spongiform encephalopathies (TSE) or prion diseases, it has distinctive features [2–4]. Most remarkably, vCJD presents at an average age of 26 years [4]; histopatholog- ically is characterized by the presence of plaques containing prion protein surrounded by vacuoles referred to as ‘‘florid’’ or ‘‘daisy’’ plaques [1]. Furthermore, the abnormal and pathogenic prion protein isoform (hereafter identified as PrPres) associated with vCJD has features that are unique among non-inherited human prion diseases [5]. PrPres is thought to be the major or lone component of the infectious agent of prion disease, the so called The distinctive features of vCJD, along with its detection in the UK following the peak of the British epidemic of the prion disease bovine spongiform encephalopathy (BSE), pointed to the con- sumption of prion-contaminated beef products as the possible source of infection [1,7]. Successful transmission to non-human primates and transgenic mice expressing the human prion protein (human PrP) with replication of major features of the vCJD phenotype, provided overwhelming evidence supporting the notion of cattle-to-human transmission [8–10]. These findings established vCJD as the first Western world prion disease to be acquired by oral infection. Kuru, discovered in the 1950s, was endemic among New Guinea tribes practicing ritualistic canni- balism [11–13]. PLoS ONE \| www.plosone.org January 2010 \| Volume 5 \| Issue 1 \| e8765 January 2010 \| Volume 5 \| Issue 1 \| e8765 January 2010 \| Volume 5 \| Issue 1 \| e8765 1 Multiorgan Involvement in vCJD The oral route of prion infection in vCJD raised the possibility that tissues and organs, beside the central nervous system (CNS), might also be affected. To date, PrPres has been reported in several tissues and organs outside the CNS of vCJD patients (Table 1) [14–19, P. Brown, unpublished data]. Preparation of Tissue Homogenates Although the amount of PrPres in non-neural tissues is small compared to that in the brain, the risk posed by the spread of even small amounts of PrPres has been underscored by the iatrogenic transmission of vCJD from blood donors in the preclinical phase of the disease [20]. Tissue homogenates (TH) (10%, wt/vol) were prepared at 4uC in phosphate buffered saline (PBS) lacking Ca2+ and Mg2+, 1% Sarkosyl (pH 7.4), followed by centrifugation at 10006g for 5 minutes to remove cellular debris. Excess of collagen was eliminated by homogenizing tissue and removing the white and dense fraction containing mainly collagen from the fraction rich in parenchymal tissue. Contamination of non-nervous tissue with brain tissue that might have occurred at autopsy was controlled by sampling the depth of the organs and discarding the tissue at the surface. Dura mater where this procedure was unsuitable was rinsed extensively with PBS before homogenization. We examined the main characteristics and tissue distribution of PrPres in a case of vCJD, in which the disease was most likely acquired in the UK but which is officially referred to as an American case because illness onset occurred in the US [21]. In an extensive autopsy examination, sodium phosphotungstate (NaPTA) precipitation, a highly sensitive method of PrPres detection [14,22], was used to establish the presence and estimate the relative amounts of PrPres in several organs and tissues made available to the National Prion Disease Pathology Surveillance Center (NPDPSC). Collection and Processing of Tissues A whole body autopsy was performed within 20 hours from death. The National Prion Disease Pathology Surveillance Center (NPDPSC) received frozen and fixed tissue samples. Frozen tissue included slices from one cerebral and cerebellar hemisphere, portions of pituitary gland and dura mater as well as samples from the trachea, breast, heart, lung, esophagus, stomach, duodenum, jejunum, ileum, colon, liver, spleen, pancreas, adrenal gland, kidney, urinary bladder, uterus, ovary, mesenteric lymph nodes, diaphragm and skin. The skin was taken from the chest wall. In addition, paraffin blocks or sections from the same tissues were also received. Frozen tissues were stored at 280uC. Histopathology and Prion Protein Immunohistochemistry Histopathology and Prion Protein Immunohistochemistry Histology and immunohistochemistry were carried out as previously described [23] on brain sections from frontal, temporal and parietal neocortices (the occipital cortex was unavailable), neo- striatum, thalamus, cerebellar hemisphere and on sections from all received tissues. Immunohistochemistry was carried out with the monoclonal antibody 3F4 to the PrP residues 109–112 [24]. p p m g y Immunoblot. Aliquots of TH or NaPTA-precipitated samples were either examined untreated or after treatment for 1 hour at 37uC with proteinase K (PK) (specific activity 44 units(U)/mg, Sigma Aldrich) at the concentration of 2 U/ml (1 U/ml corresponds to 23 mg/ml when PK specific activity is 44 U/mg) for 60 minutes at 37uC while constantly agitated. The reaction was terminated by addition of 3 mM of phenylmethylsulfonyl fluoride. Samples were diluted in sample buffer (final concentration: 3% sodium dodecyl sulfate [SDS], 4% ß-mercaptoethanol, 10% glycerol, 2 mM EDTA, 62.5 mM Tris, pH 6.8) and boiled for 10 minutes before loading. For deglycoyslation of the protein, samples were denatured and incubated in the presence of recombinant peptide N glycosidase F (PNGase F) according to the manufacturer’s protocol (New England Biolabs). Protein samples were separated in 15% Tris-Glycine SDS-PAGE gels using gel electrophoresis apparatus holding running gels of different lengths (Criterion 7 cm and home made 15 cm high-resolution system, Bio-Rad). Proteins were transferred to Immobilon P (Millipore) for 2 h at 65 V, blocked in 5% (w/v) non-fat milk powder in TBS containing 0.1% (v/v) Tween-20 (TBST) (blocking solution), and incubated overnight at 4uC with selected antibodies. After several washes in TBST, membranes where incubated with a 1:4,000 dilution of a peroxidase-conjugated secondary antibody in TBST for 60 minutes at room temperature, washed in TBST and visualized by enhanced chemiluminescence (Amersham ECL Plus, GE Healthcare) on Kodak BioMax XAR films (Eastman Kodak). Two antibodies to human PrP were used: the monoclonal antibody 3F4 (to residues 109–112) and the rabbit antisera 2301 (to residues 220–231). p p m g y Immunoblot. Aliquots of TH or NaPTA-precipitated samples were either examined untreated or after treatment for 1 hour at 37uC with proteinase K (PK) (specific activity 44 units(U)/mg, Sigma Aldrich) at the concentration of 2 U/ml (1 U/ml corresponds to 23 mg/ml when PK specific activity is 44 U/mg) for 60 minutes at 37uC while constantly agitated. The reaction was terminated by addition of 3 mM of phenylmethylsulfonyl fluoride. Ethics Statement This study was conducted according to the principles expressed in the Declaration of Helsinki. No Institutional Review Board review was required because federal regulations do not require approval of research on deceased patients by the Board. Written informed consent for use of patient information/tissue specimens for research purposes has been obtained. Clinical History y Clinical data on the present patient have been reported in detail [21]. Briefly, the patient lived in Britain until the age of 13 and immigrated to the US in 1992. In early November 2001, at the age of 22 years, the patient was evaluated for depression, emotional instability and memory loss, followed one month later by involuntary movements, gait disturbances and incontinence. During the ensuing three months, the patient’s motor and cognitive deficits worsened, and confusion, hallucination, dysar- thria, bradykinesia, and spasticity also occurred. The diagnosis of vCJD was made following brain magnetic resonance imaging and confirmed by immunoblot and immunohistochemistry of tonsil tissue. She received an experimental treatment with quinacrine for 3 months, but showed only minimal and transitory improvement. The patient died in June 2004, 32 months after the clinical onset. Histopathology and Prion Protein Immunohistochemistry Samples were diluted in sample buffer (final concentration: 3% sodium dodecyl sulfate [SDS], 4% ß-mercaptoethanol, 10% glycerol, 2 mM EDTA, 62.5 mM Tris, pH 6.8) and boiled for 10 minutes before loading. For deglycoyslation of the protein, samples were denatured and incubated in the presence of recombinant peptide N glycosidase F (PNGase F) according to the manufacturer’s protocol (New England Biolabs). Protein samples were separated in 15% Tris-Glycine SDS-PAGE gels using gel electrophoresis apparatus holding running gels of different lengths (Criterion 7 cm and home made 15 cm high-resolution system, Bio-Rad). Proteins were transferred to Immobilon P (Millipore) for 2 h at 65 V, blocked in 5% (w/v) non-fat milk powder in TBS containing 0.1% (v/v) Tween-20 (TBST) (blocking solution), and incubated overnight at 4uC with selected antibodies. After several washes in TBST, membranes where incubated with a 1:4,000 dilution of a peroxidase-conjugated secondary antibody in TBST for 60 minutes at room temperature, washed in TBST and visualized by enhanced chemiluminescence (Amersham ECL Plus, GE Healthcare) on Kodak BioMax XAR films (Eastman Kodak). Two antibodies to human PrP were used: the monoclonal antibody 3F4 (to residues 109–112) and the rabbit antisera 2301 (to residues 220–231). Table 1. Peripheral tissues shown to contain PrPres in vCJD1. Adrenal gland Pituitary gland Appendix Rectum Autonomic ganglia Retina Blood vessels Skeletal muscle Colon Spinal ganglia Ileum Spleen Jejunum Thymus Lymph nodes Tonsil Optic nerve Trigeminal ganglia Peripheral nerves 1Data obtained from immunoblotting and immunohistochemistry studies. Brown P., unpublished data; WHO Expert Committee Meeting, Baden, Austria, 18 May 2007 (updated through Jan 2009). doi:10.1371/journal.pone.0008765.t001 1Data obtained from immunoblotting and immunohistochemistry studies. Brown P., unpublished data; WHO Expert Committee Meeting, Baden, Austria, 18 May 2007 (updated through Jan 2009). doi:10.1371/journal.pone.0008765.t001 January 2010 \| Volume 5 \| Issue 1 \| e8765 PLoS ONE \| www.plosone.org 2 Multiorgan Involvement in vCJD Multiorgan Involvement in vCJD Figure 1 Histological and immunohistochemical features A Multiorgan Involvement in vCJD Histopathological Examination Many mono-centric plaques, often large and occasionally surrounded by ‘‘pseudo vacuoles’’, were present preferentially in the deep cerebral cortex and superficial white matter as well as, to a lesser extent, in the cerebellar cortex and white matter (Fig. 1B). All the organs that had been examined (see Material and Methods for details) were unremarkable except for the kidney and the descending colon which evidenced lymphocytic inflammatory infiltrates (data not shown). In the kidney the infiltrates displayed a focal follicular pattern consistent with interstitial nephritis whereas in the descending colon the lymphocytic infiltrates were linear and located in the sub-mucosa. Figure 1. Histological and immunohistochemical features. A and B. The cerebral cortex is markedly disrupted with prominent astrogliosis and presence of amyloid plaques (arrows in B); frontal cortex; H.E. C and D: PrP immunostaining shows the presence of many plaques (arrows) and plaque-like aggregates in superficial and deep cortical regions with a punctate background staining; frontal cortex. E: Distinctive spot-like PrP immunostaining in the molecular layer and many plaques especially evident in the Purkinje cell layer of the cerebellum. F: Plaques, often in a row are present in the superficial white matter; cerebellum. C–F: Monoclonal antibody (mAb) 3F4. doi:10.1371/journal.pone.0008765.g001 Histopathological Examination p g Both gray and white matter structures were severely atrophic with nearly total loss of neurons and replacement of the neuropil with prominent gemistocytic astrogliosis (Fig. 1A and B). Thus, the typical spongiform degeneration was not observed. Instead, there were irregular extracellular spaces consistent with the astroglial scarring present in the cerebral cortex (Fig. 1B). Macrophages were also present occasionally, especially in the white matter, and probably reflected Wallerian degeneration. The cerebral and cerebellar cortices and the basal ganglia were more affected than the thalamus. Many mono-centric plaques, often large and occasionally surrounded by ‘‘pseudo vacuoles’’, were present preferentially in the deep cerebral cortex and superficial white matter as well as, to a lesser extent, in the cerebellar cortex and white matter (Fig. 1B). All the organs that had been examined (see Material and Methods for details) were unremarkable except for the kidney and the descending colon which evidenced lymphocytic inflammatory infiltrates (data not shown). In the kidney the infiltrates displayed a focal follicular pattern consistent with interstitial nephritis whereas in the descending colon the lymphocytic infiltrates were linear and located in the sub-mucosa. Immunohistochemical staining for PrP of brain sections revealed numerous well circumscribed as well as more diffuse PrP deposits consistent with unicentric plaques or early plaques (also called plaque-like) formations, which were especially prominent in the very superficial and deep cortical layers (Fig. 1C and D). Granular and ‘‘synaptic’’ immunostaining patterns were easily detectable in basal ganglia and thalamus. The cerebellum showed a leopard skin-like immunostaining and plaque-like patterns in the molecular and granule cell layer, respectively (Fig. 1E and F). Polarized light examination confirmed that the plaques contained amyloid (data not shown). No PrP immunostaining was detected in any of the tissues examined outside the brain. Both gray and white matter structures were severely atrophic with nearly total loss of neurons and replacement of the neuropil with prominent gemistocytic astrogliosis (Fig. 1A and B). Thus, the typical spongiform degeneration was not observed. Instead, there were irregular extracellular spaces consistent with the astroglial scarring present in the cerebral cortex (Fig. 1B). Macrophages were also present occasionally, especially in the white matter, and probably reflected Wallerian degeneration. The cerebral and cerebellar cortices and the basal ganglia were more affected than the thalamus. Characterization of Brain PrP Immunoblot analyses of the PK-digested total homogenate (TH) from all cerebral cortices examined displayed the characteristic electrophoretic mobility and glycoform ratios of the PrPres described in vCJD (Fig. 2A) [5,26]. In the cerebellum PrPres showed a slightly faster migration (Fig. 2B). When a high resolution gel (15%, 15 cm long) was used, the PrPres unglycosy- lated form in the cerebellum appeared to resolve into three bands, which included the band corresponding to the PrPres type 2 of 19 kDa and two additional bands that migrated about 0.5 kDa and 1 kDa faster (Fig. 2C). The upper band containing the diglycosylated PrP isoform was over-represented in all brain Genetic analysis demonstrated methionine homozygosity at codon 129 and no mutations or other variations in the open reading frame of the PrP gene. PLoS ONE \| www.plosone.org January 2010 \| Volume 5 \| Issue 1 \| e8765 January 2010 \| Volume 5 \| Issue 1 \| e8765 3 Multiorgan Involvement in vCJD Figure 2. Detection and characterization of PrPres and PK-sensitive PrP in brain. A: Immunoblot of total homogenates (TH), treated with proteinase K (PK), obtained from the frontal cortex of sCJDMM1, sCJDMM2 (representing PrPres types 1 and 2, respectively) and the present case showing the over-representation of the upper band (Diglyc.) containing the diglycosylated form, and the co-migration of the lowest band (Unglyc.), containing the unglycosylated form, with the corresponding band of sCJDMM2. B: Immunoblot of TH from the four regions of the cerebral cortex and the cerebellum, treated with PK as indicated. The cerebellar unglycosylated PrPres isoform generates a thicker and overall slightly faster migrating band than the corresponding PrPres from the cerebral cortex. C: A high-resolution immunoblot (15%, 15 cm long gel) confirms that the monoglycosylated and unglycosylated PrPres isoforms from the cerebellum have a faster electrophoretic mobility than the corresponding forms from the cerebral cortex, and shows that the cerebellar unglycosylated isoform resolves into three fragments including a 19 kDa band, corresponding to PrPres type 2, and two additional bands of slightly lower relative molecular weight (arrowheads); T: Temporal; Cb: Cerebellum. In A–C membranes were probed with the mAb 3F4. D and E: Ratios of the PrPres glycoforms (D) and of the total PrP and PrPres (E) obtained from the same brain regions examined in panel B. Each bar represents the mean 6 SD of three densitometric determinations on each of two tissue samples. doi:10.1371/journal.pone.0008765.g002 Figure 2. Characterization of Brain PrP Detection and characterization of PrPres and PK-sensitive PrP in brain. A: Immunoblot of total homogenates (TH), treated with proteinase K (PK), obtained from the frontal cortex of sCJDMM1, sCJDMM2 (representing PrPres types 1 and 2, respectively) and the present case showing the over-representation of the upper band (Diglyc.) containing the diglycosylated form, and the co-migration of the lowest band (Unglyc.), containing the unglycosylated form, with the corresponding band of sCJDMM2. B: Immunoblot of TH from the four regions of the cerebral cortex and the cerebellum, treated with PK as indicated. The cerebellar unglycosylated PrPres isoform generates a thicker and overall slightly faster migrating band than the corresponding PrPres from the cerebral cortex. C: A high-resolution immunoblot (15%, 15 cm long gel) confirms that the monoglycosylated and unglycosylated PrPres isoforms from the cerebellum have a faster electrophoretic mobility than the corresponding forms from the cerebral cortex, and shows that the cerebellar unglycosylated isoform resolves into three fragments including a 19 kDa band, corresponding to PrPres type 2, and two additional bands of slightly lower relative molecular weight (arrowheads); T: Temporal; Cb: Cerebellum. In A–C membranes were probed with the mAb 3F4. D and E: Ratios of the PrPres glycoforms (D) and of the total PrP and PrPres (E) obtained from the same brain regions examined in panel B. Each bar represents the mean 6 SD of three densitometric determinations on each of two tissue samples. doi:10.1371/journal.pone.0008765.g002 (Table 2 and Fig. 3A and B). Detection in the skin required doubling the TH concentration (equivalent to 4 mg of wet tissue) but this procedure failed to reveal PrPres in other organs (Fig. 3B, 3C, and data not shown). regions examined including the cerebellum (Fig. 2B and D). Total PrP and PrPres were best represented in the temporal cortex and cerebellum while they were present in the least amount least amount in the occipital cortex (Fig. 2B and E). In addition, we confirmed the presence of a 17 kDa PrPres fragment matching the anchorless PrPres type 2 fragment previously described in sporadic CJD (sCJD) and vCJD [27], whereas the 12/13 C-terminal fragment commonly present in sCJD was not detected (data not shown) [28]. These two findings are in agreement with the previously reported molecular characteristics of PrPres from vCJD [27]. Characterization of Brain PrP To assess whether PrPres types 1 and 2 co-occurred in brain as previously reported [29], we digested the TH with a high concentration (32 U/ml) of PK and used high resolution gels (15%, 15 cm long), a technique that allows for the detection of even small amounts of PrPres type 1 and 2 (up to 3–5% of total PrPres) when they co-exist [30]. This procedure failed to demonstrate PrPres type 1 in the brain regions examined in this case (data not shown). With NaPTA precipitation, we easily detected PrPres in the mesenteric lymph nodes, spleen, ileum, pancreas, skin, and to a lesser extent, in the descending colon, liver, ovary and kidney (Table 2 and Fig. 3D). The unequivocal identification of PrPres in the kidney required multiple sampling and an additional two-fold loading of the gel but these procedures failed to reveal PrPres in the ascending colon (Fig. 3C and D). Compared to direct TH blotting, NaPTA preparations often revealed a slower electrophoretic migration of up to 0.5 kDa (Fig. 3C, 3D and data not shown), as previously reported [14]. A significant over-representation of the diglycosylated form with a ratio comparable to that of the brain was apparently maintained in all the organs except the pituitary gland, the skin and some of the TH preparations from the uterus where diglycosylated and monoglycosylated isoforms had nearly the same concentration (Table 2, Fig. 3). Generally, in the NaPTA preparations the unglycosylated form was less well represented than in the TH preparations (Table 2, Fig. 3 and data not shown). Detection of PrPres in Non-Nervous Tissues PrPres could be easily detected in the dura mater, the pituitary and adrenal glands, and the uterus using direct blotting of the TH PLoS ONE \| www.plosone.org January 2010 \| Volume 5 \| Issue 1 \| e8765 January 2010 \| Volume 5 \| Issue 1 \| e8765 4 Multiorgan Involvement in vCJD Table 2. Synopsis of PrPres analyses in the brain and other tissues. POSITIVE TISSUES TH2 NaPTA3 PrPres4 Glyc. ratio NEGATIVE TISSUES Brain + + 100 47:33:20 Other Tissues Dura mater1 + + 8.6 53:34:13 Asc. Colon Pituitary gland + + 5.3 40:41:19 Breast Adrenal gland + + 2.5 45:39:17 Diaphragm Uterus + + 6?1021 50:38:13 Duodenum Skin + + 7?1022 39:43:19 Esophagus Spleen - + ,7?1022 60:35:6 Heart Ileum - + ,7?1022 57:40:3 Jejunum Mesenteric LN - + ,7?1022 48:46:11 Left lung Pancreas - + ,7?1022 53:36:11 Right lung Liver - + ,7?1022 57:34:9 Stomach Ovary - + ,7?1022 58:37:5 Trachea Descending colon5 - + ,7?1022 62:34:5 Urinary bladder Kidney - + ,7?1022 52:35:13 1In bold italic organs where PrPres had not been detected previously. 2TH: PrPres searched in tissue homogenate. 3NaPTA: PrPres searched following enrichment with sodium phosphotungstate precipitation. 4Amount of PrPres expressed as percentage of PrPres present in the frontal cortex. Glyc. ratio: glycoform ratio expressed as percentage of the sum of the three isoforms and representing diglycosylated:monoglycosylated:unglycosylated forms. Data listed for tissue positive in both TH and NaPTA preparations were obtained from TH. 5PrPres was previously reported in colon with no specification of the segment examined. doi:10.1371/journal.pone.0008765.t002 extensive loss of neurons, in which most of the vacuoles are formed, with ensuing astroglial scar [34]. As previously reported [21], the BSE exposure most likely occurred between the early eighties, when the BSE epidemic emerged in the UK, and 1992, when the patient immigrated to the US. This assumption is consistent with an incubation period of 9 to 21 years, which correlates well with the medium incubation period of 17 years estimated for the UK cases of vCJD [35]. The brain PrPres of the present case displayed the glycoform ratio and electrophoretic mobility characteristic of the PrPres associated with vCJD [5]. One exception is the cerebellum where the monoglycosylated and unglycosylated PrPres isoform migrated slightly faster than the PrPres from other brain regions and resolved in three bands. Detection of PrPres in Non-Nervous Tissues The variation in PrPres electrophoretic character- istics between the cerebellum and the cerebral cortex is not surprising for it has also been observed in sCJD [36]. Yet to our knowledge it has never been reported in vCJD. Finally, contrary to previous reports [29], PrPres type 1 did not co-occur with type 2. This discrepancy might stem from our rigorous PrP digestion with PK and from the use of different antibodies, an approach that rules out the possibility that partially cleaved fragments derived by the incomplete digestion of PrPSc be misinterpreted as the type 1 fragment [30,37]. The major finding of the present study is the demonstration that PrPres is present in a number of non-CNS tissues and organs which previous studies had reported as free of PrPres (Table 1 and 2) [14– 19, P. Brown, unpublished data]. These tissues include the dura mater, skin, liver, kidney, pancreas, descending colon, uterus and ovary (Table 2 and Fig. 3). The use of NaPTA, along with the long disease duration, may both have contributed to the undisputed detection of PrPres in these organs in this case. The glycoform ratio of the brain PrPres was not retained in every peripheral organ examined (Fig. 4). In the pituitary gland and the skin the diglycosylated and monoglycosylated PrPres isoforms were about equally represented thus the diglycosylated isoform was not dominant. On the other hand, electrophoretic mobility appeared to match that of the brain. Variations in the glycoform ratio could be assessed only on the TH because the glycoform ratio, as well the electrophoretic mobility, is affected by NaPTA enrichment [14]. doi:10.1371/journal.pone.0008765.t002 Notably, the antibody 2301 to the PrP C-terminal region revealed that in contrast to findings in the brain, most of the PK- sensitive PrP had the electrophoretic mobility of approximately 18 kDa (after deglycosylation) in all non-neural tissues, whereas the full length isoform appeared to be underrepresented (Fig. 4, data not shown). This finding was particularly prominent in the uterus but least evident in the pituitary gland (Fig. 4). Epitope mapping indicated that the 18 kDa fragment was truncated at the N-terminus matching the characteristics of the fragment identified as C1 (Fig. 4) [31]. In addition to being PK-sensitive, C1 could be easily distinguished from the unglycosylated form of PrPres detectable in PK untreated samples, named C2, which electro- phoretically migrated to 19 kDa as in other organs (Fig. 4). Detection of PrPres in Non-Nervous Tissues The presence of prion in the human dura mater is not surprising because sCJD has been transmitted following transplantation of dura obtained from sCJD-affected cases [38]. However, to our knowledge this is the first immunoblot demonstration of PrPres in the dura mater in any prion disease. The detection of relatively large amounts of PrPres in the dura mater raises the possibility of contamination with brain tissue at autopsy. Although this possibility cannot be completely ruled out, extensive rinses in PBS were performed before homogenization in some experiments without observing a reduction in the amount of the PrPres detected. Prion infectivity of kidney and liver has been demonstrated by bioassay in other human prion diseases [39], and PrPres has been observed in the kidney of scrapie infected sheep [40]. The presence of PrPres has also been reported in kidney, liver and pancreas of scrapie infected mice in association with lymphofolli- cular proliferation [41]. This last finding is relevant to the present case in which multiple lymphocytic infiltrates with follicular pattern were present in the kidney. However, contrary to this report, we observed no significant inflammatory reaction in any of the other tissues which contained PrPres. A puzzling finding of our study is the presence of PrPres albeit in small amounts in the kidney but not in the urinary bladder. This apparent discrepancy is relevant to the recent demonstrations of prion infectivity in urine PLoS ONE \| www.plosone.org Discussion D: PrPres from mesenteric lymph nodes and other visceral organs recovered following NaPTA precipitation and compared with TH from the frontal cortex (1:120 dilution) and sCJDMM1 following two film exposures. All organs but ascending colon are positive. Of note, unglycosylated isoform is underrepresented in all NaPTA precipitated samples as compared with that of the TH preparations (see panel A, lanes 3 and 4 and panel B). A–D: Membranes were probed with the mAb 3F4. doi:10.1371/journal.pone.0008765.g003 completed to delivery has been reported in sCJD, iatrogenic CJD and vCJD [50,51]; however, transmission to the progeny has not been examined in detail or confirmed in any of these cases. The first detailed determination of PrPC and PrPres in the reproductive and gestational tissues from a sCJD patient has been carried out only recently [51]. Although this study failed to detect PrPres, remarkably it showed that, in uterine tissue obtained at biopsy, most of the PK-sensitive PrP is truncated at the N-terminus and matches the C-terminal PrPC fragment C1 which is generated during normal PrPC metabolism [51]. Similarly, in the present case we observed that the C1-like fragment was largely predominant over the full-length PrPC in the uterus, and it was easily digested by PK but it was present along with a significant amount of characteristic vCJD PrPres (Fig. 4). Since the N- terminus of the PrPres type 2 associated with vCJD is at residues 92–99, the uterine PrPres must have formed from the full length PrPC rather than from C1, the N-terminus of which is at residues 111–112 [31,52]. These findings raise the question of the origin of the PrPres found in the uterus, a question that is currently unanswered. A similar question may be raised for the urine, in of animals carrying experimental or naturally occurring prion diseases [42–46]. It would indicate that prion infectivity in urine is acquired from the kidney while the urinary bladder acts as a bystander. However the amount of PrPres we observed in the kidney was minimal, and might have not been sufficient to infect the urine and to propagate to the bladder in detectable amounts. Indeed we failed to demonstrate PrPres in the urine in the present case even after hundred-fold urine concentration (data not shown). Obviously more studies are needed to clarify this issue. The present study also demonstrates for the first time the presence of PrPres in the skin in a human prion disease. PLoS ONE \| www.plosone.org Discussion Our study confirms the diagnosis of vCJD in the present case, based on the characteristics of the PrPres and the methionine homozygosity at codon 129 of the PrP gene, the last feature being invariably present in vCJD [32]. However, we also observed two unusual features in this case. The first is the long disease duration of 32 months, which is more than twice the 14 month mean duration of the British cases of vCJD [3]. However, cases of up to 40 months duration after the diseases onset have been reported [3,33]. The second unusual feature is the absence of typical spongiform degeneration which likely stemmed from the long duration of the disease. The long disease duration likely led to PLoS ONE \| www.plosone.org January 2010 \| Volume 5 \| Issue 1 \| e8765 January 2010 \| Volume 5 \| Issue 1 \| e8765 5 Multiorgan Involvement in vCJD Figure 3. Detection of PrPres in non-nervous tissues. A: PrPres from dura mater and frontal cortex (1:24 dilution) from the present case is compared to PrPres of the frontal cortex from sCJDMM1 (type 1) and sCJDMM2 (type 2). B: Two film exposures of immunoblots from non-nervous tissues compared with that of the frontal cortex (1:10 dilution). Pituitary gland, adrenal gland and uterus are clearly positive while the bands in the remaining preparations are considered to be non specific. C: PrPres from skin (double TH loading, equivalent to 4 mg of wet tissue) is barely detectable in TH (Lane 3) compared with frontal cortex TH, which is diluted 1:4 (lane 1) or 1:130 (lane 2). Skin PrPres is better detectable along with the kidney PrPres after sodium phosphotungstate (NaPTA) precipitation of PrPres (Lanes 4 and 5), especially after long exposure (Lanes 4L and 5L) (kidney TH loaded in double amount; probed with mAb 3F4). D: PrPres from mesenteric lymph nodes and other visceral organs recovered following NaPTA precipitation and compared with TH from the frontal cortex (1:120 dilution) and sCJDMM1 following two film exposures. All organs but ascending colon are positive. Of note, unglycosylated isoform is underrepresented in all NaPTA precipitated samples as compared with that of the TH preparations (see panel A, lanes 3 and 4 and panel B). A–D: Membranes were probed with the mAb 3F4. doi:10.1371/journal.pone.0008765.g003 Figure 3. Detection of PrPres in non-nervous tissues. Discussion A: PrPres from dura mater and frontal cortex (1:24 dilution) from the present case is compared to PrPres of the frontal cortex from sCJDMM1 (type 1) and sCJDMM2 (type 2). B: Two film exposures of immunoblots from non-nervous tissues compared with that of the frontal cortex (1:10 dilution). Pituitary gland, adrenal gland and uterus are clearly positive while the bands in the remaining preparations are considered to be non specific. C: PrPres from skin (double TH loading, equivalent to 4 mg of wet tissue) is barely detectable in TH (Lane 3) compared with frontal cortex TH, which is diluted 1:4 (lane 1) or 1:130 (lane 2). Skin PrPres is better detectable along with the kidney PrPres after sodium phosphotungstate (NaPTA) precipitation of PrPres (Lanes 4 and 5), especially after long exposure (Lanes 4L and 5L) (kidney TH loaded in double amount; probed with mAb 3F4). D: PrPres from mesenteric lymph nodes and other visceral organs recovered following NaPTA precipitation and compared with TH from the frontal cortex (1:120 dilution) and sCJDMM1 following two film exposures. All organs but ascending colon are positive. Of note, unglycosylated isoform is underrepresented in all NaPTA precipitated samples as compared with that of the TH preparations (see panel A, lanes 3 and 4 and panel B). A–D: Membranes were probed with the mAb 3F4. doi:10.1371/journal.pone.0008765.g003 Figure 3. Detection of PrPres in non-nervous tissues. A: PrPres from dura mater and frontal cortex (1:24 dilution) from the present case is compared to PrPres of the frontal cortex from sCJDMM1 (type 1) and sCJDMM2 (type 2). B: Two film exposures of immunoblots from non-nervous tissues compared with that of the frontal cortex (1:10 dilution). Pituitary gland, adrenal gland and uterus are clearly positive while the bands in the remaining preparations are considered to be non specific. C: PrPres from skin (double TH loading, equivalent to 4 mg of wet tissue) is barely detectable in TH (Lane 3) compared with frontal cortex TH, which is diluted 1:4 (lane 1) or 1:130 (lane 2). Skin PrPres is better detectable along with the kidney PrPres after sodium phosphotungstate (NaPTA) precipitation of PrPres (Lanes 4 and 5), especially after long exposure (Lanes 4L and 5L) (kidney TH loaded in double amount; probed with mAb 3F4). Acknowledgments which although the prion infectivity has been demonstrated in animals by bioassay [42–46], the only detected form of PrP under normal condition in animals and humans, is a fragment matching the C1 [53, 54, Notari et al., unpublished data]. We are grateful to Dr Stephen Emancipator for the valuable help in the study of the kidney histology, Dr. Jose´ Luis Velajos for his valuable support, Dr. Kim Hummel for editorial assistance, and Mmes Diane Kofskey and Phyllis Scalzo, who provided the histological and immunochemical preparations. All these considerations notwithstanding, the widespread pres- ence of PrPres in visceral organs that we observed in the present case further reinforces the concerns over iatrogenic transmission of vCJD. These concerns are already compelling given the multiple reports of vCJD transmission by blood transfusion. Author Contributions Conceived and designed the experiments: SN FJM PG. Performed the experiments: SN FJM IC. Analyzed the data: SN FJM PB WQZ PG. Contributed reagents/materials/analysis tools: SBH WJS SZ PG. Wrote the paper: SN FJM SBH EDB LBS IC PP WJS PB SZ WQZ PG. Conceived and designed the experiments: SN FJM PG. Performed the experiments: SN FJM IC. Analyzed the data: SN FJM PB WQZ PG. Contributed reagents/materials/analysis tools: SBH WJS SZ PG. Wrote the paper: SN FJM SBH EDB LBS IC PP WJS PB SZ WQZ PG. Discussion Previously, PrPres has been detected in the skin from animals with experimental or naturally occurring scrapie [47] as well as in the antler velvet of elk affected by CWD [48]. Furthermore, it is remarkable that we observed PrPres in the uterus and the ovary, a finding which implicates the reproductive system, thereby raising the possibility of maternal transmission of vCJD. Vertical transmissibility of prion infection has been demonstrated in transgenic mice infected with BSE [49]. Related literature on human prion diseases is very scanty. Pregnancy January 2010 \| Volume 5 \| Issue 1 \| e8765 6 Multiorgan Involvement in vCJD Figure 4. Characteristics of PK-sensitive PrP. Immunoblot analysis of total homogenate from brain, pituitary gland and uterus are shown. The samples, with or without previous PK treatment, were deglycosylated with PNGase F. Membranes were probed with the mAbs 3F4 and 2301 as indicated. A: The brain has relatively large amounts of full-length isoform and PrPres C2 fragment but the N-terminus truncated PrPC fragment (C1) is poorly represented. In addition, brain preparation shows a previously unreported PK-sensitive fragment with molecular weight of 25 kDa (arrow), detectable only in deglycosylated samples, of undetermined origin. B: The C1 fragment is relatively better represented in the pituitary gland C: while it is overly abundant in the uterus. doi:10.1371/journal.pone.0008765.g004 Figure 4. Characteristics of PK-sensitive PrP. Immunoblot analysis of total homogenate from brain, pituitary gland and uterus are shown. The samples, with or without previous PK treatment, were deglycosylated with PNGase F. Membranes were probed with the mAbs 3F4 and 2301 as indicated. A: The brain has relatively large amounts of full-length isoform and PrPres C2 fragment but the N-terminus truncated PrPC fragment (C1) is poorly represented. In addition, brain preparation shows a previously unreported PK-sensitive fragment with molecular weight of 25 kDa (arrow), detectable only in deglycosylated samples, of undetermined origin. B: The C1 fragment is relatively better represented in the pituitary gland C: while it is overly abundant in the uterus. doi:10.1371/journal.pone.0008765.g004 References 9. Bruce ME, Will RG, Ironside JW, McConnell I, Drummond D, et al. (1997) Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 389: 498–501. 1. Will RG, Ironside JW, Zeidler M, Cousens S, Estebeiro K, et al. (1996) A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347: 921–925. 2. Will RG, Zeidler M, Stewart GE, Macleod MA, Ironside JW, et al. (2000) Diagnosis of new variant Creutzfeldt-Jakob disease. Ann Neurol 47: 575–582. 10. Scott MR, Will R, Ironside J, Nguyen HO, Tremblay P, et al. 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Ironside JW, Head MW (2004) Neuropathology and molecular biology of variant Creutzfeldt-Jakob disease. Curr Top Microbiol Immunol 284: 133–159. 33. The National CJD Surveillance Unit (2007) Creutzfeldt-Jakob disease surveillance in the UK. Sixteenth annual report, Available: http://www.cjd. ed.ac.uk. 52. Parchi P, Zou W, Wang W, Brown P, Capellari S, et al. (2000) Genetic influence on the structural variations of the abnormal prion protein. Proc Natl Acad Sci USA (97): 10168–10172. 34. Bignami A, Forno LS (1970) Status spongiosus in Jakob-Creutzfeldt disease. Electron microscopic study of a cortical biopsy. Brain 93: 89–94. 53. Narang HK, Dagdanova A, Xie Z, Yang Q, Chen SG (2005) Sensitive detection of prion protein in human urine. Exp Biol Med (Maywood) 230: 343–349. 35. Valleron AJ, Boelle PY, Will R, Cesbron JY (2001) Estimation of epidemic size and incubation time based on age characteristics of vCJD in the United Kingdom. Science 294: 1726–1728. 54. Andrievskaia O, Algire J, Balachandran A, Nielsen K (2008) Prion protein in sheep urine. J Vet Diagn Invest (20): 141–146. PLoS ONE \| www.plosone.org January 2010 \| Volume 5 \| Issue 1 \| e8765 8
https://openalex.org/W49635032	https://hal.science/hal-01064458v2/file/BifurcationPaper9.pdf	English	null	Scaling laws for the bifurcation escape rate in a nanomechanical resonator	Physical review. E, Statistical, nonlinear and soft matter physics	2,015	cc-by-sa	5,795	Scaling laws for the bifurcation-escape rate in a nanomechanical resonator Martial Defoort, Vadim Puller, Olivier Bourgeois, Fabio Pistolesi, Eddy Collin To cite this version: Martial Defoort, Vadim Puller, Olivier Bourgeois, Fabio Pistolesi, Eddy Collin. Scaling laws for the bifurcation-escape rate in a nanomechanical resonator. Physical Review E : Statistical, Nonlinear, and Soft Matter Physics, 2015, 92 (5), pp.050903(R). 10.1103/PhysRevE.92.050903. hal-01064458v2 Martial Defoort, Vadim Puller, Olivier Bourgeois, Fabio Pistolesi, Eddy Collin To cite this version: Martial Defoort, Vadim Puller, Olivier Bourgeois, Fabio Pistolesi, Eddy Collin. Scaling laws for the bifurcation-escape rate in a nanomechanical resonator. Physical Review E : Statistical, Nonlinear, and Soft Matter Physics, 2015, 92 (5), pp.050903(R). 10.1103/PhysRevE.92.050903. hal-01064458v2 PACS numbers: 85.85.+j, 05.40.-a, 05.10.Gg, 05.70.Ln Transition from a metastable to a stable state is a phenomenon of ubiquitous interest in science: in ther- mal equilibrium it is the essence of the activation law in chemistry [1, 2], it underlies nucleation in phase transi- tions, magnetization reversal in molecular magnets [3], biological switches in cells behavior [4] or RNA dynam- ics [5], transitions of Josephson junctions [6] or ﬂuc- tuations in SQUIDs [7], the list being obviously non- exhaustive. More recently the study of escape statistics has been possible also for out-of-equilibrium dynamical systems like Penning traps [8], Josephson junctions [9], and nano-electromechanical systems [10–14]: the state- switching eﬀect is extensively used in bifurcation ampli- ﬁers, with for instance state-of-the-art quantum bit read- out schemes [15]. In most of these cases the escape time distribution is exponential and the rate Γ characterizes completely the phenomenon. Analytical solutions [16] of the dynamical equations show that its value depends ex- ponentially on a parameter D−1, that coincides with the (inverse of the) temperature for equilibrium systems and more generally is related to the power spectrum of the relevant ﬂuctuations. One can then write: Direct experimental measurement of the escape time and study of the dependence of Ea and Γ0 over a wide range of a system’s parameters is not a trivial task, since the exponential dependence of the escape time makes it either too long or too short for a reasonable observation protocol. For dynamical systems the resonating period ﬁxes a lower bound on the time. Nano-mechanical res- onators with resonance frequency in the MHz range are thus a prominent choice to investigate the bifurcation in- stability of Duﬃng oscillators: they are high frequency dynamical systems with a high quality factor for which the distance to the bifurcation point can be directly con- trolled. In the analysis of switching and reaction rates, three problems can thus be distinguished: obtaining the expo- nent Ea, the prefactor Γ0, and their respective scalings for systems away from thermal equilibrium. The expo- nent has been the ﬁrst subject of interest, with the early work of Arrhenius [1]. The prefactor has then been ad- dressed by Kramers later on [2], while ﬁnally the scaling of both for dynamical systems has been derived by Dyk- man [17]. Scaling laws for the bifurcation-escape rate in a nanomechanical resonator M. Defoort,1 V. Puller,2 O. Bourgeois,1 F. Pistolesi,2 and E. Collin1 1 Universit´e Grenoble Alpes, CNRS Institut N´EEL, BP 166, 38042 Grenoble Cedex 9, France 2 Univ. Bordeaux, LOMA, UMR 5798, F-33400 Talence, France CNRS, LOMA, UMR 5798, F-33400 Talence, France M. Defoort,1 V. Puller,2 O. Bourgeois,1 F. Pistolesi,2 and E. Collin 1 Universit´e Grenoble Alpes, CNRS Institut N´EEL, BP 166, 38042 Grenoble Cedex 9, France 2 Univ. Bordeaux, LOMA, UMR 5798, F-33400 Talence, France CNRS, LOMA, UMR 5798, F-33400 Talence, France M. Defoort,1 V. Puller,2 O. Bourgeois,1 F. Pistolesi,2 and E. Collin1 1 Universit´e Grenoble Alpes, CNRS Institut N´EEL, BP 166, 38042 Grenoble Cedex 9, France 2 Univ. Bordeaux, LOMA, UMR 5798, F-33400 Talence, France CNRS, LOMA, UMR 5798, F-33400 Talence, France We report on experimental and theoretical studies of the ﬂuctuation-induced escape time from a metastable state of a nanomechanical Duﬃng resonator in cryogenic environment. By tuning in situ the non-linear coeﬃcient γ we could explore a wide range of the parameter space around the bifurcation point, where the metastable state becomes unstable. We measured in a relaxation process the distribution of the escape times. We have been able to verify its exponential distribution and extract the escape rate Γ. We investigated the scaling of Γ with respect to the distance to the bifurcation point and γ, ﬁnding an unprecedented quantitative agreement with the theoretical description of the stochastic problem. Simple power scaling laws turn out to hold in a large region of the parameter’s space, as anticipated by recent theoretical predictions. These unique ﬁndings, implemented in a model dynamical system, are relevant to all systems experiencing under-damped saddle-node bifurcation. PACS numbers: 85.85.+j, 05.40.-a, 05.10.Gg, 05.70.Ln HAL Id: hal-01064458 https://hal.science/hal-01064458v2 Submitted on 23 Nov 2015 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 - ShareAlike 4.0 International License ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) FIG. 1: (Color online) Top panel: Schematic of the ex- perimental setup with the nano-resonator structure. Bottom panel: Linear and Duﬃng resonances (respectively grey and black points, with top-right and bottom-left axes). The lines show the ﬁt. The nonlinear resonance is for Vg = 9.4 V, which shifts the resonance frequency and opens an hysteresis (thin green arrows highlight upward and downward sweeps). The relaxations occur at a detuning ω −ωb from the bifurcation frequency (red point and vertical arrow). Inset: Gaussian dis- tribution histogram of the measured intrinsic frequency ﬂuc- tuations. with ω0/2π=7.07 MHz the resonance frequency, ∆ω/2π=1.84 kHz the linewidth, and fd and fn the drive and noise forces divided by the mass of the resonator. We ﬁx the drive force so that mfd = 65 pN, leading to a constant maximal displacement amplitude of 100 nm. As can be deduced from our characterizations [20], this amplitude is small enough to guarantee that nonlinear damping mechanisms such as discussed in Refs. [24, 25] are small (see comment in the discussion section). The noise force signal is ﬁltered so that the force spectrum R dteiωt⟨fn(t)fn(0)⟩ω = 2D is constant over a bandwidth of 1 MHz around 7 MHz. The Duﬃng coeﬃcient γ scales as V 2 g and is for us negative [22]. At ﬁxed driving force, the system admits two amplitudes of oscillation for suﬃciently large \|γ\| as shown in Fig. 1 (bistability). By ﬁtting with the standard Duﬃng expressions [26] the parameters ∆ω, ω0 and γ together with the bifurcation frequency ωb can be obtained with a good accuracy. The experiment is then performed by sweeping ω from the stable regime (ω > ω0) down to the edge of the hysteresis at a given value of ω−ωb in the high amplitude state (see Fig. 1). The sweeping rate (a few Hz/s) is an important parameter which should both guarantee adiabaticity of the sweep and high accuracy in the measurement [33]. Finally, the escape time from the metastable state is detected when the measured displacement amplitude falls below an appropriate threshold value. Typically 103 escape events are recorded for each set of parameters. PACS numbers: 85.85.+j, 05.40.-a, 05.10.Gg, 05.70.Ln It is actually in micro and nano-mechanical systems that a measurement of the power law depen- dence of Ea with respect to the distance from the bi- furcation point has been performed, giving the predicted value within experimental error [10, 11]. Nevertheless, the activation energy has been claimed to match theory at best within a factor of 2 due to injected noise calibra- tion [10]. To our knowledge no attempts have been done to obtain a more quantitative veriﬁcation of the predic- tions of Dykman and Krivoglaz [17], in particular for the scaling law of the prefactor Γ0 and the dependence to the Duﬃng non-linear coeﬃcient γ of both Γ0 and Ea. An- swering the three above mentioned problems together is thus the aim of our work, using a unique nano-mechanical implementation of the bifurcation phenomenon. Γ = Γ0 exp(−Ea/D), (1) (1) where the prefactor Γ0 is assumed to depend very weakly on D, and Ea in analogy with a potential system can be called activation energy: it parametrizes the distance to the unstable point. For out-of-equilibrium systems a central theoretical result is the paper by Dykman and Krivoglaz [17], that found an explicit expression for Ea and Γ0 for a generic dynamical system close to the bi- furcation point, where the line of metastable states joins the line of unstable ones. It predicts universal power laws dependence of Ea and Γ0 on the distance from the bifurcation point in terms of \|ω −ωb\|, where ω is the driving frequency of the dynamical system and ωb is its bifurcation value. In this Letter we report on experimental and theoreti- 2 B < 1 T and a gate electrode is also capacitively cou- pled to the nanomechanical device (gap about 100 nm) [20]. The resonator admits large distortions (in the hun- dred nm range) to be attained while remaining intrinsi- cally extremely linear [22], while a well-controlled non- linearity can be generated by means of a DC gate volt- age bias Vg [23]. This distinctive feature enables to tune the global non-linearity of our device without changing the displacement amplitude. Using an adder we apply both a sinusoidal drive and a noise voltage from a voltage source generator. PACS numbers: 85.85.+j, 05.40.-a, 05.10.Gg, 05.70.Ln The resulting electric signal together with a 1 kOhm bias resistor is used to inject an AC cur- rent through the goalpost and generates both driving and controllable (zero average) noise forces on the resonator. More information on the calibration and experimental details can be found in Refs. [20, 22]. The resulting equa- tion of motion for the resonator displacement x reads: 7.05 7.06 7.07 0 25 50 75 100 7.20 7.21 7.22 7.23 0 25 50 75 100 x (nm) for V g = 0 V 2 (MHz) for V g = 0 V b )/2 V g x (nm) for V g = 9.4 V 2 (MHz) for V g = 9.4 V -40 -20 0 20 40 0 500 1000 1500 2000 Intrinsic noise (Hz) Counts b /2 B + 1 k 4 K FIG. 1: (Color online) Top panel: Schematic of the ex- perimental setup with the nano-resonator structure. Bottom panel: Linear and Duﬃng resonances (respectively grey and black points, with top-right and bottom-left axes). The lines show the ﬁt. The nonlinear resonance is for Vg = 9.4 V, which shifts the resonance frequency and opens an hysteresis (thin green arrows highlight upward and downward sweeps). The relaxations occur at a detuning ω −ωb from the bifurcation frequency (red point and vertical arrow). Inset: Gaussian dis- tribution histogram of the measured intrinsic frequency ﬂuc- tuations. 7.05 7.06 7.07 0 25 50 75 100 7.20 7.21 7.22 7.23 0 25 50 75 100 x (nm) for V g = 0 V 2 (MHz) for V g = 0 V b )/2 V g x (nm) for V g = 9.4 V 2 (MHz) for V g = 9.4 V -40 -20 0 20 40 0 500 1000 1500 2000 Intrinsic noise (Hz) Counts b /2 B + 1 k 4 K V g ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) The experiment has been repeated for three diﬀerent values of the noise forces fn, three diﬀerent detunings ω −ωb (up to 5% of the hysteresis), and ﬁve diﬀerent values of Vg (and thus of γ), for a total of 45 escape histograms. The resulting settings are summarized in Fig. 2. cal investigations of the dependence of Ea and Γ0 on the system parameters for a driven nano-mechanical oscilla- tor in the non-linear regime in presence of a controlled noise force. It is well known that for a suﬃciently strong non-linear term the system admits for some values of the driving frequency a metastable solution. By measuring the escape rate for a wide range of parameters we could verify the validity of the power scaling laws predicted by Dykman and Krivoglaz for both Ea and Γ0. Remark- ably, we found that the scaling holds experimentally in a much larger region of the parameter space than the one for which the theory of Ref. [17] has been derived. Con- cerning the Ea dependence on detuning, the possibility of an extended region of scaling was discussed in Refs. [18, 19]. Performing the full numerical simulation of the stochastic problem adapted to our device parameters we found that experiment and theory are in excellent quan- titative agreement. The experiment is performed on a unique goalpost (depicted in top graph of Fig. 1) aluminum-coated sili- con nano-electro-mechanical resonator. It consists in two cantilever feet of length 3 µm linked by a paddle of length 7 µm, all about 250 nm wide and 150 nm thick for a total mass m = 1.25 10−15 kg [20]. The experiment is per- formed at 4.2 K in cryogenic vacuum (pressure < 10−6 mbar). The motion is actuated and detected by means of the magnetomotive scheme [21], with a magnetic ﬁeld For each data measurement, the experimental value of ωb might slightly diﬀer from the one obtained by the initial ﬁt. This problem is detected by sweeping rela- tively rapidly ω (tens of Hz/sec) through the bifurca- tion point and measuring the escape value ωb prior to 3 FIG. 2: (Color online) Bifurcation parameter space (normal- ized driving force versus Ω= 2\|ω −ω0\|/∆ω). ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) On the other hand when ω is such that the amplitude is max- imal, the two eigenvalues coincide, inducing fully two- dimensional ﬂuctuations. Thus beyond this point the approximation used to obtain Eq. (4) breaks down. This condition reads 4Ωb\|Ω−Ωb\| ≪1. P(t) = Γe−Γt Z dǫ σ √ 2π e−ǫ2/(2σ2)−Γ′ǫt . (3) (3) Fitting it to the data with the method of Kolmogorov- Smirnov [30], to avoid losses of information due to his- togram binning, the two independent parameters of the distribution, Γ and the product Γ′σ, can be obtained. A typical curve is shown in the inset of Fig. 2. Note that this procedure does not need any hypothesis on the explicit functional dependence of Γ on ωb. On the other hand the procedure breaks down for too small detunings, and we thus need to drop the data for four values of the detuning. We can then verify the validity of Eq. (1) for the system at hand by plotting log Γ as a function of 1/D (see Fig. 3). The linear ﬁt gives Ea and Γ0. The absolute experimental deﬁnition of the noise level is diﬃcult, and we introduce a calibration factor C (close to 1) between D and the nominal injected noise power. Note that it simply amounts to multiply Ea by C, thus leaving the Fitting it to the data with the method of Kolmogorov- Smirnov [30], to avoid losses of information due to his- togram binning, the two independent parameters of the distribution, Γ and the product Γ′σ, can be obtained. A typical curve is shown in the inset of Fig. 2. Note that this procedure does not need any hypothesis on the explicit functional dependence of Γ on ωb. On the other hand the procedure breaks down for too small detunings, and we thus need to drop the data for four values of the detuning. We can then verify the validity of Eq. (1) for the system at hand by plotting log Γ as a function of 1/D (see Fig. 3). The linear ﬁt gives Ea and Γ0. The absolute experimental deﬁnition of the noise level is diﬃcult, and we introduce a calibration factor C (close to 1) between D and the nominal injected noise power. ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) The escape exponential distribution has thus to be averaged over these ﬂuctuations. For \|ω−ωb\| ≫σ one can expand this dependence: Γ(ω−ωb−ǫ) = Γ+Γ′ǫ+. . . , where ǫ is the Gaussian-distributed shift of ωb. This gives the following distribution for the escape times: In order to extract the scaling dependence of Ea and Γ0 on the detuning and the non-linear parameter γ it is convenient to recall the predictions that can be obtained following Ref. [17]. Let us rescale the detuning by deﬁn- ing Ω= 2\|ω −ω0\|/∆ω with Ωb = 2\|ωb −ω0\|/∆ω. For Ωb ≫ √ 3 (that holds for all the data of our experiment) one obtains that Ωb ≈3\|γ\|f 2 d/(4ω2∆ω2) with the param- eters in Eq. (1) reading [34]: Ea = 2f 2 d 3∆ω \|Ω−Ωb\|3/2 Ω5/2 b , Γ0 = ∆ω 2 \|Ω−Ωb\|1/2Ω1/2 b 2π . (4) (4) The basic assumptions to obtain these expressions are that Ea/D ≫1 in order to keep the escape a rare event, and to be able to reduce this two-dimensional problem (amplitude and phase) into a one-dimensional one. This second condition (much less appreciated in the literature) is only veriﬁed when the driving frequency ω is in a tiny region close to the bifurcation point ωb and far from the frequency for which the amplitude is maximum. In this region, one of the eigenvalues of the linearized dynamical equations of motion vanishes, which induces a slow mo- tion in the direction of the relative eigenvector. On the other hand when ω is such that the amplitude is max- imal, the two eigenvalues coincide, inducing fully two- dimensional ﬂuctuations. Thus beyond this point the approximation used to obtain Eq. (4) breaks down. This condition reads 4Ωb\|Ω−Ωb\| ≪1. The basic assumptions to obtain these expressions are that Ea/D ≫1 in order to keep the escape a rare event, and to be able to reduce this two-dimensional problem (amplitude and phase) into a one-dimensional one. This second condition (much less appreciated in the literature) is only veriﬁed when the driving frequency ω is in a tiny region close to the bifurcation point ωb and far from the frequency for which the amplitude is maximum. In this region, one of the eigenvalues of the linearized dynamical equations of motion vanishes, which induces a slow mo- tion in the direction of the relative eigenvector. ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) The grey area is the NEMS bistability regime where the right edge is the transition from a high amplitude oscillation to a low one (the left edge is the opposite) and K is the spinode point where hysteresis starts to open. We show within the bistability the data points at diﬀerent voltages Vg. Inset: typical low Vg re- laxation curve obtained with about 1000 relaxations, and ﬁt with and without ﬂuctuations on ωb. 0.0 0.5 1.0 10 -1 10 0 10 1 10 2 700 Hz 400 Hz 100 Hz Exponential Fit -1 s) D -1 (N -2 /kg -2 Hz -1 ) FIG. 3: (Color online) Escape time as a function of D−1 for Vg = 9.4 V at diﬀerent detunings ω −ωb from the bifurcation point. 0.0 0.5 1.0 10 -1 10 0 10 1 10 2 700 Hz 400 Hz 100 Hz Exponential Fit -1 s) D -1 (N -2 /kg -2 Hz -1 ) FIG. 2: (Color online) Bifurcation parameter space (normal- ized driving force versus Ω= 2\|ω −ω0\|/∆ω). The grey area is the NEMS bistability regime where the right edge is the transition from a high amplitude oscillation to a low one (the left edge is the opposite) and K is the spinode point where hysteresis starts to open. We show within the bistability the data points at diﬀerent voltages Vg. Inset: typical low Vg re- laxation curve obtained with about 1000 relaxations, and ﬁt with and without ﬂuctuations on ωb. FIG. 3: (Color online) Escape time as a function of D−1 for Vg = 9.4 V at diﬀerent detunings ω −ωb from the bifurcation point. scaling dependence unmodiﬁed. The value of Γ0 is not aﬀected by this calibration either. More experimental details can be found in Ref. [31]. each relaxation-time acquisition. A typical histogram of the distribution of ωb is shown in the inset of Fig. 1 for Vg = 9.4 V. It has Gaussian form with a half-width σ in the range of tens of Hz. This tiny spread (10−6 to 10−5 of ωb) is due to low-frequency intrinsic ﬂuctuations of the resonating frequency, which actual origin is still under debate [27–29]. Even if extremely small, due to the high sensitivity of the bifurcation phenomenon the ﬂuctuations of ωb modify slightly the value of Γ at each measurement, and we have to take this eﬀect into ac- count. ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) A remarkable scaling is then observed in all the experimental range, with a ﬁtted slope as a function of the detuning of 1.53±0.04 and 0.55±0.2, for Ea and Γ0 respectively. This matches the analytic predictions by Dykman and Krivoglaz, and we use this good agreement to deﬁne the noise source calibration fac- tor C: scaling D by C the prediction of Eq. (4) coincides with the ﬁtted value for Ea (dashed line in Fig. 4 left panel). The dependence on the non-linear parameter Ωb could also be tested for both quantities. It is shown in the insets of Fig. 4 and gives ﬁtted slopes of −2.43±0.05 and 0.6 ± 0.1, again in excellent agreement with Eq. (4). One can see that the exact (numerical) result has the same power law dependence as the analytical results (dashed line), even where the approximate theory is not supposed to hold. Quantitative agreement between ex- periment and theory on Ea is obtained with C ≈1.3, thus validating the experimental noise amplitude calibra- tion to within 15 % which is remarkable. Note that the simulation does not contain any other free parameter, which are all experimentally known to better than 5 %. Concerning Γ0, we are not aware of previous attempts to compare this quantity to the theoretical predictions. The agreement with the full theory is within a factor of about 3, which is remarkable given the logarithmic pre- cision on this parameter. We speculate that these dis- crepancies could arise from the actual algorithm used to extract Γ0, or from more fundamental reasons like ex- tra (non-Duﬃng) nonlinearities appearing in Eq. (2) (i.e. non-linear damping, or non-cubic restoring force terms) [35]. To better understand this remarkable scaling in such a large parameter region we solved numerically the stochas- tic problem. This can be done by introducing the com- plex slow amplitude z(t) deﬁned as x(t) = z(t)eiωt + z(t)∗e−iωt and then convert the Langevin Eq. (2) to a Fokker-Planck equation ∂τP = LP for the probabil- ity density P(u, v, τ) of the real and imaginary part of z = (3\|γ\|/∆ω)1/2(u + iv) as a function of the dimension- less time τ = t∆ω. The escape rate from a given domain can be calculated by solving the equation L†τ(u, v) = −1 with zero boundary condition at the border of the domain [16]. ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) Note that it simply amounts to multiply Ea by C, thus leaving the In the experiment we performed this quantity ranged uniformly between 0.13 to 71, thus a part of the data where well outside the range of the expected validity of Eq. (4), enabling to investigate the behavior of Γ in a region where no present analytical prediction exists. As explained, the expressions for Ea and Γ0 in Eq. (4) de- 4 4 5x10 -4 5x10 -3 10 1 10 2 10 3 5 15 10 -2 10 -1 10 0 E a (N 2 /kg 2 Hz) b 5/3 b b E a b \| -3 /2 0.1 1 10 10 1 10 2 10 3 10 4 3 10 10 1 10 2 0 (Hz) \| b \| b b 0 \| b \| -1 /2 FIG. 4: (Color online) Scaling plots for Ea (left) and Γ0 (right) with respect to detuning. The full circles indicate the experimental points, the open (blue) triangles the prediction of the full numerical simulation, the (red) full lines the linear ﬁt to the data, and the dashed (blue) lines the prediction of Eq. (4). Insets: scaling with the non-linear parameter Ωb. 5x10 -4 5x10 -3 10 1 10 2 10 3 5 15 10 -2 10 -1 10 0 E a (N 2 /kg 2 Hz) b 5/3 b b E a b \| -3 /2 0.1 1 10 10 1 10 2 10 3 10 4 3 10 10 1 10 2 0 (Hz) \| b \| b b 0 \| b \| -1 /2 FIG. 4: (Color online) Scaling plots for Ea (left) and Γ0 (right) with respect to detuning. The full circles indicate the experimental points, the open (blue) triangles the prediction of the full numerical simulation, the (red) full lines the linear ﬁt to the data, and the dashed (blue) lines the prediction of Eq. (4). Insets: scaling with the non-linear parameter Ωb. sought Poissonian rate. The numerical results for Ea and Γ0 are shown in Fig. 4 in open (blue) triangles. pend only on the detuning and the non linear coeﬃcient (through Ωb), the other parameters being the same for all data points. To test the validity of Dykman-Krivoglaz ex- pressions, we produce a scaling plot, where the logarithm of Ea and Γ0 are plotted as a function of \|Ω−Ωb\|/Ω5/3 b and \|Ω−Ωb\|Ωb (see Fig. 4). ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) Rev. Lett. 94, 156403 (2005). [32] F. Pistolesi, Y. Blanter, and I. Martin, Physical Review B 78, 085127 (2008). y , ( ) [11] C. Stambaugh and H. B. Chan, y ( ) [11] C. Stambaugh and H. B. Chan, Phys. Rev. B 73, 172302 (2006). [33] We can estimate adiabaticity using theoretical expres- sions from Ref. [24]. Eqs. (50), (53) and (57) give the shift in bifurcation frequency δerr induced by ﬁnite sweep rate. We obtain δerr = 2π 0.5 Hz at most with our exper- imental parameters, which means that the error in the resonance position is less than 20 ppm of the Duﬃng fre- quency shift itself. At the same time, the critical slowing down time τsd can be estimated from Eq. (52). We ob- tain τsd smaller than 8 ms for all our settings, which shall be compared to the smallest recorded bifurcation time of order 40 ms. ( ) [12] H. B. Chan and C. Stambaugh, Phys. Rev. Lett. 99, 060601 (2007). ( ) [12] H. B. Chan and C. Stambaugh, Ph R L tt 99 060601 (2007) [ ] g , Phys. Rev. Lett. 99, 060601 (2007). [13] H. B. Chan, M. I. Dykman, and C. Stambaugh, Phys. Rev. Lett. 100, 130602 (2008). ( ) [14] Q. P. Unterreithmeier, T. Faust, and J. P. Kotthaus, Phys. Rev. B 81, 241405 (2010). Phys. Rev. B 81, 241405 (2010). [15] N. Boulant, G. Ithier, P. Meeson, F. Nguyen, D. Vion, [15] N. Boulant, G. Ithier, P. Meeson, F. Nguyen, D. Vion, D. Esteve, I. Siddiqi, R. Vijay, C. Rigetti, F. Pierre, and M. Devoret, Phys. Rev. B 76, 014525 (2007). y ( ) [16] P. H¨anggi, P. Talkner, and M. Borkovec, Rev. Mod. Phys. 62, 251 (1990). [34] These expressions are obtained following the method of Ref. [17]. Note that in our work the bifurcation is anal- ysed as a function of frequency detuning and nonlinear parameter (not applied force). [17] M. I. Dykman and M. A. Krivoglaz, Sov. Phys. JETP , 60 (1979). [18] M. Dykman, I. Schwartz, and M. Shapiro, Phys. Rev. E 72, 021102 (2005). [35] We tried to quantify the impact of nonlinear damping and of gate coupling non-Duﬃng contributions on the dynam- ics equation. Using a quadratic ﬁt to describe nonlinear damping [25, 26], one can estimate the p parameter of Ref. [24] to be at worst about 0.06. ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) Mastering of the bifurcation escape technique by having a reliable theory and experimental We gratefully acknowledge discussions with M. Dyk- man, K. Hasselbach, E. Lhotel and A. Feﬀerman. We thank J. Minet and C. Guttin for help in setting up the experiment. We acknowledge support from MI- CROKELVIN, the EU FRP7 grant 228464 and of the French ANR grant QNM n◦0404 01. [1] S. Arrhenius, Z. Physik. Chem. 4, 226 (1889). [25] E. Collin, T. Moutonet, J.-S. Heron, O. Bourgeois, Y. M. Bunkov, and H. Godfrin, Phys. Rev. B 84, 054108 (2011). [ ] ( ) [2] H. A. Kramers, Physica 7, 284 (1940). [ ] [2] H. A. Kramers, Physica 7, 284 (1940). [3] M. A. Novak, R. Sessoli, A. Caneschi, and D. Gatteschi, J. of Magn. and Magn. Mater. 146, 211 (1995). [ ] , , , , J. of Magn. and Magn. Mater. 146, 211 (1995). ( ) [26] E. Collin, Y. M. Bunkov, and H. Godfrin, Phys. Rev. B 82, 235416 (2010). [4] E. M. Ozbudak, M. Thattai, H. N. 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Frunzio, and M. H. Devoret, Phys. Rev. Lett. 93, 207002 (2004). [31] M. Defoort, PhD thesis: Non-linear dynamics in nano- electromechanical systems at low temperature (CNRS et Universit´e Grenoble Alpes, unpublished, 2014). [10] J. S. Aldridge and A. N. Cleland, Phys. ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) This gives the average time needed to reach the bor- der starting at (u, v). The equation reads explicitly: In conclusion, we have investigated the escape dynam- ics close to the bifurcation point for a nanomechanical resonator in the Duﬃng non-linear regime measured at cryogenic temperatures. Using a single ideally tunable system, we have: (i) Measured the escape rate Γ as a function of the noise amplitude D, the detuning to the bifurcation point ω −ωb, and the nonlinear parameter γ. (ii) Extracted Ea and Γ0 as deﬁned by Eq. (1). (iii) Veriﬁed that the universal scaling of Ea and Γ0 initially predicted for a tiny region around the bifurcation point holds actually in a region up to two orders of magnitude larger than the original one. (iv) Veriﬁed by solving nu- merically the exact problem, that the observation is in quantitative agreement with the behavior expected for a driven Duﬃng oscillator. The scaling of Ea as a func- tion of \|Ω−Ωb\| is consistent with the predictions of Refs. [D(∂2 u + ∂2 v) −fu∂u −fv∂v]τ = −1 , (5) (5) with D = 3\|γ\|D/(8ω3∆ω), fu = u + v(u2 + v2) −Ω, fv = v−u(u2+v2)−Ω−Fd, and Fd = fd(3\|γ\|)1/2/[2(ω∆ω)3/2]. Eq. (5) can be solved numerically [32] to obtain the av- erage escape time that coincides with the inverse of the 5 [18, 19]. Due to the generality of the Duﬃng model, these results are of interest for a wide class of systems. Even beyond the fundamental interest in the scaling laws we point out that the device acts as a very sensitive am- pliﬁer: it allows the detection of tiny variations of the resonator frequency. Understanding the frequency ﬂuc- tuations in mechanical resonators is a current challenge of the ﬁeld [27–29]. Mastering of the bifurcation escape technique by having a reliable theory and experimental veriﬁcation of the scaling of the rates is a crucial step towards the study of modiﬁcations induced by other phe- nomena. [18, 19]. Due to the generality of the Duﬃng model, these results are of interest for a wide class of systems. Even beyond the fundamental interest in the scaling laws we point out that the device acts as a very sensitive am- pliﬁer: it allows the detection of tiny variations of the resonator frequency. Understanding the frequency ﬂuc- tuations in mechanical resonators is a current challenge of the ﬁeld [27–29]. ¨x + ∆ω ˙x + ω2 0x + γx3 = fd cos(ωt) + fn(t) (2) Following the cal- culation procedure of Ref. [17], the alteration of the en- ergy potential Ea is then at worst about 20 %. From the gate capacitance Taylor series coeﬃcients of Ref. [20], we calculate that the ”eﬀective” Duﬃng nonlinear parame- ter measured in a frequency-sweep experiment could be modiﬁed by about 4 % with respect to the value com- puted from the actual x3 restoring force term, which is small. [19] O. Kogan, arXiv:0805.0972v2 (2008). [20] E. Collin, M. Defoort, K. Lulla, T. Moutonet, J.-S. Heron, O. Bourgeois, Y. M. Bunkov, and H. Godfrin, Review of Scientiﬁc Instruments 83, 045005 (2012). [21] A. Cleland and M. Roukes, Sensors and Actuators 72, 256 (1999). [22] E. Collin, T. Moutonet, J.-S. Heron, O. Bour- geois, Y. M. Bunkov, and H. Godfrin, J Low Temp Phys 162, 653 (2011). ( ) [23] I. Kozinsky, H. W. C. 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https://openalex.org/W3091565660	https://lirias.kuleuven.be/bitstream/123456789/661567/2/2020184.pdf	English	null	Stability of the Virome in Lab- and Field-Collected Aedes albopictus Mosquitoes across Different Developmental Stages and Possible Core Viruses in the Publicly Available Virome Data of <i>Aedes</i> Mosquitoes	MSystems	2,020	cc-by	10,903	Stability of the Virome in Lab- and Field-Collected Aedes albopictus Mosquitoes across Different Developmental Stages and Possible Core Viruses in the Publicly Available Virome Data of Aedes Mosquitoes on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from Chenyan Shi,a,b,c Lu Zhao,d,e Evans Atoni,d,e Weifeng Zeng,f Xiaomin Hu,g Jelle Matthijnssens,c Zhiming Yuan,d,e Han Xiad,e enyan Shi,a,b,c Lu Zhao,d,e Evans Atoni,d,e Weifeng Zeng,f Xiaomin Hu,g Jelle Matthijnssens,c Zhim Han Xiad,e ,c Lu Zhao,d,e Evans Atoni,d,e Weifeng Zeng,f Xiaomin Hu,g Jelle Matthijnssens,c Zhiming Yuan,d,e aCenter Lab of Longhua Branch and Department of Infectious Disease, The Second Clinical Medical College, Jinan University (Shenzhen People's Hospital), Shenzhen, China bPost-doctoral Scientiﬁc Research Station of Basic Medicine, Jinan University, Guangzhou, China on October 13, 2020 at 26989832 ttp://msystems.asm.org/ cDepartment of Microbiology, Immunology and Transplantation, Laboratory of Clinical and Epidemiological Virology, Laboratory of Viral Metagenomics, Rega Institute, KU Leuven, Leuven, Belgium ey Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China niversity of Chinese Academy of Sciences, Beijing, China dKey Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China eUniversity of Chinese Academy of Sciences, Beijing, China dKey Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China fLiwan Center for Disease Control and Prevention, Guangzhou, Guangdong, China gCollege of Life Science, South-Central University for Nationalities, Wuhan, China gCollege of Life Science, South-Central University for Nationalities, Wuhan, China Chenyan Shi and Lu Zhao contributed equally to this article. Author order was determined on the basis of contribution level. on October 13, 2020 at 26989832 asm.org/ ABSTRACT Aedes mosquitoes can efﬁciently transmit many pathogenic arboviruses, placing a great burden on public health worldwide. In addition, they also carry a number of insect-speciﬁc viruses (ISVs), and it was recently suggested that some of these ISVs might form a stable species-speciﬁc “core virome” in mosquito popula- tions. However, little is known about such a core virome in laboratory colonies and if it is present across different developmental stages. In this study, we compared the viromes in eggs, larvae, pupae, and adults of Aedes albopictus mosquitoes collected from a lab colony and compared each to the virome of different developmental stages collected in the ﬁeld. The virome in lab-derived A. albopictus was very stable across all stages, consistent with a vertical transmission route of these viruses, and formed a possible “vertically transmitted core virome.” The different stages of ﬁeld- collected A. RESEARCH ARTICLE Host-Microbe Biology crossm RESEARCH ARTICLE Host-Microbe Biology September/October 2020 Volume 5 Issue 5 e00640-20 Citation Shi C, Zhao L, Atoni E, Zeng W, Hu X, Matthijnssens J, Yuan Z, Xia H. 2020. Stability of the virome in lab- and ﬁeld-collected Aedes albopictus mosquitoes across different developmental stages and possible core viruses in the publicly available virome data of Aedes mosquitoes. mSystems 5:e00640-20. https://doi.org/10.1128/mSystems.00640-20. Editor Rup Lal, University of Delhi Copyright © 2020 Shi et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Zhiming Yuan, yzm@wh.iov.cn, or Han Xia, hanxia@wh.iov.cn. Received 10 July 2020 Accepted 15 September 2020 Published 29 September 2020 Stability of the Virome in Lab- and Field-Collected Aedes albopictus Mosquitoes across Different Developmental Stages and Possible Core Viruses in the Publicly Available Virome Data of Aedes Mosquitoes albopictus mosquitoes also contained this stable vertically transmitted core virome, as well as another set of viruses (e.g., viruses distantly related to Gua- deloupe mosquito virus, Hubei virga-like virus 2, and Sarawak virus) shared by mos- quitoes across different stages, which might represent an “environment-derived core virome.” To further study this core set of ISVs, we screened 48 publicly available SRA viral metagenomic data sets of mosquitoes belonging to the genus Aedes, showing that some of the identiﬁed ISVs were identiﬁed in the majority of SRAs and provid- ing further evidence supporting the core-virome concept. IMPORTANCE Our study revealed that the virome was very stable across all devel- opmental stages of both lab-derived and ﬁeld-collected Aedes albopictus. The data representing the core virome in lab A. albopictus proved the vertical transmission route of these viruses, forming a “vertically transmitted core virome.” Field mosqui- toes also contained this stable vertically transmitted core virome as well as addi- tional viruses, which probably represented “environment-derived core virome” and which therefore were less stable over time and geography. By further screening publicly available SRA viral metagenomic data sets from mosquitoes belonging to the genus Aedes, some of the identiﬁed core ISVs were shown to be present in the msystems.asm.org 1 September/October 2020 Volume 5 Issue 5 e00640-20 Shi et al. majority of SRAs, such as Phasi Charoen-like phasivirus and Guadeloupe mosquito vi- rus. How these core ISVs inﬂuence the biology of the mosquito host and arbovirus infection and evolution deserves to be further explored. KEYWORDS Aedes mosquito, Guadeloupe mosquito virus, Phasi Charoen-like phasivirus, core virome, environment-derived core virome, vertically transmitted core virome M o M osquitoes are highly diverse and widely disseminated. They occupy numerous biotopes and are potential vectors for several pathogenic arboviruses. Speciﬁ- cally, Aedes spp. pose a great threat to global public health. This is due to their traits of ecological plasticity that include egg diapause (1), versatility in using natural and/or urban breeding spots (2), and opportunistic feeding patterns (3, 4), which might have promoted their dispersion and adaptation to new, uncolonized territories that range from tropical to temperate regions (5). Moreover, a large number of pathogenic arboviruses, such as dengue virus (DENV), yellow fever virus (YFV), Zika virus (ZIKV), and chikungunya virus (CHIKV), are efﬁciently transmitted by Aedes mosquitoes, mainly A. aegypti and A. albopictus (6). Stability of the Virome in Lab- and Field-Collected Aedes albopictus Mosquitoes across Different Developmental Stages and Possible Core Viruses in the Publicly Available Virome Data of Aedes Mosquitoes In particular, DENV epidemics are a major public health concern in Guangdong Province of southern China, especially in its capital city, Guang- zhou (GZ). The dengue fever cases in Guangzhou represented 50% of the national incidence between 1978 and 2011 (7), and there was an explosive outbreak in 2014 with 45,224 reported dengue fever cases (8). A. albopictus is among the most invasive mosquitoes and is widely distributed in China. It is the main vector for DENV in China and the sole vector in Guangzhou (9, 10). on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from In light of the holobiont concept developed in recent years, mosquito-associated microbial communities play an important role in host biology, which may provide new strategies for mosquito and arbovirus control (11). Mosquitoes are holometabolous insects whose life cycle goes through four separate stages, including egg, larva, and pupa stages in an aquatic habitat and a subaerial adult stage. A continuum of bacteria from the aquatic environment to immature stages and adult mosquitoes has been suggested (12), but bacterial clearance occurs during mosquito metamorphosis from pupae to adults (13). In addition, the nutrients produced by symbiotic bacteria are very important for larval development (14) and larval bacteria can inﬂuence immune responses and vector competence in adult mosquitoes (15). However, little is known about the virome community dynamics in mosquitoes during holometabolous development. Some studies have performed viral metagenom- ics on ﬁeld mosquitoes collected from different countries (including the United States, Puerto Rico, China, Kenya, Australia, Sweden, etc.), mainly focusing on novel virus discovery (16–24). Some newly discovered mosquito-speciﬁc viruses (MSVs) have been proposed as future biological control agents against arboviruses (25–27) or as novel vaccine platforms (27) or used in diagnostic assays in chimeric virus formation with structural protein genes from arboviruses (27). In a recent study, we showed the presence of a relatively stable “core eukaryotic virome” in adult ﬁeld A. aegypti and Culex quinquefasciatus mosquitoes collected in Guadeloupe (28). However, the pres- ence of insect-speciﬁc viruses (ISVs) in mosquito lab colonies is poorly studied, which limits our understanding of their role in the development and physiology of mosqui- toes. September/October 2020 Volume 5 Issue 5 e00640-20 RESULTS Comparison of the eukaryotic viromes in lab- and ﬁeld-collected A. albopictus. Pools of eggs, larvae, pupae, and (male/female) adults from a lab colony in Wuhan, as well as pools of larvae, pupae, and adults from the ﬁeld in Guangzhou underwent metagenomic sequencing. Averages of 26.2 million trimmed reads were obtained per pool (Table 1) and were de novo assembled into 1,657,229 contigs in total. The clustering of 71,303 contigs longer than 500 bp from all samples at 95% nucleotide (nt) identity over 80% of the read length resulted in 56,419 representative contigs. Accord- ing to BLASTx annotation results obtained using DIAMOND, the majority (93%) of the representative contigs belonged to Eukaryota (mainly derived from the mosquito host genome) and 179 contigs were assigned as eukaryotic viruses. Eukaryotic viral reads in each pool occupied 0.2% to 1.8% of the trimmed reads, except for the 17-GZ-larva pool, which contained 15.8% viral reads. Most of the eukaryotic viral contigs belonged to 80 different viruses (including several viruses with segmented genomes), although some of these had very low similarities to known viruses in the database (Fig. 1A). No known pathogenic arboviruses were identiﬁed, and the closest relatives of the identiﬁed viruses were generally found in insects or plants. Only 20 of the 80 viral species belonged to an established viral genus or family (e.g., Flavivirus, Iﬂavirus, Phasivirus, Quaranjavirus, Rhabdoviridae, Virgaviridae, Totiviridae, Nodaviridae), whereas the re- maining viral genomes were most closely related to viruses discovered in recent years that current lack a formal taxonomical classiﬁcation. The heat map shown in Fig. 1A displays the number of reads mapped against each of the representative (partial) viral genomes as well as the percentage of coverage of each virus per sample (see Table S3 and Fig. S1 in the supplemental material). The virome in ﬁeld-collected A. albopictus from both 2017 and 2018 showed signiﬁcantly higher richness and diversity than that in lab-derived mosquito pools. The adjusted P values of the Wilcoxon test determined for the Chao1 and Shannon indices were 0.012 and 0.008, respectively (Fig. S1). The virome proﬁles of the different pools of lab colonies were relatively stable, except that several viral species were absent from or were present in low abundance in the larva pool (Fig. 1A). The ﬁeld-collected larva, pupa, and adult pools collected in GZ in 2018 and the adult pool collected in 2017 also displayed very similar viral communities. Stability of the Virome in Lab- and Field-Collected Aedes albopictus Mosquitoes across Different Developmental Stages and Possible Core Viruses in the Publicly Available Virome Data of Aedes Mosquitoes No known pathogenic arboviruses were identiﬁed, and the closest relatives of the identiﬁed viruses were generally found in insects or plants. Only 20 of the 80 viral species belonged to an established viral genus or family (e.g., Flavivirus, Iﬂavirus, Phasivirus, Quaranjavirus, Rhabdoviridae, Virgaviridae, Totiviridae, Nodaviridae), whereas the re- maining viral genomes were most closely related to viruses discovered in recent years that current lack a formal taxonomical classiﬁcation. The heat map shown in Fig. 1A displays the number of reads mapped against each of the representative (partial) viral genomes as well as the percentage of coverage of each virus per sample (see Table S3 and Fig. S1 in the supplemental material). The virome in ﬁeld-collected A. albopictus from both 2017 and 2018 showed signiﬁcantly hi h i h d di it th th t i l b d i d it l Th dj t d P TABLE 1 Aedes albopictus from China used for viral metagenomic sequencing Sample name Mosquito species Yr of collection Habitat Location No. of mosquitoes used for sequencing No. of trimmed reads obtained 17-Lab-egg A. albopictus 2017 Lab Wuhan, Hubei, China 200 23,801,510 17-Lab-larva A. albopictus 2017 Lab Wuhan, Hubei, China 250 22,990,930 17-Lab-pupa A. albopictus 2017 Lab Wuhan, Hubei, China 250 18,066,298 17-Lab-adultF A. albopictus 2017 Lab Wuhan, Hubei, China 250 20,109,482 17-Lab-adultM A. albopictus 2017 Lab Wuhan, Hubei, China 250 19,227,638 17-GZ-larva A. albopictus 2017 Field Liwan district, Guangzhou, China 2,000 29,564,646 17-GZ-adult A. albopictus 2017 Field Liwan district, Guangzhou, China 1,000 37,766,072 18-GZ-larva A. albopictus 2018 Field Liwan district, Guangzhou, China 2,000 35,580,984 18-GZ-pupa A. albopictus 2018 Field Liwan district, Guangzhou, China 500 25,744,126 18-GZ-adult A. albopictus 2018 Field Liwan district, Guangzhou, China 1,100 29,644,914 on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from TABLE 1 Aedes albopictus from China used for viral metagenomic sequencing Sample name Mosquito species Yr of collection Habitat Location No. of mosquitoes used for sequencing No. of trimmed reads obtained 17-Lab-egg A. albopictus 2017 Lab Wuhan, Hubei, China 200 23,801,510 17-Lab-larva A. albopictus 2017 Lab Wuhan, Hubei, China 250 22,990,930 17-Lab-pupa A. albopictus 2017 Lab Wuhan, Hubei, China 250 18,066,298 17-Lab-adultF A. albopictus 2017 Lab Wuhan, Hubei, China 250 20,109,482 17-Lab-adultM A. albopictus 2017 Lab Wuhan, Hubei, China 250 19,227,638 17-GZ-larva A. albopictus 2017 Field Liwan district, Guangzhou, China 2,000 29,564,646 17-GZ-adult A. albopictus 2017 Field Liwan district, Guangzhou, China 1,000 37,766,072 18-GZ-larva A. Stability of the Virome in Lab- and Field-Collected Aedes albopictus Mosquitoes across Different Developmental Stages and Possible Core Viruses in the Publicly Available Virome Data of Aedes Mosquitoes albopictus 2018 Field Liwan district, Guangzhou, China 2,000 35,580,984 18-GZ-pupa A. albopictus 2018 Field Liwan district, Guangzhou, China 500 25,744,126 18-GZ-adult A. albopictus 2018 Field Liwan district, Guangzhou, China 1,100 29,644,914 TABLE 1 Aedes albopictus from China used for viral metagenomic sequencing on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from mately 1,300 kilometers apart. Third, we conducted a comparative meta-analysis using comparisons between the viruses identiﬁed in this study and 48 publicly available virome SRA data sets of Aedes mosquitoes. Finally, phylogenetic analyses were per- formed on the following three selected viruses: Aedes phasmavirus (APV), which was highly present in both ﬁeld and lab A. albopictus from China; Guadeloupe mosquito virus (GMV)-related viruses; and Phasi Charoen-like phasivirus (PCLPV), which was found in the majority of investigated virome data sets. September/October 2020 Volume 5 Issue 5 e00640-20 Stability of the Virome in Lab- and Field-Collected Aedes albopictus Mosquitoes across Different Developmental Stages and Possible Core Viruses in the Publicly Available Virome Data of Aedes Mosquitoes On the other hand, knowledge of the normal healthy background virome as a reference will allow us to distinguish between inherent vertically transmitted compo- nents and components acquired from the environment of ﬁeld mosquitoes, which will enable the identiﬁcation of potential viral pathogens and improve the reliability of risk assessment using lab mosquitoes. The ﬁrst aim of this study was to analyze the virome structure of A. albopictus mosquitoes during distinct developmental stages in a laboratory-derived colony orig- inally obtained in Jiangsu Province, China. Second, we compared these lab colony- derived viromes with the viromes of ﬁeld-collected A. albopictus mosquitoes from Guangzhou (Guangdong Province, China), representing locations that are approxi- msystems.asm.org 2 September/October 2020 Volume 5 Issue 5 e00640-20 A Possible Conserved Core Virome in Aedes Mosquitoes mately 1,300 kilometers apart. Third, we conducted a comparative meta-analysis using comparisons between the viruses identiﬁed in this study and 48 publicly available virome SRA data sets of Aedes mosquitoes. Finally, phylogenetic analyses were per- formed on the following three selected viruses: Aedes phasmavirus (APV), which was highly present in both ﬁeld and lab A. albopictus from China; Guadeloupe mosquito virus (GMV)-related viruses; and Phasi Charoen-like phasivirus (PCLPV), which was found in the majority of investigated virome data sets. RESULTS Comparison of the eukaryotic viromes in lab- and ﬁeld-collected A. albopictus. Pools of eggs, larvae, pupae, and (male/female) adults from a lab colony in Wuhan, as well as pools of larvae, pupae, and adults from the ﬁeld in Guangzhou underwent metagenomic sequencing. Averages of 26.2 million trimmed reads were obtained per pool (Table 1) and were de novo assembled into 1,657,229 contigs in total. The clustering of 71,303 contigs longer than 500 bp from all samples at 95% nucleotide (nt) identity over 80% of the read length resulted in 56,419 representative contigs. Accord- ing to BLASTx annotation results obtained using DIAMOND, the majority (93%) of the representative contigs belonged to Eukaryota (mainly derived from the mosquito host genome) and 179 contigs were assigned as eukaryotic viruses. Eukaryotic viral reads in each pool occupied 0.2% to 1.8% of the trimmed reads, except for the 17-GZ-larva pool, which contained 15.8% viral reads. Most of the eukaryotic viral contigs belonged to 80 different viruses (including several viruses with segmented genomes), although some of these had very low similarities to known viruses in the database (Fig. 1A). msystems.asm.org RESULTS All 48 available sets of viral metagenomic data representing Aedes sp. from the public database (SRA) (see Table S1 in the supplemental material) were further analyzed to determine the conserved prevalence of ISVs in various Aedes mosquitoes. These samples were collected from locations on different continents, including the United States, Puerto Rico, Australia, Thailand, Guadeloupe, China, and Kenya. The mosquito species in the majority of the samples was A. aegypti, except for one sample from China (A. albopictus) and ﬁve from southwestern Australia (A. alboannulatus or A. camptorhynchus). In order to investigate the potential presence of a “core virome,” we analyzed the presence/absence of each virus across the geographic origins reﬂected in the available SRA entries. The number of viral species shared among different locations is displayed in Fig. 2A, and virus names corresponding to each overlapping category can be found in Table S5. Twenty viruses belonging to the families Totiviridae, Phenuiviridae, Ortho- myxoviridae, Xinmoviridae, Virgaviridae, Rhabdoviridae, Phasmaviridae, and Flaviviridae and six unclassiﬁed viruses (Chuvirus Mos8Chu0, Kaiowa virus, Hubei odonate virus 15, Guadeloupe mosquito virus, Humaita-Tubiacanga virus, Trichoplusia ni TED virus) were identiﬁed from at least ﬁve locations. Notably, the total number of viral species present in samples from the United States and Puerto Rico was much lower than that in samples from other locations, which could have been due to the presence of different viruses in these samples, less optimal VLP enrichment procedures used, and/or the lower sequencing depths. The presence or absence of each virus in each of the analyzed virome data sets is shown in the heat map in Fig. 2B. Only the 42 viral species present in a minimum of three locations and containing at least one contig longer than 1.5 kb are displayed in the ﬁgure, and they were grouped by collection locations and viral families. Totiviridae was the most prevalent viral family, containing two highly abundant species (Aedes aegypti totivirus and Australian Anopheles totivirus) with positivity rates across samples of 63.8% and 60.3%, respectively. Phasi Charoen-like phasivirus belonging to the Phenuiviridae was present in all four Aedes species and at all locations (except SRAs from the United States, in which only three of the investigated viruses were identiﬁed). Among the unclassiﬁed viral species, Chuvirus Mos8Chu0 virus and Kaiowa virus were present in 40 and 35 of 58 samples, respectively, and were found in all four Aedes species. RESULTS However, the larval pool collected in GZ in 2017 contained more than 30 additional unique viral species with almost 100% length msystems.asm.org 3 September/October 2020 Volume 5 Issue 5 e00640-20 Shi et al. on Oc http://msystems.asm.org/ Downloaded from Shi et al. FIG 1 Eukaryotic viral genomes in wild and lab-derived Aedes albopictus pools. (A) The heat maps show the total number of mapped reads on a log10 scale (upper panel) and length coverage (lower panel) of assembled contigs of each virus. The hierarchical clustering is based on the Bray-Curtis distance matrix (Continued on next page) Shi et al. September/October 2020 Volume 5 Issue 5 e00640-20 msystems.asm.org 4 on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from on October 13, 2020 at 26989832 asm.org/ FIG 1 Eukaryotic viral genomes in wild and lab-derived Aedes albopictus pools. (A) The heat maps show the total number of mapped reads on a log10 scale (upper panel) and length coverage (lower panel) of assembled contigs of each virus. The hierarchical clustering is based on the Bray-Curtis distance matrix (Continued on next page) September/October 2020 Volume 5 Issue 5 e00640-20 msystems.asm.org A Possible Conserved Core Virome in Aedes Mosquitoes coverage that were almost completely absent in the adults collected at the same time and in the same place. Furthermore, the lab and ﬁeld A. albopictus pools had 20 viral species in common. Among them, some contigs distantly related (47% BLASTx identity) to Yongsan bunyavirus 1 (YBV1) were present in all lab- and ﬁeld-derived mosquito pools with high abundance and length coverage (Fig. 1). Some viruses such as Yongsan tombus-like virus 1 and Phasi Charoen-like phasivirus appeared to be present at higher levels in the lab-derived A. albopictus samples. In contrast, reads of several viruses appeared to be present in higher numbers in the ﬁeld samples, as was the case for Guato virus and Guadeloupe mosquito mononega-like virus (Fig. 1A). Furthermore, ﬁeld mosquito pools contained many more viruses which were absent in lab-derived samples (Fig. 1A), as exempliﬁed by Wenzhou sobemo-like virus 4 and Hubei mosquito virus 2 (both with close relationship to Guadeloupe mosquito virus) (Fig. 1B). on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from Prevalence of viruses in public SRA virome data sets derived from Aedes mosquitoes. FIG 1 Legend (Continued) calculated from the number of log10 reads. The viral species names shown in the heat map are from the taxonomic annotation by DIAMOND and KronaTools. For each of the contigs assigned to a particular species, the open reading frame (ORF) with the highest BLASTx identity to a reference sequence was taken, and the median identity of these different ORFs is shown in the shaded green bar. (B) Relative abundances of the viral species in each pool based on the number of reads. September/October 2020 Volume 5 Issue 5 e00640-20 msystems.asm.org RESULTS Guadeloupe mosquito virus was detected in ﬁeld A. aegypti and A. albopictus mosquitoes from Puerto Rico, Thailand, Guadeloupe, Kenya, and China. Humaita- Tubiacanga virus (absent in lab-derived and wild A. albopictus from this study) was found in ﬁeld A. albopictus from Yunnan (China) and ﬁeld A. aegypti from 5 locations. g ( ) calculated from the number of log10 reads. The viral species names shown in the heat map are from the taxonomic annotation by DIAMOND and KronaTools. For each of the contigs assigned to a particular species, the open reading frame (ORF) with the highest BLASTx identity to a reference sequence was taken, and the median identity of these different ORFs is shown in the shaded green bar. (B) Relative abundances of the viral species in each pool based on the number of reads. September/October 2020 Volume 5 Issue 5 e00640-20 5 msystems.asm.org FIG 2 Conservation of viral species in Aedes sp. virome data sets. (A) The number of shared viral species among Aedes mosquitoes from different locations. (B) The viral species shown in the heat map present in samples from at least three locations and containing at least one contig that is longer than 1,500 bp. Shi et al. on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from Shi et al. http://msystems Downloaded from on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from FIG 2 Conservation of viral species in Aedes sp. virome data sets. (A) The number of shared viral species among Aedes mosquitoes from different locations. (B) The viral species shown in the heat map present in samples from at least three locations and containing at least one contig that is longer than 1,500 bp. The speciﬁc viral species was considered to be present in the sample as long as the sample had one contig (500 bp) assigned to the species. , g FIG 2 Conservation of viral species in Aedes sp. virome data sets. (A) The number of shared viral species among Aedes mosquitoes from different locations. (B) The viral species shown in the heat map present in samples from at least three locations and containing at least one contig that is longer than 1,500 bp. The speciﬁc viral species was considered to be present in the sample as long as the sample had one contig (500 bp) assigned to the species. on October 13, 2020 at 26989832 asm.org/ FIG 2 Conservation of viral species in Aedes sp. virome data sets. (A) The number of shared viral species among Aedes mosquitoes from different locations. (B) The viral species shown in the heat map present in samples from at least three locations and containing at least one contig that is longer than 1,500 bp. The speciﬁc viral species was considered to be present in the sample as long as the sample had one contig (500 bp) assigned to the species. September/October 2020 Volume 5 Issue 5 e00640-20 msystems.asm.org A Possible Conserved Core Virome in Aedes Mosquitoes TABLE 2 Distribution of APV-, PCLPV-, and GMV-related viruses Source Region Origin APV (related to YBV1) PCLPV GMV-related virus Lab China 2017-Wuhan-lab Belgium 2018-Belgium-lab cell line United Kingdom 2017-Bristol-lab cell line Field China 2016-Zhejiang 2017-Guangzhou 2018-Guangzhou Puerto Rico 2014-Puerto Rico Thailand 2008-Thailand 2015-Thailand Kenya 2018-Kenya1 2018-Kenya2 United States 2014-United States 2015-United States Australia 2014-Australia 2015-Australia 2016-Australia Korea 2016-Korea Guadeloupe 2016-Guadeloupe 2017-Guadeloupe Brazil 2012-Brazil TABLE 2 Distribution of APV-, PCLPV-, and GMV-related viruses on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from Phylogeny of three selected viruses. on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from Considering the abundance, variance, uni- versality, and number of complete coding genomes of viruses obtained, phylogenetic analysis was conducted on the following three selected viruses: (i) Aedes phasmavirus (APV; see below), a novel virus distantly related to YBV1 with high abundance in both ﬁeld and lab A. albopictus samples from China; (ii) Phasi Charoen-like phasivirus (PCLPV), present in all four Aedes species and all locations (except the United States) of screened virome data sets; and (iii) Guadeloupe mosquito virus (GMV), occupying the majority of reads in the ﬁeld-collected A. albopictus samples from Guangzhou, widely distributed in SRA data sets and showing a high level of variance. Complete coding viral genomes of APV, PCLPV, and GMV recovered from analyzed virome data sets of Aedes mosquitoes as well as corresponding reference genomes from GenBank were used for phylogeny. YBV1 is a newly identiﬁed virus reported in 2019 that showed high prevalence in A. vexans nipponii from Korea (29) and is classiﬁed in the family Phasmaviridae. We identiﬁed a novel virus, APV, in this family in both lab and ﬁeld A. albopictus samples from China whose closest reference was YBV1. PCLPV, a widely distributed mosquito virus, has been identiﬁed from lab colonies, mosquito cell lines, and wild Aedes mosquitoes in eight counties or regions (China, Puerto Rico, Thailand, Kenya, the United States, Australia, Guadeloupe, and Brazil) (Table 2). It has been suggested that it is maintained in nature through vertical transmission (30). A recent study has reported that GMV is closely related to Wenzhou sobemo-like virus 4 and Hubei mosquito virus 2 (28). GMV-related viruses were detected only in mosquito samples collected from the ﬁeld as listed in Table 2 (i.e., were not detected in the mosquito cell lines or lab colonies), indicating that it was likely acquired horizontally from the environment. Phylogeny of Aedes phasmavirus. In both lab- and ﬁeld-derived A. albopictus sequencing data from samples collected in China in 2017 and 2018, three contigs of APV in each sample were found to share the highest amino acid identities of 50.8%, 48.1%, and 43.6% with the S, M, and L segments of YBV1 (GenBank accession no. MH703047.1, MH703046.1, and MH703045.1), respectively. In total, 10 viral genomes of APV with three segments were recovered from all sequenced A. albopictus samples (17-lab-egg, 17-lab-larva, 17-lab-pupa, 17-lab-adultM, 17-lab-adultF, 17-GZ-larva, 17- GZ-adult, 18-GZ-larva, 18-GZ-pupa, and 18-GZ-adult). September/October 2020 Volume 5 Issue 5 e00640-20 on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from aegypti mosquitoes collected in Thailand in 2008, in the United States in 2015, in Australia in 2016, in Zhejiang Province of China in 2016, in Guadeloupe in 2017, and in Aag2 cells from the United Kingdom), and on two more genomes obtained from lab Aag2 cell lines in Belgium (Fig. 4). It was interesting that all of the PCLPVs collected in distant geographic locations, in different years, and from ﬁeld mosquitoes versus lab cell lines clustered very closely in the three maximum-likelihood (ML) trees of each segment. The nucleotide similarities among the genomes of PCLPVs were very high, ranging from 94% to 98% for the S segment, from 95% to 99% for M segment, and from 94% to 99% for L segment. on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from Phylogeny of Guadeloupe mosquito virus-related viruses. Twelve viral genomes (with complete coding regions) similar to GMV, Wenzhou sobemo-like virus 4, and Hubei mosquito virus 2 were obtained from the analyzed virome data sets, which included six pools of ﬁeld A. albopictus collected in China (17-GZ-larvae, 17-GZ-adult, 18-GZ-larva, 18-GZ-pupa, 18-GZ-adult, and SRR7204303-CN-2016), three pools of an A. aegypti and Cx. quinquefasciatus mixture collected in Puerto Rico (SRR3168916-PR-2014, SRR3168921-PR-2014, and SRR3168924-PR-2014), and two A. aegypti pools collected in Kenya (SRR12102797-KE-2018 and SRR12102798-KE-2018) and one collected in Thai- land (SRR6155879-TH-2015). The viral genomes contained two segments encoding an RdRp and one hypothetical protein on segment 1 (1,500 to 1,600 nt) and a capsid and one hypothetical protein encoded by segment 2 (3,000 to 3,200 nt). GMV, Wenzhou sobemo-like virus 4, and Hubei mosquito virus 2 are all newly described and currently unclassiﬁed viruses with a distant relationship to the families Luteoviridae and Sobe- moviridae (28). The phylogenetic analysis performed on the basis of segments 1 and 2 indicated that these sequences fell into three clades (Fig. 5). The genomes identiﬁed in samples collected in Thailand in 2015, Puerto Rico in 2014, and Kenya in 2018 clustered together with the Guadeloupe mosquito viruses found in Guadeloupe in 2016 and 2017, forming clade 1, with the nucleotide identity in this clade ranging from 90% to 99% for both segments. Five genomes recovered from A. albopictus collected in Guangzhou in 2017 and 2018, one recovered from the same host collected in Yunnan in 2016, and Wenzhou sobemo-like virus 4 clustered together in clade 2, with nucleotide identities among them ranging from 94% to 98%. on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from The longest lengths were of 1,185, 2,022, and 6,468 nt, which corresponded to the nucleocapsid, glycoprotein, and RNA-dependent RNA polymerase (RdRp) genes, respectively. For all three segments, the nucleotide identity among the 10 assembled viral genomes ranged between 96% and Phylogeny of Aedes phasmavirus. In both lab- and ﬁeld-derived A. albopictus sequencing data from samples collected in China in 2017 and 2018, three contigs of APV in each sample were found to share the highest amino acid identities of 50.8%, 48.1%, and 43.6% with the S, M, and L segments of YBV1 (GenBank accession no. MH703047.1, MH703046.1, and MH703045.1), respectively. In total, 10 viral genomes of APV with three segments were recovered from all sequenced A. albopictus samples (17-lab-egg, 17-lab-larva, 17-lab-pupa, 17-lab-adultM, 17-lab-adultF, 17-GZ-larva, 17- GZ-adult, 18-GZ-larva, 18-GZ-pupa, and 18-GZ-adult). The longest lengths were of 1,185, 2,022, and 6,468 nt, which corresponded to the nucleocapsid, glycoprotein, and RNA-dependent RNA polymerase (RdRp) genes, respectively. For all three segments, the nucleotide identity among the 10 assembled viral genomes ranged between 96% and September/October 2020 Volume 5 Issue 5 e00640-20 msystems.asm.org 7 Shi et al. 100%, which indicated that they represent closely related variants. In a separate phylogenetic analysis of the three segments, the 10 viral genomes of APV clustered together within the genus Orthophasmavirus in the family Phasmaviridae (Fig. 3). Their closest relative was YBV1, which was identiﬁed in A. vexans mosquitoes collected in South Korea in 2016, but with low levels of nucleotide similarity (ranging from 52% to 57%) in the coding regions of the three segments. Thus, the APVs present in the lab and ﬁeld A. albopictus samples collected in China appeared to represent a novel species in the genus Orthophasmavirus. Phylogeny of Phasi Charoen-like phasivirus. Since the abundances of PCLPV reads in the A. albopictus samples from Guangzhou and lab-derived mosquitoes were relatively low, no complete genome was recovered from these pools. All three seg- ments of PCLPV with a complete coding region were successfully recovered in one A. aegypti sample from Puerto Rico collected in 2014 and two A. aegypti samples from Kenya collected in 2018. The phylogenetic analysis was performed on these newly identiﬁed PCLPVs, on all other PCLPV genomes in GenBank (including those identiﬁed in A. on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from The third clade consisted of Hubei mosquito virus 2 and one sequence from a sample collected in Kenya in 2018, with nucleotide identities ranging from 70% to 100%. The nucleotide similarities for the two segments among these three clades ranged from 54% to 69%. September/October 2020 Volume 5 Issue 5 e00640-20 msystems.asm.org 10 DISCUSSION on October 13 http://msystems.asm.org/ Downloaded from on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from FIG 5 Maximum-likelihood phylogenetic tree based on the nucleotide sequence of the complete coding regions in segments 1 and 2 of Guadeloupe mosquito virus (GMV)-related viruses. The red, blue, yellow, brown, and green squares after the sequence names highlight the strains from Guadeloupe, Puerto Rico, Thailand, Kenya, and China, respectively. Motts Mill virus identiﬁed from ticks was used as the outgroup. that the rearing conditions were stable in the lab and that the colony has been reared in the lab for many years, we collected lab-derived samples only in 2017 as no scientiﬁcally signiﬁcant changes over time were anticipated. To take potential yearly virome ﬂuctuations for ﬁeld mosquitoes into account, they were collected in two consecutive years (2017 and 2018). The proportion of the eukaryotic viral reads in each sample (ranging from 0.2% to 15.8%) was comparable to that reported from previous mosquito virome studies (24, 28), suggesting that the sequencing depth was sufﬁcient for virome analysis. As expected, the virome diversity in lab A. albopictus was lower than that in ﬁeld mosquitoes (Fig. 1), probably due to the less complex environment and the availability of clean water and food resources. Unlike the bacterial communities seen in ﬁeld- collected mosquitoes (13, 31–35), it is interesting that the virome in lab-derived A. albopictus was very stable across all developmental stages, consistent with vertical transmission of these viruses. For the larvae only, relatively few viral reads were identiﬁed. This ﬁnding suggested that the virus might remain dormant at a very low concentration without or with very low rates of replication. The fact that these viruses were also present in ﬁeld mosquitoes suggested that A. albopictus seems to contain a “vertically transmitted core virome” as was described before for A. aegypti and Culex quinquefasciatus from Guadeloupe (28), for which stable distinct core eukaryotic vi- romes were identiﬁed which possessed nearly identical viruses across time and space (28). In addition, another set of viruses was found to be shared by the ﬁeld-collected A. albopictus mosquitoes across different stages, suggesting that they have a “core virome” with higher richness and diversity (Fig. S1). Whether these additional viruses were acquired from the environment or the lab-derived mosquitoes lost these viruses in captivity remains to be determined. All these vertically transmitted core viromes in A. DISCUSSION In this study, we ﬁrst characterized the eukaryotic virome across different develop- mental stages of lab-derived and ﬁeld-collected A. albopictus from China. Five pools from lab mosquitoes (containing 1,250 individuals in total) and ﬁve pools from ﬁeld- collected mosquitoes (6,600 individuals in total) were analyzed (Table 1). Considering msystems.asm.org 8 September/October 2020 Volume 5 Issue 5 e00640-20 A Possible Conserved Core Virome in Aedes Mosquitoes FIG 3 Maximum-likelihood phylogenetic tree based on the nucleotide level of complete coding region in the S, M, and L segments of the newly found Aedes phasmaviruses (APVs) in Aedes mosquitoes from China (highlighted with green squares) and other representative members in the family Phasmaviriade. Representatives in the genus Orthobunyavirus belonging to the family Peribunyaviridae were used as the outgroup. September/October 2020 Volume 5 Issue 5 e00640-20 on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from FIG 3 Maximum-likelihood phylogenetic tree based on the nucleotide level of complete coding region in the S, M, and L segments of the newly found Aedes phasmaviruses (APVs) in Aedes mosquitoes from China (highlighted with green squares) and other representative members in the family Phasmaviriade. Representatives in the genus Orthobunyavirus belonging to the family Peribunyaviridae were used as the outgroup. September/October 2020 Volume 5 Issue 5 e00640-20 msystems.asm.org 9 Shi et al. G 4 Maximum-likelihood phylogenetic tree based on the nucleotide sequence of complete co gions of the S, M, and L segments of Phasi Charoen-like phasivirus (PCLPV) identiﬁed from A (Continued on next p ptember/October 2020 Volume 5 Issue 5 e00640-20 on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from FIG 4 Maximum-likelihood phylogenetic tree based on the nucleotide sequence of complete coding regions of the S, M, and L segments of Phasi Charoen-like phasivirus (PCLPV) identiﬁed from Aedes (Continued on next page) msystems.asm.org 10 msystems.asm.org 10 September/October 2020 Volume 5 Issue 5 e00640-20 September/October 2020 Volume 5 Issue 5 e00640-20 A Possible Conserved Core Virome in Aedes Mosquitoes FIG 5 Maximum-likelihood phylogenetic tree based on the nucleotide sequence of the complete coding regions in segments 1 and 2 of Guadeloupe mosquito virus (GMV)-related viruses. The red, blue, yellow, brown, and green squares after the sequence names highlight the strains from Guadeloupe, Puerto Rico, Thailand, Kenya, and China, respectively. Motts Mill virus identiﬁed from ticks was used as the outgroup. September/October 2020 Volume 5 Issue 5 e00640-20 mosquitoes and cells, together with other representative members of the family Phenuiviriade. The strains from Guadeloupe are highlighted with red squares, strains derived from Belgian Aag2 cells with black diamonds, strains from Kenya with brown squares, and strains from Puerto Rico with blue squares. Representative Orthonairovirus strains belonging to the family Nairoviridae were used as the outgroup. DISCUSSION albopictus deserve more attention with respect to their effects on vector competence for important medically relevant arboviruses. Due to the mosquito samples having originated from only one lab colony or one location in the ﬁeld, further surveillance over a larger geographic range and longer periods of time will be needed to conﬁrm the stability of the virome proﬁle across different developmental stages. In addition, the larval pool collected in the wild in 2017 was an outlier, as it contained many unique msystems.asm.org 11 FIG 4 Legend (Continued) FIG 4 Legend (Continued) mosquitoes and cells, together with other representative members of the family Phenuiviriade. The strains from Guadeloupe are highlighted with red squares, strains derived from Belgian Aag2 cells with black diamonds, strains from Kenya with brown squares, and strains from Puerto Rico with blue squares. Representative Orthonairovirus strains belonging to the family Nairoviridae were used as the outgroup. September/October 2020 Volume 5 Issue 5 e00640-20 msystems.asm.org 11 Shi et al. viruses, which probably originated from the particular water environment they lived in, which could have been contaminated by other coinhabiting insects or larvae. Alterna- tively, it could be the case that a number of mosquitoes other than A. albopictus were included in the pool by accident. These results further suggest a signiﬁcant inﬂuence of the breeding site ecology on the mosquito viral community. Since some viruses (e.g., PCLPV, GMV, and Aedes aegypti totivirus) identiﬁed in A. albopictus from China were also identiﬁed as part of the core virome in A. aegypti from Guadeloupe (28), we were interested in determining how these core viruses in Aedes mosquitoes were further distributed around the world. Therefore, we explored 48 Aedes sp. viral metagenomic data sets from a public database (SRA). The samples were from different countries on different continents (China, the United States, Australia, Kenya, Thailand, Puerto Rico, and Guadeloupe) and from different mosquito species (A. aegypti, A. albopictus, A. alboannulatus, and A. camptorhynchus) and were treated with different wet-lab and sequencing procedures using different amounts of pooled mosquitoes and sequencing depths, but a highly prevalent set of widely distributed viruses, such as Totiviridae, Orthomyxoviridae, Xinmoviridae, Flaviviridae, PCLPV, and GMV, were able to be identiﬁed on the family or species level (Fig. 2). How these conserved viruses in Aedes mosquitoes interact with or inﬂuence an arbovirus infection is an interesting point for further studies. A previous study explored the effect of coinfections of PCLPV and cell-fusing agent virus (CFAV) on the replication of arboviruses in cell line Aa23 derived from A. albopictus (36). CFAV-PCLPV-positive Aa23 cells strongly inhibited the growth of two ﬂaviviruses (ZIKV and DENV) and completely blocked infection by La Crosse virus (bunyavirus). Although the exact blocking mechanism was not known, on the one hand, the results suggested that the data generated from laboratory cell lines persistently infected by mosquito-speciﬁc viruses (MSVs) should be interpreted with caution. FIG 4 Legend (Continued) On the other hand, the intra-MSV interactions need to be considered when the inﬂuence of MSVs on vector competence is explored. on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from Although many viruses were very prevalent in Aedes mosquitoes, such as Chuvirus Mos8Chu0, Kaiowa virus, Wuhan mosquito virus 6, Whidbey virus, Aedes aegypti toti- virus, and Australian Anopheles totivirus, phylogenetic analysis was further performed on GMV and PCLPV, which had the greatest number of complete coding genomes, and on APV, which was highly abundant in both ﬁeld and lab A. albopictus from China. APV was found to be distantly related to the YBV1 identiﬁed from A. vexans from South Korea forming a separate clade. All the Chinese strains from both lab- and ﬁeld- collected mosquitoes were very similar (Fig. 3). Also, for PCLPV, the genomes found in samples from different years, locations, and habitats were very similar for all three segments (Fig. 4). Interactions between bunyaviruses and arthropods occurring over 20 million years had been previously demonstrated (37–41). The ﬁndings indicating that APV and PCLPV seem to be very closely related were puzzling and might suggest a rather relatively recent spread of this virus or a very low evolutionary rate, which would be unexpected for RNA viruses. However, the effects of these viruses on their host are very poorly understood. A recent study revealed that preexisting infection of Aag2 cells with PCLPV did not affect the infection and growth of the mosquito-borne viruses in genus Flavivirus, Alphavirus, and Rhabdovirus in cell culture (42). GMV is a recently described two-segmented, currently unclassiﬁed virus. Three separate clades were observed for GMVs and GMV-related viruses, largely clustering according to location. This group of viruses should be proposed as a new family, and their role in arbovirus infection needs to be further studied. In summary, our results reveal that the virome proﬁle was very stable across different stages in both lab- and ﬁeld-derived Aedes albopictus and that a number of possible core viruses exist in Aedes mosquitoes of at least four species in multiple locations in seven countries. Since the number of available Aedes virome data sets is still rather limited and since wet-lab procedures, sequencing depths, and pool sizes differed greatly among the analyzed data sets, the core viruses need to be further conﬁrmed by next-generation sequencing (NGS) or PCR. September/October 2020 Volume 5 Issue 5 e00640-20 msystems.asm.org 12 MATERIALS AND METHODS Sample collection. An A. albopictus colony was established in our laboratory (Wuhan, China) in 2017 which originated from a stable colony from the National Institute for Communicable Disease Control and Prevention (China CDC; Beijing, China). The adult mosquitoes were maintained at 27 to 30°C with 60% to 85% relative humidity using a photoperiod of a 12-h:12-h light-dark cycle. The larvae were fed with a mixture of yeast powder and wheat mill. Adult mosquitoes were placed into cages (30 cm by 30 cm by 30 cm), and the males were fed with 10% sucrose solution, while the females were fed with blood from mice. Egg, larva, pupa, and male and female adult samples of lab colony mosquitoes were collected in August 2017 (Table 1). on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from Field larva, pupa, and adult samples of A. albopictus were trapped in Guangzhou (Liwan district), Guangdong Province, China, in July to November of 2017 and 2018. In Kenya, adult mosquitoes of A. aegypti were trapped during July and August of 2018 in Ukunda and Kisumu. All samples were collected from residential quarters in urban areas, transported to the laboratory using an appropriate cold chain, and stored at 80°C. Mosquito species were determined by morphological identiﬁcation, and samples were assigned into pools according to the date of collection (Table 1). Sample preparation for NGS. The collected mosquitoes were pooled according to developmental stage for sequencing. The pooled samples were triturated by the use of a Tgrinder OSE-Y30 electric tissue grinder (Tiangen, China) on ice using sterile pestles with 200 l of RPMI medium. Mosquito homogenates were clariﬁed by centrifugation at 20,000 g (4°C for 30 min) and ﬁltered through a 0.45-m-pore-size membrane ﬁlter (Millipore, Billerica, USA). Supernatants were collected and stored at 80°C. RNA was extracted from the supernatants with an RNeasy minikit (Qiagen, Germany) according to the manufac- turer’s instructions. Then, strand-speciﬁc libraries were prepared using a TruSeq stranded total RNA sample preparation kit (Illumina, USA). TruSeq PE (paired-end) cluster kit v3 (Illumina, USA) and TruSeq SBS kit v3-HS (Illumina, USA) (300 cycles) were used for sequencing with 150 bp per read (PE 2 150 bp), which was performed on an Illumina HiSeq 2500 platform (Illumina, USA) by Shanghai Biotechnology Corporation. Bioinformatic analysis of viral metagenomic data. MATERIALS AND METHODS The obtained raw paired-end reads were trimmed for quality and adapters using Trimmomatic (43), and the remaining reads were de novo assembled into contigs using metaSPAdes (44). Contigs from all pools longer than 500 bp were ﬁltered for redundancy at 95% nucleotide identity over 80% of the length using ClusterGenomes (45). The collection of representative contigs was classiﬁed using DIAMOND (46) against the nr database (updated 29 September 2019) using the sensitive mode for taxonomic annotation. KronaTools (47) was used to parse the output ﬁle of DIAMOND, which was used to ﬁnd the least common ancestor of the best 25 DIAMOND hits (based on BLASTx score) for each contig. All contigs annotated as eukaryotic viruses were extracted using an in-house Python script. The abundance and length coverage of eukaryotic virus contigs in individual pools were obtained by aligning trimmed and decontaminated reads of each sample to the collection of representative contigs using BBMap (48). Abundance tables were extracted from eukaryotic viruses and further used for making heat maps in R with the ComplexHeatmap (49), ggplot2 (50), and phyloseq (51) packages. The length coverage of each viral species per sample was calculated by the following formula: contig length covered by at least one read/the total length of the corresponding contig. Eukaryotic virus screening of Aedes mosquito virome data in SRA. To investigate the level of conservation of the eukaryotic core viruses identiﬁed in Chinese samples from this study with those of other Aedes mosquitoes worldwide, we retrieved 48 published SRA data sets (including 25 data sets of Aedes sp. from Guadeloupe 28], 8 from Puerto Rico [16], 4 from the United States [16], 7 from Australia [52, 53], 2 from Kenya, 1 from Thailand [53], and 1 from China [54]) (Fig. 6; see also Table S1 in the supplemental material). The world map displaying the geographic origin of all Aedes virome data sets used in this study was drawn with ArcGI (ArcMap 10.5). The raw reads of the SRA data sets were de novo assembled using SKESA (55) with default settings, which was less computationally intensive than metaSPAdes. These obtained contigs were taxonomically annotated using DIAMOND against the nr database (updated 29 September 2019) in sensitive mode. KronaTools (47) and an in-house Python script were used to parse the output ﬁle of DIAMOND and extract eukaryotic virus contigs. The presence of eukaryotic viruses in all Aedes virome data sets. FIG 4 Legend (Continued) To fully characterize and understand the genetic and phenotypic diversity of mosquito-speciﬁc viruses, samples from individual msystems.asm.org 12 A Possible Conserved Core Virome in Aedes Mosquitoes Aedes mosquitoes of additional species collected from additional locations and at additional time points, processed and sequenced with the same method, should be analyzed. September/October 2020 Volume 5 Issue 5 e00640-20 msystems.asm.org 13 Shi et al. on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from FIG 6 Global distribution of Aedes mosquitoes virome data sets used in this study. on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from on October 13, 2020 at 26989832 http://msystems.asm.org/ Downloaded from FIG 6 Global distribution of Aedes mosquitoes virome data sets used in this study. FIG 6 Global distribution of Aedes mosquitoes virome data sets used in this study. PCLPV; and (iii) 18-GZ-larva-seg1 (MT361057) and 18-GZ-pupa-seg2 (MT361060) for GMV. The consensus sequences of these viruses were generated from the bam ﬁles using SAMtools and bcftools (58). PCLPV; and (iii) 18-GZ-larva-seg1 (MT361057) and 18-GZ-pupa-seg2 (MT361060) for GMV. The consensus sequences of these viruses were generated from the bam ﬁles using SAMtools and bcftools (58). The nucleotide sequences of the complete genomes or complete coding regions in the genomes (segments) of these viruses were used to determine their evolutionary history together with appropriate reference strains from GenBank. Alignments of the sequences were performed with MAFFT v7.222 (59) using the most accurate algorithm, L-INS-I, with 1,000 cycles of iterative reﬁnement. Ambiguously aligned regions were removed by trimAl v1.2 (60) using an automated trimming heuristic, which was optimized for maximum-likelihood (ML) phylogenetic tree reconstruction. The phylogenetic trees for each segment were reconstructed from 1,000 ultrafast bootstrap ML tree replicates using IQ-TREE v1.6.11 (61) with best-ﬁt model selection by ModelFinder (62). FigTree v1.4.3 (63) was used for phylogenetic tree visualization. Data availability. Accession numbers of the viruses obtained from our data set are listed in Table S2, and viral genome sequences recovered from the SRA data sets can be found at https://github.com/ Matthijnssenslab/AedesVirome. All viral sequences used to construct phylogenetic trees can be found at https://github.com/Matthijnssenslab/AedesVirome. SUPPLEMENTAL MATERIAL Supplemental material is available online only. FIG S1, PDF ﬁle, 0.2 MB. TABLE S1, XLSX ﬁle, 0.01 MB. TABLE S2, XLSX ﬁle, 0.01 MB. TABLE S3, XLSX ﬁle, 0.01 MB. TABLE S4, XLSX ﬁle, 0.01 MB. TABLE S5, CSV ﬁle, 0 MB. msystems.asm.org 14 MATERIALS AND METHODS Taxonomically identiﬁed eukaryotic virus contigs longer than 500 bp were extracted from all 58 Aedes virome data sets and placed together in a single ﬁle. Viral species that contained at least one contig longer than 1,500 bp were used for visualization purposes. A viral species was considered present in the sample as long as the sample had one contig (500 bp) assigned to the species. 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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of the People’s Republic of China (2018ZX10101004); the National Health Commission of the People’s Republic of China (2018ZX10711001-006); the Chinese Academy of Sciences (153211KYSB20160001); the Wuhan Institute of Virology, China (WIV-135-PY2); and the Health Commission of Hubei Province (WJ2019Q060). C.S. performed the bioinformatics analysis of NGS data with support from J.M. L.Z., H.X., and A.E. designed the experiments, conducted the wet lab experiments, and msystems.asm.org 14 September/October 2020 Volume 5 Issue 5 e00640-20 A Possible Conserved Core Virome in Aedes Mosquitoes performed the phylogenetic analysis. W.Z. collected the ﬁeld mosquito samples. C. and H.X. drafted the manuscript. L.Z., A.E., X.H., J.M., and Z.Y. reviewed the manuscrip performed the phylogenetic analysis. W.Z. collected the ﬁeld mosquito samples. C.S. and H.X. drafted the manuscript. 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https://openalex.org/W4380563235	https://zenodo.org/records/8037180/files/TAFPS1317.pdf	Swahili	null	3- SINF ONA TILI OʻQITISH SAVODXONLIGI DARSLIGIDAGI "KULOL CHOLNING HIYLASI" MAVZUSIDAGI HIKOYANING BOSHLANGʻICH SINF OʻQUVCHILARI UCHUN TARBIYAVIY AHAMIYATI	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,430	THEORETICAL ASPECTS IN THE FORMATION OF PEDAGOGICAL SCIENCES International scientific-online conference Taʼlimda texnologiyalardan foydalanish hamda ona-tili taʼlimda hamkorlikda oʻqitish texnologiyalari, modulli oʻqitish texnologiyalaridan unumli foydalanib, tanqidiy tahlil qilish va uning tarbiyaviy ahamiyatlarini kundalik hayotga, talabalar, oʻquvchilar, yoshlar hayotiga tadbiq etish masalalari ham hikoyaning ahamiyatliligini, dolzarbligini oshirib beradi. ,,Kulol cholning hiylasiʼʼ nomli hikoyada keltirilishicha atrofi suvliklar bilan oʻralgan orolliklarning hiyla- nayranglarga uchishi va oʻz yerlarining muqaddas ekanligini unutgan holatda kulolchilik buyumlarini yasashlari natijasida yurtlarining suv bosib ketishi kuzayilgan. Ushbu holatlarda ham qanday chora va vaziyatlar koʻrish borasida tarbiyaviy hamiyat koʻzga tashlanadi. Oʻqituvchi taʼlim jarayonida pedagogik innovatsion texnologiyalaridan, axborot kommunikatsiyalaridan foydalanar ekan darsning ilmiyligi, tushunarlilik darajasini oshirish uchun turli xil ko rgazmali qurollardan foydalanadi. Misol uchun: “Kulol cholning hiylasi” hikoyasini tahlil etish uchun avvalo, tayyorlangan videoroliklar yoki slayt kabi mahsulotlardan foydalanish oʻquvchilarning axborot texnologiyalari bilan qiziqishlarini oshirish hamda hikoyaning tub maqsadi - vatanparvarlik, vatanni sevish, vatanni har bir neʼmatini asrash tamoyillarni oʻquvchi yoshlarning qalbiga jo qilish kabi masalalar yotadi. Ushbu hikoyaning tarbiyaviy jihati shundan iboratki, har qanday sinov va ijtimoiy vaziyatlar nuqtai negizidan vatanni himoya qilish, uni asrab-avaylash yovlarning hujumlari, hiyla nayranglariga uchmasdan vatan tuprogʻini koʻzga turish lozim ekanligi anglashiladi. Toʻrt tomoni suvdan iborat boʻlgan orolliklar vatan tuproqlaridan yasagan koʻzalaei va ularning ortidan orttirgan boyliklari natijasida sahnadan arzimagan bir yilni sotib olib muqaddasi yillarni ajralgandan soʻng, oddiy bir sahroda yashar, vatanning qadriga etishadi. Ushbu hikoyaning tarbiyaviy maqsadi vatani asrab-avaylash, vatanga boʻlgan muhabbat va ushbu jarayonidan vatanga hissa qoʻshish kabi tushunchalarni ochib berishda yordam beradi. Taʼlim berish va oʻqitish turlarining turli xususiyatlari mavjud boʻlib, yaʼni hikoyalar bilan ishlash texnologiyasi ham tarbiyaviy jihatdan quyidagicha dolzarbligini koʻrsatadi. Oʻquvchi-yoshlarda hikoya natijasida kuzatuvchanlik va darsda oʻquvchilarning asar qahramonlari bilan oʻzaro intensiv sifatlarini shakllantirish ularning ijobiy va salbiy qahramon ekanliklarini tasvirlash kabilar ham yuzaga keladi. “50-90-yillar mobaynida boshlangʻich sinflarda ona tili oʻqitish metodikasi sohasida anchagina qoʻllanmalar yaratildi. Bu yillar mobaynida ona tili oʻqitish metodikasi fan sifatida rivojlana boshladi, umumiy pedagogik, didaktik va psixologik xarakterdagi ilmiy tekshirishlarning natijalari Taʼlimda texnologiyalardan foydalanish hamda ona-tili taʼlimda hamkorlikda oʻqitish texnologiyalari, modulli oʻqitish texnologiyalaridan unumli foydalanib, tanqidiy tahlil qilish va uning tarbiyaviy ahamiyatlarini kundalik hayotga, talabalar, oʻquvchilar, yoshlar hayotiga tadbiq etish masalalari ham hikoyaning ahamiyatliligini, dolzarbligini oshirib beradi. ,,Kulol cholning hiylasiʼʼ nomli hikoyada keltirilishicha atrofi suvliklar bilan oʻralgan orolliklarning hiyla- nayranglarga uchishi va oʻz yerlarining muqaddas ekanligini unutgan holatda kulolchilik buyumlarini yasashlari natijasida yurtlarining suv bosib ketishi kuzayilgan. Ushbu holatlarda ham qanday chora va vaziyatlar koʻrish borasida tarbiyaviy hamiyat koʻzga tashlanadi. 3- SINF ONA TILI OʻQITISH SAVODXONLIGI DARSLIGIDAGI “KULOL CHOLNING HIYLASI” MAVZUSIDAGI HIKOYANING BOSHLANGʻICH SINF OʻQUVCHILARI UCHUN TARBIYAVIY AHAMIYATI Olimboyeva Malohat Qudrat qizi Urganch davlat universiteti Boshlang‘ich ta’lim yo‘nalishi 2-bosqich talabasi https://doi.org/10.5281/zenodo.8037180 ANNOTATSIYA: Ushbu maqolada taʼlim tizimini isloh qilish jarayonida amalga oshiriladigan ishlar va ularning ahamiyati albatta, oliy taʼlim muassasalari, maktab taʼlimi va maktabgacha taʼlimning oʻziga xos xususiyatlarini ochib berishga yordam beradi. Bunda, oʻqituvchilarning kompetentligidan foydalanishi ularni adabiyot oʻqitish metodikasining mazmun- mohiyati ona tili oʻqitish borasidagi uslub va xususiyatlarini oʻzlarida shakllantirishlarida katta yordam beradi. Shuningdek, maktab oʻquvchilari uchun darsliklarda keltirilgan hikoyalar, qissalarning mazmun-mohiyatini ochib berish, ularda tarbiyaviy oʻziga xoslikni shakllantirishning mazmun-mohiyati ham taʼlim jarayonining bir ajralmas qismini tashkil etadi. KALIT SO‘ZLAR: Hikoya elementlari, estetik zavq, modulli ta’lim texnologiyasi, axborot kommunikatsiyalari, fanlararo integratsiya, hikoyachilik. KALIT SO ZLAR: Hikoya elementlari, estetik zavq, modulli ta lim texnologiyasi, axborot kommunikatsiyalari, fanlararo integratsiya, hikoyachilik. Boshlangʻich sinf oʻquvchilarida hikoyachilik va hikoyachilikka boʻlgan tarbiyaviy ahamiyatning oʻzga xosligini ochib berishda muhim dasturiy elementlardan foydalanib, ularning kundalik hayoti kelib chiqish voqealar rivoji va voqealar, yechimning didaktik jihatdan oʻyinlar bilan voqealar bilan ahamiyatliligi ham tahlil jarayonida yuzaga keladi. Uchinchi sinf ona tili oʻqitish savodxonligining darslikdagi. “Kulol cholning hiylasi” mavzusida hikoyaning boshlangich sinf oʻquvchilari uchun tarbiyaviy ahamiyatga katta. Shuningdek, hikoyaning tahlil qilinish metodlari, usullari shakllari, oʻzaro chambarchastlikni tashkil etadi voqealar rivojining qanday yoʻsinda tashkil etilishi oʻquvchilar tomonidan qayta hikoya qilish usulida tenglanishi va ulardan bilimlarning salohiyat darajasida shakllanishida yordam beradi. “Har bir fan oʻzining oʻganish obyekti va predmetiga ega boʻlganidek, hozirgi kunda boshlangʻich sinflarda oʻqish metodikasi adabiy taʼlim metodikasi sifatida ish koʻrishi lozimligi gʻoyasi ilgari surilmoqda. Haqiqatan ham, adabiyot oʻqitish sistematik kursiga tayyorlovchi predmet sifatida oʻqish metodikasi bolalar adabiyoti namunalari asosida oʻz maqsad va vazifalarini belgilaydi. Shundan kelib chiqqan holda boshlangʻich sinfda oʻqish darslarining oʻrganish obyekti – oʻquvchilarning adabiy taʼlimni egallash jarayoni deb belgilanishi maqsadga muvofiqdir”.[1] 80 THEORETICAL ASPECTS IN THE FORMATION OF PEDAGOGICAL SCIENCES International scientific-online conference Oʻqituvchi taʼlim jarayonida pedagogik innovatsion texnologiyalaridan, axborot kommunikatsiyalaridan foydalanar ekan darsning ilmiyligi, tushunarlilik darajasini oshirish uchun turli xil ko rgazmali qurollardan foydalanadi. Misol uchun: “Kulol cholning hiylasi” hikoyasini tahlil etish uchun avvalo, tayyorlangan videoroliklar yoki slayt kabi mahsulotlardan foydalanish oʻquvchilarning axborot texnologiyalari bilan qiziqishlarini oshirish hamda hikoyaning tub maqsadi - vatanparvarlik, vatanni sevish, vatanni har bir neʼmatini asrash tamoyillarni oʻquvchi yoshlarning qalbiga jo qilish kabi masalalar yotadi. Ushbu hikoyaning tarbiyaviy jihati shundan iboratki, har qanday sinov va ijtimoiy vaziyatlar nuqtai negizidan vatanni himoya qilish, uni asrab-avaylash yovlarning hujumlari, hiyla nayranglariga uchmasdan vatan tuprogʻini koʻzga turish lozim ekanligi anglashiladi. Toʻrt tomoni suvdan iborat boʻlgan orolliklar vatan tuproqlaridan yasagan koʻzalaei va ularning ortidan orttirgan boyliklari natijasida sahnadan arzimagan bir yilni sotib olib muqaddasi yillarni ajralgandan soʻng, oddiy bir sahroda yashar, vatanning qadriga etishadi. Ushbu hikoyaning tarbiyaviy maqsadi vatani asrab-avaylash, vatanga boʻlgan muhabbat va ushbu jarayonidan vatanga hissa qoʻshish kabi tushunchalarni ochib berishda yordam beradi. Taʼlim berish va oʻqitish turlarining turli xususiyatlari mavjud boʻlib, yaʼni hikoyalar bilan ishlash texnologiyasi ham tarbiyaviy jihatdan quyidagicha dolzarbligini koʻrsatadi. Oʻquvchi-yoshlarda hikoya natijasida kuzatuvchanlik va darsda oʻquvchilarning asar qahramonlari bilan oʻzaro intensiv sifatlarini shakllantirish ularning ijobiy va salbiy qahramon ekanliklarini tasvirlash kabilar ham yuzaga keladi. “50-90-yillar mobaynida boshlangʻich sinflarda ona tili oʻqitish metodikasi sohasida anchagina qoʻllanmalar yaratildi. Bu yillar mobaynida ona tili oʻqitish metodikasi fan sifatida rivojlana boshladi, umumiy 81 THEORETICAL ASPECTS IN THE FORMATION OF PEDAGOGICAL SCIENCES International scientific-online conference THEORETICAL ASPECTS IN THE FORMATION OF PEDAGOGICAL SCIENCES ona tili oʻqitish metodikasi fanini takomillashtirish va yangi metodika yaritshga imkon berdi”.[2] ona tili oʻqitish metodikasi fanini takomillashtirish va yangi metodika yaritshga imkon berdi”.[2] Haqiqatdan ham, hikoyani oʻqib chiqqandan kulol cholning hilasiga qoyil qolmasdan iloj qolmaydi. Bunda, kulollarning oʻz vatan tuproqlaridan kulolchilik buyumlarini yasashi, pulga mol-dunyoga berilishi natijasida vatan taraqqiyotini unutish kabi holatlar oʻʻquvchi-yoshlarga erish tuyulishi mumkin. Bunda, hikoyadan kelib chiqqan holda har kim oʻzining shaxsiy individual fikrlarni bildirishi, vatan taraqqiyotiga munosib hissa qoʻshish yoʻlidagi qarama- qarshiliklar, xatti-harakatlarning umumiy dasturlar asosida amalga oshirilishi koʻzga tashlanadi. Ona tili oʻqitish fanidan beriladigan uyga vazifalar ham pedagoglarning oʻquvchilar bilan ishlashish, texnologik bilimlarning oshishi, shuningdek, hikoyalarda berilgan mazmun-mohiyatning toʻgʻri talqin qilinishi bilan belgilanadi. Oʻqish turlari va ularning xususiyatlari hikoyalar bilan bevosita aloqadorlikni bogʻliq etib, uchinchi sinf oʻquvchilarining shaxsiy fikrlarida individual tarzda ishlash qobiliyatlari bilan yuzaga keladi. Ona tili oʻqitishda faqatgina taʼlim tarbiya berish kasb-hunargs yoʻnaltirishdan tashqari oʻquvchilarni turli oʻrinlarda vaziyatga, muhitga mos ravishda mustaqil fikrlashlarining oʻstirilishi va ahamiyati ham dolzarblikni keltirib chiqaradi. Bunda, oʻquvchilarning maʼnaviy kamolotini shakllantirish va ularga taʼlim jarayonida turli xil islohotlar, texnologiyalardan foydalanish metodlarning umummilliy birligi sifatida bilim, axborot olish kabilar keltiriladi. Boshlangʻich sinf oʻquvchilarining savodxonligini oshirishda axborot- texnologiyalaridan foydalanishning ham oʻrni katta boʻlib, ekran va saytlarda keltirilgan hikoya haqidagi qisqacha jumlalar ifodalarda ular oʻzlarining koʻrish orqali, eshitish orqali, yaʼni fikr-mulohaza yuritish orqali bilimlarini mustahkamlab boradi. Oʻquvchilarda xulosaviylik va xulosa qismini shakllantirish, didaktik maslahatlar, modulli taʼlim texnologiyalaridan foydalanish lozim boʻlib, ona tili oʻqitish metodikasining tarixiy asoslari va shakllanishida fanlararo integratsiyaning oʻrni va roli katta roʻl oʻynaydi. Bunda, boshlangʻich sinf oʻquvchilarning savodxonligini oshirishda ularga bilim berish, axborot berish va maʼlumotlarni jamlash xususiyatlari nuqtai nazardan integratsiya, fanlararo integratsiyani shakllantirish ham muhimdir. Fanlararo integratsiyaning shakllantirishda “kulol choling hiylasi” nomli hikoya bilan oʻzaro geografik jihatdan aloqadorlikda geografiya fanining oʻrni katta boʻlib, ushbu fanda taʼlimda hamkorlikda oʻqitish texnologiyasi fanlararo integratsiyaning oʻzaro rivojlanishi va taʼlim jarayoni tashkil etishda katta va muhim rol oynaydi. Shunday qilib, boshlangʻich sinf oʻquvchilarning 82 THEORETICAL ASPECTS IN THE FORMATION OF PEDAGOGICAL SCIENCES International scientific-online conference savodxonligini oshirishda modulli taʼlim texnologiyalarida “Kulol cholning hiylasi” nomli hikoyadan foydalanish har bir fuqarolarning vatanga muhabbati, uning asrab avaylanishi, vatan tuproqlarning koʻzga surtilishi bilan qadrlanadi. Foydalanilgan adabiyotlar: 1. Gʻulomova X. Boshlangʻich sinfda oʻqish darslarini tashkil etish metodikasi. Academic research in educational sciences - 2020 2. Izzatullayeva Nafisa Hikmatilloyevna. “Boshlangʻich taʼlimda ona tili oʻqitish metodikasi” maqola. THEORETICAL ASPECTS IN THE FORMATION OF PEDAGOGICAL SCIENCES International scientific-online conference “Science and education” scientific journal savodxonligini oshirishda modulli taʼlim texnologiyalarida “Kulol cholning hiylasi” nomli hikoyadan foydalanish har bir fuqarolarning vatanga muhabbati, uning asrab avaylanishi, vatan tuproqlarning koʻzga surtilishi bilan qadrlanadi. mova X. Boshlangʻich sinfda oʻqish darslarini tashkil etish metodikas mic research in educational sciences - 2020 1. Gʻulomova X. Boshlangʻich sinfda oʻqish darslarini tashkil etish metodikasi. Academic research in educational sciences - 2020 2. Izzatullayeva Nafisa Hikmatilloyevna. “Boshlangʻich taʼlimda ona tili oʻqitish metodikasi” maqola “Science and education” scientific journal 2. Izzatullayeva Nafisa Hikmatilloyevna. “Boshlangʻich taʼlimda ona tili oʻqitish metodikasi” maqola. “Science and education” scientific journal 83
https://openalex.org/W4360922117	https://sciforum.net/paper/download/13865/manuscript	English	null	The Geosite of Travertine Waterfall of El Ksiba (Morocco): A Heritage to Enhance and Preserve	null	2,022	cc-by	2,779	Proceedings The Geosite of Travertine Waterfall of El ksiba (Morocco), a Heritage to Enhancement and Preserve dellAh AIT BARKA , Jamila Rais , Ahmed BARAKAT , Elhassan LOUZ , Samir NADEM Geomatic, Georesources and Environment Laboratory, Faculty of Sciences and Techniques, Sultan My Slimane University, Béni Mellal, Morocco * Correspondence: author: AbdellAh AIT BARKA; a.aitbarka@usms.ma Abstract: The travertines of El Ksiba forming cliffs with an extension of about 8 km and a variable height that can reach about thirty meters. They are developed on lacustrine limestones and con- glomerates of Early Quaternary age. The results reveal the high scientific (≃0.88) and aesthetic (≃0.88) values related to the strong representativeness of the regional geological phenomena. The assessment also shows the high economic value (≃0.75) and cultral (≃0.81). In this work, we pre- sented the strategies of valorization and protection of this heritage in the framework of rural socio- economic development through activities related to geo-tourism and geo-education. Keywords: travertines; inventory; geo-tourism 1-. Introduction Geotourism is an activity that today qualified as innovative, which could lead to the sustainable development of society, and could contribute to the popularization of geolog- ical and geomorphological information through education, awareness of the public to ge- oconservation and protection of their heritage [1–2]. The travertines of El ksiba are depos- its of calcium and magnesium carbonate of great interest for identify and refine infor- mation about climatic, hydrological and vegetation cover variations through paleo-envi- ronment changes. These travertines have Quaternary age, forming cliffs with an extension of about 8 km and a variable height that can reach about thirty meters. They are developed on lacustrine limestones and conglomerates of Early Quaternary age. At the level of these travertine there are cavities, caves and shelters of variable dimension formed by the ad- vance of the top of the waterfall and others by the phenomena of the karstification. These formations are unique in the study area, containing remains of plants and animals (leaves, trunks, mollusks) [3], which allow to reconstruct the bioclimatic conditions of their estab- lishment. It has long been the preferred rock for construction and ornamental and aggre- gate in the region [4]. The abundance of resurgences, waterfalls, caves with an importance cultural value (Hyena Cave), exceptional ruiniforms landscapes cliffs and stone arch and fascinating panoramic views, make this territory a suitable tourist destination for excur- sions, hiking and climbing. Despite all these opportunities, this heritage remains un- known to the general public and little exploited by managers, while it could be a signifi- cant natural resource for local socio-economic development. To address this gap, an in- ventory and assessment was conducted to provide a database to support decision makers in any geo-heritage promotion project in the region which is the subject of previous stud- ies such as [3,5,6], This inventory-builder utilized Reynard (2016) method [7]. Beside the elevated aesthetic values of the identified geosites our approach also took the scientific values and some additional values into account to identify geosites. On the basis of geosite identification, a geotourism facilities have also been proposed to promote this rich geo- heritage. This promotion can popularize the geosciences and create income generating Citation: BARKA, A.A.; Rais , J.; BARAKAT , A.; LOUZ , E.; NADEM, S. The Geosite of Travertine Water- fall of El ksiba (Morocco), a Heritage to Enhancement and Preserve. 2022, 69, x. 1-. Introduction https://doi.org/10.3390/xxxxx Academic Editor(s): Received: date Accepted: date Published: date Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Copyright: © 2022 by the authors. Submitted for possible open access publication under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https://cre- ativecommons.org/licenses/by/4.0/). www.mdpi.com/journal/proceedings www.mdpi.com/journal/proceedings Proceedings 2022, 69, x. https://doi.org/10.3390/xxxxx Proceedings 2022, 69, x FOR PEER REVIEW 2 of 6 activities, which will improve the socio-economic situation of the local communities of the region. activities, which will improve the socio-economic situation of the local communities of the region. 2. Study Area The study area is located in the north of the province of Beni Mellal, in the region of Beni Mellal-Khenifra (Figure 1), limited to the Atlas of El Ksiba, of which the quarries of materials represent one of the important economic activities. It is characterized by a com- plex geology mainly of Mesozoic age, with a mountainous topography. The dominant climate is continental; very cold in winter and very hot in summer. The study area is char- acterized by a dense vegetation cover represented by holm oak (Quercus ilex), Zeen oak (Quercus canariensis), Juniper (Juniperus) and Dwarf palm (Chamaerops humilis). The study area characterized by an important hydrographic network, represented by valleys and rivers, and an important groundwater table. 3. Geological Setting The Triassic and Jurassic form most of the outcrops in the study area (Figure 1). The Triassic is represented by red clays and altered basalts, with intercalations of shale and quartzites of Paleozoic age. The lias is formed by massive limestones and dolomites, they constitute the most dominant geological formation at the scale of the study area. The up- per lias corresponds to an essentially marly episode of Toarcian - Aalenian. The Dogger is essentially limestone. Cretaceous formations represented by marls and lenticular con- glomerates. The Cretaceous formations are continued by sandstones of the pink piedmont molasse and conglomerates of the Mio-Pliocene which are concentrated in the piedmont area. The Quaternary is also well represented by alluvium and travertine deposits that lie unconformably with the formations of the Dir. Proceedings 2022, 69, x FOR PEER REVIEW 3 of 6 Figure 1. (a). The geological map of the study area. Extracted by the geological map of Morocc 1:100,000. Sheet of Kasba Tadla: (b) geographical location of study area. Figure 1. (a). The geological map of the study area. Extracted by the geological map of Morocco 1:100,000. Sheet of Kasba Tadla: (b) geographical location of study area. 5. Results and Discussion The travertine of El ksiba is one of the particular geosites of the region, as a result of the waterfall abundance. It is chosen in this study as a geosite for its large surface area (Figure 2a, b), its richness in resurgences, seasonal waterfalls, caves (Figure 2d) with an importance cultural value (Hyena Cave), exceptional ruiniforms landscapes (Figure 2c), cliffs and stone arch and fascinating panoramic views. Despite all these wonderful natural features, undesirable pressures affect this castle, such as the quarrying of building mate- rials. These travertines is very porous by holes of size millimeter to several meters orna- mented by stalagmites and stalactites. These formations related to the rapid precipitation of carbonates are caused by the release of CO2 from karstic water, it revealed on all its height of abundant remains of plants and animals (leaves, trunks, molluscs...), which make it possible to reconstruct the bioclimatic conditions of their establishment. The study of these travertine formations has several scientific interests (≃0.88), including infor- mation on the past functioning, paleo-climat and its paleo- topography. Therefore, a ge- osite tells the history value (≃0.81) in the region by the abundance of caves. This caves were called Tighramt (a castle) [10], formed naturally in a very rugged terrain. Moreover, these caves look like collective granaries intended for the protection of everything precious during the war at that time (herds, cereals, children and women) because they are invisible to their enemies. Today most of the berber families live in these caves in the region. These caves are a priceless treasure in the study area, and are among the most important tourist assets, especially since they lead to underground galleries [3], and play an important role in spe- leology. These caves can also play a role in encouraging ecotourism by transforming these caves into ecological shelters. They play a major role in tourism development and partic- ipate in the local development of rural areas. In addition, this area is rich in cultural at- tractions (≃0.81); the Hyena Cave that show the power of a former leader of the tridus in Atlas. In other hand, the travertine of El ksiba was an important economic value (≃0.75),This is reflected in the increase in the number of local tourists due to the paving of roads and the acquisition of local products such as oil, pomegranate and other local clothing that express the local identity and heritage. 4. Materials and Methods In this study, we have selected the geosites by assessing their scientific, aesthetic, ecological, and cultural relevance [7]. This approach focuses on inventory the representa- tive geodiversity sites to select the most representative ones with high overall value. The inventory was conducted in two stages: selection of geosites and their evaluation. The selection was based on literature reviews and field visits, and uses a code to locate the geosite. This identification code consists of three parts [8],: (1) the abbreviation of the re- gion in capital letters, (2) the processes responsible for the genesis of the geomorphic form in lower case letters, and (3) a numerical identifier for the site. The geosite was evaluate according to criteria [9],: the central value (scientific value): representativeness, integrity, rarity and paleogeographic value and additional values (ecological, Aesthetic, cultural, economic, and rarity value. Each of these criteria was independently evaluated by a nu- merical score ranging from 0 (none), 0.25 (low), 0.5 (medium), 0.75 (high) to 1 (very high). Proceedings 2022, 69, x FOR PEER REVIEW 4 of 6 The final value of the object was obtained by the average the four criteria that compose it in central value and in additional values (Table 1). 5. Results and Discussion Travertines of El ksiba: (a) area occupied by travertines (google earth image, october 20, 2022); (b) view on the travertines; (c) ruiniforms landscapes; (d) the Hyena Cave. . Figure 2. Travertines of El ksiba: (a) area occupied by travertines (google earth image, october 20, 2022); (b) view on the travertines; (c) ruiniforms landscapes; (d) the Hyena Cave. . 6. Conclusion The quantitative evaluation shows that the travertines of El ksiba have an important scientific value (0.88); it presents an open-air museum rich in paleontological, speleologi- cal and paleoclimatic data which helps us to reconstruct the old environment. In this sense, we propose, as a tool to valorize this heritage, to install a plaque explaining the mode of origin of this travertine in its different forms, integrating the site in areas of sci- entific, economic and cultural importance, and directing local tourism to this area to un- derstand these forms and thus preserve them. Developing tourist infrastructures such as ecological guest houses and creating new outlets to sell local products. In addition to stop- ping the installation of quarries of building materials in this area. Acknowledgments: The authors wish to express their great gratitude to all those who participated in the realization of this work, also to the anonymous reviewers for their valuable and constructive suggestions during the development of this article. 1. Guerra, V., Lazzari, M. Geoheritage Assessment and Potential Geotouristic Enhancement in Mountain Environments: a Test- Site in the Northern Apennines (Italy). Geoheritage 14, 97 (2022). https://doi.org/10.1007/s12371-022-00729-1 2. Valentini, L.; Guerra, V.; Lazzari, M. Enhancement of Geoheritage and Development of Geotourism: Comparison and Inferences from Different Experiences of Communication through Art. Geosciences 2022, 12, 264. https://doi.org/10.3390/geosciences12070264 5. Results and Discussion It is also an opportunity for hunting and fishing enthusiasts to practice their hobby to sing this region with different types of animals and birds, in addition to its wealth with different types of trees and medicinal plants (Ecological value). p g The scientific, economic, cultural and ecological importance of the travertines of El Ksiba give it a remarkable attraction, which must be integrated into the regional develop- ment activities. As well as its ease of access and its coolness, that contrasts with the over- whelming heat of the region. These travertines is a lever for sustainable development of the city and contributes to socio-economic development through the creation of new in- come-generating activities (ecological guest houses, traditional crafts, natural local prod- ucts, ...). Table 1. Quantitative assessment of the scientific and additional value of El ksiba’s Travertine. Code Name Scientific Value Additional Value Int Rep Rar Pal Sc V Ecol Aes Cul Eco Ad V ELKhyd001 the Hyena Cave 1 0.75 1 0.75 0.88 0.75 0.75 0.75 0.75 0.75 ELKkar002 Spring 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 ELKhyd003 Panoramic view 1 1 0.75 1 0.94 1 1 1 0.75 0.94 ELKkar004 ruiniforms landscapes 0.75 1 1 1 0.94 0.75 1 0.75 0.75 0.812 Travertine Value 0.75 0.88 0.75 0.63 0.88 0.81 0.88 0.81 0.75 0.81 Table 1. Quantitative assessment of the scientific and additional value of El ksiba’s Travertine. Proceedings 2022, 69, x FOR PEER REVIEW 5 of 6 Note: Int means integrity; Rep means representativity; Rar means Rarity; Pal means Paleogeograph- ical value; Sc V means Scientific value; Ecol means ecological value; Aes means Aesthetic value; Cul means cultural value; Eco means economical value; Ad V means Additional value. Note: Int means integrity; Rep means representativity; Rar means Rarity; Pal means Paleogeograph- ical value; Sc V means Scientific value; Ecol means ecological value; Aes means Aesthetic value; Cul means cultural value; Eco means economical value; Ad V means Additional value. Note: Int means integrity; Rep means representativity; Rar means Rarity; Pal means Paleogeograph- ical value; Sc V means Scientific value; Ecol means ecological value; Aes means Aesthetic value; Cul means cultural value; Eco means economical value; Ad V means Additional value. Figure 2. Travertines of El ksiba: (a) area occupied by travertines (google earth image, october 20, 2022); (b) view on the travertines; (c) ruiniforms landscapes; (d) the Hyena Cave. . Figure 2. g p gy) 9. Grandgirard V., 1999. l’évaluation des géotopes. Geologia Insubrica 4: 59–66 6. Rais J., Barakat A., Louz E., Ait Barka A., 2021. Geological heritage in the M’Goun Geopark: A proposal of geo-itineraries around the Bine El Ouidane dam (Central High Atlas, Morocco). International Journal of Geoheritage and Parks 9(2): 242–263. DOI 10.1016/j.ijgeop.2021.02.006. 5. Louz E, Rais J, Ait Barka A, Nadem S, Barakat A., 2022. Geological heritage of the Taguelft syncline (M'Goun Geopark): Inventory, assessment, and promotion for geotourism development (Central High Atlas, Morocco) International Journal of Geoheritage and Parks 10 (2022) 218–239. https://doi.org/10.1016/j.ijgeop.2022.04.002 j jg p 7. Reynard E., Perret A., Bussard J., Grangier l., Martin S., 2016. Integrated Approach for the Inventory and Management of Geomorphological Heritage at the regional Scale. Geoheritage 8(1): 43–60. DOI 10.1007/s12371-015-0153-0. 10. Gautier F. F., 1925. les cavernes du Dir. Hespéris (5)4: 383-414. 3. Ait barka A., Rais J., Barakat A., Louz E., Nadem S., 2022. The karst landscapes of Beni Mellal Atlas (central Morocco): identification for promoting geoconservation and tourism. Quaestiones Geographicae 41(3), Bogucki Wydawnictwo Naukowe, Poznań, pp. 87–109. 13 figs, 2 tables. https://doi.org/10.2478/quageo-2022-0027 p g g g g ( ) 8. Grandgirard, V., & Szepesi, A. (1997). Geomorphology and management of natural heritage (the p new task in geomorphology). Noosfera, 3, 59–65. pp g p g q g akat A., El Baghdadi M., Rais J., 2015. A GIS-Based Inventory of Ornamental Stone and Aggregate Opera ellal region (Morocco). Arabian Journal For Science and Engineering 40(7): 2021–2031. DOI 10.1007/ s13369 g g g zepesi, A. (1997). Geomorphology and management of natural heritage (the protection of the geotopes, a phology). Noosfera, 3, 59–65. References Proceedings 2022, 69, x FOR PEER REVIEW 6 of 6 3. Ait barka A., Rais J., Barakat A., Louz E., Nadem S., 2022. The karst landscapes of Beni Mellal Atlas (central Morocco): identification for promoting geoconservation and tourism. Quaestiones Geographicae 41(3), Bogucki Wydawnictwo Naukowe, Poznań, pp. 87–109. 13 figs, 2 tables. https://doi.org/10.2478/quageo-2022-0027 5. Louz E, Rais J, Ait Barka A, Nadem S, Barakat A., 2022. Geological heritage of the Taguelft syncline (M'Goun Geopark): Inventory, assessment, and promotion for geotourism development (Central High Atlas, Morocco) International Journal of Geoheritage and Parks 10 (2022) 218–239. https://doi.org/10.1016/j.ijgeop.2022.04.002 6. Rais J., Barakat A., Louz E., Ait Barka A., 2021. Geological heritage in the M’Goun Geopark: A proposal of geo-itineraries around the Bine El Ouidane dam (Central High Atlas, Morocco). International Journal of Geoheritage and Parks 9(2): 242–263. DOI 10.1016/j.ijgeop.2021.02.006. j jg p 7. Reynard E., Perret A., Bussard J., Grangier l., Martin S., 2016. Integrated Approach for the Inventory and Management of Geomorphological Heritage at the regional Scale. Geoheritage 8(1): 43–60. DOI 10.1007/s12371-015-0153-0. 8. Grandgirard, V., & Szepesi, A. (1997). Geomorphology and management of n new task in geomorphology). Noosfera, 3, 59–65. g p gy 9. Grandgirard V., 1999. l’évaluation des géotopes. Geologia Insubrica 4: 59–66 10. Gautier F. F., 1925. les cavernes du Dir. Hespéris (5)4: 383-414.
https://openalex.org/W3168019641	https://ri.conicet.gov.ar/bitstream/11336/183786/2/CONICET_Digital_Nro.63b6e3b3-a20d-4d0b-921e-c4d287b43f78_B.pdf	English	null	New asteroid species from the Eocene of Seymour Island, Antarctica	Acta Palaeontologica Polonica	2,021	cc-by	12,103	A new zoroasterid asteroid from the Eocene of Seymour Island, Antarctica Received 6 December 2019, accepted 12 December 2020, available online 7 June 2021. Copyright © 2021 E.E. Palópolo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (for details please see http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. alternately carinate, and tube feet in four rows becoming two rows distally (Blake 1987; Mah 2000, 2007; Mah and Foltz 2011; Mah and Blake 2012; Fau and Villier 2018). A new zoroasterid asteroid from the Eocene of Seymour Island, Antarctica EVANGELINA E. PALÓPOLO, SOLEDAD S. BREZINA, SILVIO CASADIO, MIGUEL GRIFFIN and SERGIO SANTILLANA LÓPOLO, SOLEDAD S. BREZINA, SILVIO CASADIO, MIGUEL GRIFFIN, Palópolo, E.E., Brezina, S.S., Casadio, S., Griffin, M., and Santillana, S. 2021. A new zoroasterid asteroid from the Eocene of Seymour Island, Antarctica. Acta Palaeontologica Polonica 66 (2): 301–318. New, well-preserved fossil starfish material is recorded from the Eocene La Meseta Formation exposed in Seymour Island, Antarctica. The use of new technology (i.e., microCT) on several fragments enabled the visualization of new characters and the differentiation of a new species, Zoroaster marambioensis sp. nov., which was previously identified as Zoroaster aff. Z. fulgens. Diagnostic characters of Z. marambioensis sp. nov. are (i) central disc plate enlarged, lobate and flattened, (ii) disc ring with enlarged, tumid radials and polygonal, flattened inter-radials, (iii) primary spines on disc only present on radials, (iv) oral armature with 1–3 primary spines and 1–2 secondary spines for each prominent adambulacral. The depositional setting represents the outer zone of an estuary dominated by marine processes affected by long lived hyperpycnal flows. We argue that zoroasterids colonized a distal part of the estuary under normal marine salinity and were killed by the input of freshwater carried by a hyperpycnal flow, and immediately buried by fine grained sandstone. Sedimentological data suggest that Z. marambioensis sp. nov. lived in shallow-water environments, it seems possible that they were adapted to higher temperatures than other Recent species of the genus, which inhabit cold, deep marine environments. Key words: Asteroidea, Zoroasteridae, palaeoenvironment, Paleogene, La Meseta Formation, Antarctic Peninsula. Evangelina E. Palópolo [eepalopolo@unrn.edu.ar] and Silvio Casadio [scasadio@unrn.edu.ar], Universidad Nacio nal de Río Negro, Instituto de Investigación en Paleobiología y Geología, Río Negro, Argentina; and IIPG. UNRN. Consejo Nacional de Investigaciones científicas y Tecnológicas (CONICET), Av. Roca 1242, (R8332EXZ) General Roca, Río Negro, Argentina. d S. Brezina [sbrezina@unrn.edu.ar], Universidad Nacional de Río Negro, Instituto de Investigación en Paleo a y Geología, Río Negro, Argentina. Miguel Griffin [mgriffin@fcnym.unlp.edu.ar], Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Edificio Anexo Laboratorios Museo (Laboratorio 110), Avenida 122 y 60, La Plata, Buenos Aires, Argentina. Sergio Santillana [ssantillana@dna.gov.ar], Instituto Antártico Argentino, 25 de Mayo 1143, San Martín, provincia de Buenos Aires, Argentina. Received 6 December 2019, accepted 12 December 2020, available online 7 June 2021. Received 6 December 2019, accepted 12 December 2020, available online 7 June 2021. Introduction Zoroasterids (the family Zoroasteridae Sladen, 1889) com- prise a group of starfishes with five rays, a small disc, long and tapering arms, and skeletal plates arranged in series (both transverse and longitudinal) covered by primary and secondary spines (McKnight 2006). Species of this family also have a single marginal row, papular pores arranged on longitudinal and transverse series, imbricated or reticulated disc and arm plate arrangement, actinolateral spines larger than other primary spines, adpressed or facing abactinally, straight pedicellariae (except in Pholidaster), adambulacrals The family was originally defined by Sladen (1889), and includes eight genera (one of them with only fossil repre- sentatives) and 35 nominal species inhabiting abyssal and bathyal environments of the Atlantic, Pacific and Indian oceans (Mah 2007). The fossil record of the family Zoroasteridae is scarce. Relatively few species are known, including one from the Jurassic of Europe (Hess 1974; Villier et al. 2009), two from the Eocene of Antarctica and New Zealand (Blake and Zinsmeister 1979, 1988; Blake and Aronson 1998; Eagle Acta Palaeontol. Pol. 66 (2): 301–318, 2021 https://doi.org/10.4202/app.00714.2019 302 ACTA PALAEONTOLOGICA POLONICA 66 (2), 2021 2006; this article), and one from the Miocene of Japan (Kato and Oji 2013). These records are highly biased because of their body plan (i.e., the small disc, the very long arms and weak ossicle connection). characters intermediate between the Paleozoic and post-Pa- leozoic Asteroidea, such as a single marginal series and the arrangement of arm ossicles and spines (Blake 1987, 1990; Mah 2000, 2007; Mah and Foltz 2011; Mah and Blake 2012). Among the Zoroasteridae, the genus Zoroaster Wyville Thomson, 1873, shows more derived skeletal characters (e.g., imbricate and well-armored skeleton) than Myxoderma Fisher, 1905, and Sagenaster Mah, 2007, which have reticu- lated and open skeletons (Mah 2007). ) Echinoderm ossicles are connected by muscles, liga- ments, interlocking stereom, cement or a combination of these (Ausich et al. 2001). Because starfishes have a large coelomic cavity that extends into each arm (Ferguson 1992; Brusca and Brusca 2003) and most show a weakly articu- lated body skeleton, they are prone to complete disarticu- lation within few days after death (Brett et al. 1997) due to soft tissue decomposition and skeletal collapse. The fact that ossicle fusion in asteroids is far from that achieved in echi- noids is a key feature contributing to disarticulation. Introduction Yet, in zoroasterids ossicles are quite firmly tied together relative to other asteroids, a fact that could somewhat enhance their preservation potential. However, we consider that a simple stratum containing several specimens of almost complete asteroids in living position would still be considered an ex- ceptionally preserved deposit (Brett et al. 1997), even in the case of zoroasterids in which ossicles are more closely tied together than in other asteroids. This type of preservation provides important information about paleobiology, paleo- ecology, sedimentary environment and taphonomic history of the remains (Brett 1978). Primary spines, secondary spines and pedicellariae, to- gether with soft tissues, have been widely used in Recent asteroid systematics. Shape, size and arrangement of these structures are important for identification of zoroasterid species (Downey 1970; Blake 1987; Clark and Downey 1992, among others). Nevertheless, some species of the fam- ily have different morphotypes that render taxonomic iden- tification difficult (Howell et al. 2004). Specimens analyzed herein are preserved in detail; both oral and aboral surface characters can be identified, spines and pedicellariae are often in life position. This evidence allowed us to identify dependable characters and to describe a new species of Zoroaster, previously reported by Blake and Zinsmeister (1979) as Zoroaster aff. Z. fulgens, from the La Meseta For mation (Eocene, Antarctic Peninsula). Institutional abbreviations.—IAA, Instituto Antártico Argentina, San Martín, Buenos Aires, Argentina; IAA-Pi, Colección Paleontología de Invertebrados, Repositorio Antártico de Colecciones Paleontológicas y Geológicas del Instituto Antártico Argentino, San Martín, Buenos Aires, Argentina; IIPG, Instituto de Investigación en Paleobiología y Geología, General Roca, Río Negro, Argentina; RAA, Repositorio Antártico de Colecciones Paleontológicas y Geológicas del IAA, San Martín, Buenos Aires, Argentina; The systematics of the group has been studied deeply based on Recent species but many questions remain regard- ing its origin and evolution because of its poor fossil record. Previous authors discussed the possible origin of the fam- ily in the Wedellian Province and its subsequent biogeo- graphical history (Blake 1987, 1990; Mah 2007; Villier et al. 2009; Gale 2011; Mah and Foltz 2011; Mah and Blake 2012). Phylogenetic studies of the Asteoidea place the Zoroasteridae as a basal clade within the order Forcipulatida, based on Fig. 1. Geologic map (A) and stratigraphic column (B) of Seymour Island, Antarctica (modified from Montes et al. 2013). The star shows the place of discovery. Abbreviatons: M, Middle; U, Upper. Fig. 1. Geological setting The La Meseta Formation, exposed in Seymour Island (Fig. 1) off the northern tip of the Antarctic Peninsula, rep- resents the upper part of the infilling of the James Ross Basin and comprises a succession of 250 m of sediments depos- ited in an incised valley (Montes et al. 2019). Sedimentation took place in estuarine and wave-influenced tidal-shelf en- vironments (Marenssi et al. 1998a; Porȩbski 2000). The La Meseta Formation is well known for its shell-beds dominated by molluscs but also containing a unique fauna of Antarctic Eocene terrestrial vertebrates that includes several mammals (marsupials, edentates and ungulates) and birds. The fossil associations were the subject of numerous systematic studies (Feldmann and Woodburne 1988; Stilwell and Zinsmeister 1992; Bitner 1996; Goin et al. 1999; Hara 2001). Marenssi et al. (1998a, b) subdivided the La Meseta Formation into six allomembers. From bottom to top these are the Valle de las Focas, Acantilados, Campamento, Cucullaea I, Cucullaea II and Submeseta allomembers. The depositional setting ranged from a prograding delta front to a storm-influenced subaque- ous delta plain dominated by tides after marine-flooding within the incised valley (Marenssi et al. 1998a). The new zoroasterid specimens described in this paper come from the Cucullaea I Allomember (Fig. 2). They were collected in an area measuring 20 m2 (64°14’24’’ S, 56°40’02’’ W). The Cucullaea I Allomember begins with a shell concentration of several densely- to poorly-packed and poorly-sorted laterally continuous beds, or lenses ranging from 0.5–1.5 m thick, with sharp undulating bases and sharp tops, and trough cross-bed- ding. Sadler (1988) characterized this shell bed (his Telm 4) by its high content of phosphatic teeth and bones, and suggested it is a transgressive lag distinguished by abundant phosphate pebbles and glauconite. The densely packed beds are domi- nated by the multiple specimens of bivalve Cucullaea raea Zinsmeister, 1984. This concentration represents a tidal chan- nel facies in the outermost part of an estuary (Taylor et al. 2008). In terms of sequence stratigraphy these concentrations represent a tidal ravinement surface. The age of the lower and middle part of the La Meseta Formation is middle Lutetian to Priabonian (Amenábar et al. 2020). Th iddl d t f th C ll I All Fig. 2. Detailed stratigraphic column from Cucullaea I Allomember, La Meseta Formation. Abbreviations: C, conglomerate; cS, coarse sandstone; fS, fine sandstone; mS, medium sandstone. Scale bars 100 mm. ples and massive fine sandstone. Introduction Geologic map (A) and stratigraphic column (B) of Seymour Island, Antarctica (modified from Montes et al. 2013). The star shows the place of discovery. Abbreviatons: M, Middle; U, Upper. PALÓPOLO ET AL.—A NEW EOCENE ASTEROID FROM ANTARCTICA 303 Fig. 2. Detailed stratigraphic column from Cucullaea I Allomember, La Meseta Formation. Abbreviations: C, conglomerate; cS, coarse sandstone; fS, fine sandstone; mS, medium sandstone. Scale bars 100 mm. UNAM, Universidad Nacional Autónoma de México, Ciudad de México, México; UNRN, Universidad Nacional de Río Negro, General Roca, Río Negro, Argentina; YPF, Yacimientos Petrolíferos Fiscales, Buenos Aires, Argentina. UNAM, Universidad Nacional Autónoma de México, Ciudad de México, México; UNRN, Universidad Nacional de Río Negro, General Roca, Río Negro, Argentina; YPF, Yacimientos Petrolíferos Fiscales, Buenos Aires, Argentina. Nomenclatural acts.—This published work and the nomen- clatural acts it contains, have been registered in ZooBank: urn:lsid:zoobank.org:pub:38AF70B7-F4FE-4166-9DE5- 3EC8D68A30E6. Material and methods Studied specimens are housed in Colección Paleontología de Invertebrados, Repositorio Antártico de Colecciones Paleontológicas y Geológicas, Instituto Antártico Argentino (IAA) under IAA-Pi-373 code. ( ) While the fossil starfishes were fairly complete in the out- crop, because of their brittleness they suffered some break- age during collection and transport. About 250 fragments were analyzed, most of them preserved in detail. No fur- ther treatment was required for fossils except washing and brushing to remove sediment grains attached mostly to the oral surface of skeletons. Pores of the madreporic plate are filled by sediment grains that cannot be removed. Fragments were observed under binocular microscope on both oral and aboral surfaces. Four fragments were observed using Zeiss® Scanning Electronic Microscope (SEM), model Evo MA 15, with variable pressure. MicroCT scans were made with other three fragments using MicroCT Bruker SkyScan 1173 (Pixel size: 50μm, Source Voltage: 110 kV, Source Current: 72 uA) at the Laboratorio de Química Analítica, YPF Tecnología (La Plata, Buenos Aires). Image reconstruction was made using Nrecon 1.6.9.8 software (Filter: Haming, Beam Hardening Correction: 10%, Cone-beam Angle: 17.544950°). A total of 1086, 1098, and 1111 slices were recovered from fragments 1, 2, and 3, respectively. MicroCT image sets were processed using 3D Slicer 4.8.1 (Fedorov et al. 2012) and Drishti 2.6.3 (Limaye 2012) software (SOM 2). Fig. 3. Overview of asteroid layer in the type locality, GPS POI 64°14’24” S, 56°40’02” W, Cucullaea I Allomember, La Meseta Formation (Eocene). Seymour Island, Antarctica. Arrows indicate asteroid fragments. http://app.pan.pl/SOM/app66-Palopolo_etal_SOM.pdf) and exported to TNT version 1.5 (Goloboff and Catalano 2016) and PAUP trial version 4.0a167 (Swofford 2003). Analyses were performed following the methods of Mah (2007). Support estimation by Bootstrapping and Jackknifing methods were made using PAUP, with unrooted trees, unordered charac- ters, 1000 replicates, gaps treated as “missing” and multi- state taxa interpreted as uncertainty. Bremer support was calculated using TNT software. Consistency and Retention indexes were calculated for consensus tree, boopstrappig and Jackknifing tree. On the basis of original and extended descriptions (Alcock 1893; Ludwig 1905; Fisher 1905, 1906, 1916, 1919, 1928; Clark 1913, 1916, 1920; Clark and Downey 1992; Esteban-Vasquez 2018), nine new characters were added to the zoroasterid phylogenetic matrix published by Mah (2007). These were considered by the authors as dependable characters for the species of Zoroaster. Characters of Zoroaster marambioensis sp. nov. were also added to that matrix. Geological setting This part of the section represents the outer zone of an estuary dominated by marine processes affected by long lived hyperpycnal flows. According to Zavala and Pan (2018), the key features of sustained hyperpycnal flows include: (i) an origin associated to a direct fluvial discharge, which is often characterized by long lived flows with fluctuating changes in velocity and concentration, (ii) common occurrence of associated bed- load processes, and (iii) a turbulent flow with a light inter- stitial fluid (freshwater) together with other light elements in suspension (e.g., charcoal, leaves, and trunks). During a hy- perpycnal discharge, freshwater, plant debris and charcoal, are forced to go down and to travel basinwards. The middle and upper part of the Cucullaea I Allomem ber includes facies of laminated siltstone and fine sandstone with climbing ripples, fine grained laminated sandstone and siltstone with trunks, fine grained sandstone with wave rip- Zoroasterids were collected at the top of a massive fine sandstone bed and they were covered by laminated fine 304 ACTA PALAEONTOLOGICA POLONICA 66 (2), 2021 Fig. 3. Overview of asteroid layer in the type locality, GPS POI 64°14’24” S, 56°40’02” W, Cucullaea I Allomember, La Meseta Formation (Eocene). Seymour Island, Antarctica. Arrows indicate asteroid fragments. sandstone. Individual laminae are millimeter thick and are intercalated with thin levels with abundant carbonaceous material and even charcoal (Figs. 2, 3). This observation is consistent with the facies L (facies related to flow lofting) hyperpycnal flow facies tract of Zavala et al. (2011). Lofting rhythmites that cover the zoroasterids are the result of the aggradation of fine-grained materials from suspension clouds related to the buoyant inversion of hyper- pycnal flows at flow margin areas (Zavala et al. 2012). We interpret that the zoroasterids colonized a distal part of the estuary under normal marine salinity and were killed by the input of freshwater carried by a hyperpycnal flow, and immediately buried by fine grained sandstone. The ab- sence of tractive structures in these sandstones suggests an accumulation by normal settling from a suspension cloud elevated over the depositional surface. Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E 1979 Zoroaster aff. Z. fulgens Thomson, 1873; Blake and Zinsmeister 1979: 1151–1152, pl. 2: 1–11. p 1988 Zoroaster aff. Z. fulgens Thomson, 1873; Blake and Zinsmeister, 1988: 495, figs. 3: 7–10, 4: 1–4. 1998 Zoroaster aff. Z. fulgens Thomson, 1873; Blake and Aronson, 1998: 345. Etymology: After the place of discovery, i.e., Marambio (Seymour) I l d A t ti P i l Etymology: After the place of discovery, i.e., Marambio (Seymour) Island, Antarctic Peninsula. Island, Antarctic Peninsula. Type material: Holotype: IAA-P-373-A, incomplete specimen com- prising five fragments. Paratypes: IAA-P-373-B to K, ten incomplete specimens comprising four specimens with incomplete disc and arms, three specimens with complete disc and partially preserved arms, and three almost complete arm fragments without disc structures. Type locality: GPS POI 64°14’24” S, 56°40’02” W, Seymour Island, Antarctica. Type horizon: Cucullaea I Allomember, La Meseta Formation, Eocene. Material.—Type material and twenty (including IAA-Pi- 373-L–N, Q1, R) additional arm and disc fragments. All from the same locality and layer. Almost complete or fragmented arms articulated with the disc in most cases. Arm plates articulated by proximal and distal lobes, rel- atively large spaces for papulae between plates at proximal part of the arms (Fig. 5C1, C4), becoming smaller distally. Ossicles arranged in well-defined longitudinal and trans- verse rows along arms (Figs. 5C1, C2, 9). Diagnosis.—Central disc plate enlarged, lobate, flattened or slightly depressed. Primary circlet with enlarged, lo- bate, tumid radials that abut small, polygonal and flattened inter-radials (Figs. 5A2, 7A, 9A3, B1, C2). Small abactinal disc plates between radials and central plate, and between primary circlet and marginals (Figs. 4A, B2, F2, 5A2, D). Primary spines on disc only present on radials. Marginals hexagonal, proximally with one spine every two margin- als, distally lacking spines (Fig. 4A). Four or five rows of actinolaterals proximally, the upper row polygonal, without primary spines, extending distally until the area between the last two marginals at the arm tip (Figs. 5C3, 6A2, B1, B2, 8A1, A2). Oral armature well developed (Fig. 8B1, one to three spines for each prominent adambulacral and one or two secondary spines). One or two big pedicellariae and 2–3 small pedicellariae associated to each prominent adam- bulacral plate (Fig. 6H1, H2). Each ambulacral plate with a long and well-developed furrow on actinal view (Figs. 4B1, H2, 6E). Terminals enlarged, crescent-shaped, wider than long, with a prominent notch (Fig. 8A1, A2). Systematic palaeontology sharp on actinal surface (see Table 1 for a succinct summary of the characters which distinguish this species from other zoroasterids). Systematic palaeontology Class Asteroidea Blainville, 1830 Superorder Forcipulatacea Blake, 1987 Order Forcipulatida Perrier, 1884 Family Zoroasteridae Sladen, 1889 Genus Zoroaster Thomson, 1873 Class Asteroidea Blainville, 1830 Superorder Forcipulatacea Blake, 1987 Order Forcipulatida Perrier, 1884 Family Zoroasteridae Sladen, 1889 Genus Zoroaster Thomson, 1873 Description.—Rays five. Major radius (R): 103–150 mm. Minor radius (r): 15–18 mm. R/r: 6.87–8.33. Breadth of the ray at its base: 13 mm. Eighteen marginals to first 10 cari- nals (Figs. 4F2, G1, H1, 9B1). Arms long, narrow, tapering distally (Fig. 4A, C1). Cross section of arms subcylindrical (Figs. 6B2, 7B, C). Entire body surface covered mainly by secondary spines (Fig. 4). Family Zoroasteridae Sladen, 1889 Genus Zoroaster Thomson, 1873 Type species: Zoroaster fulgens Wyville Thomson, 1873; Eocene–Re- cent; Pacific, Atlantic and Indian oceans. Disc small, tumid, interbrachial angles acute. Abactinal surface of the disc formed by a central ossicle, surrounded by a ring of five radials and five interradials, a madreporic plate, slightly modified marginals and a variable number of small abactinal disc ossicles (Figs. 5B1, 7A, 9B1). Centrale ossicle, when preserved, flattened or slightly depressed (the last character maybe as a taphonomic feature). Radials en- larged, tumid, weakly lobate, bearing a central primary spine, intercalated with smaller, flattened, polygonal in- terradials, covered by secondary spines (Figs. 5B1, 7A). Radials abut interradial ossicles (Fig. 9A3, C2). Madreporic plate relatively small (half the size of interradials), circular, slightly elevated, with multiple channels and pores radiat- ing from the center (Figs. 4B2, 5 E1, E2), not fused to the adjacent interradial ossicle (Fig. 7A), surrounded by sec- ondary spines and pedicellariae, cup-shaped basal plates. Marginal plates on interbrachial angles enlarged, raised, subtriangular in shape, separated from the radials by small irregular inset adradial ossicles (Figs. 4A, B2, F2, 5A2, D). Disc ossicles articulated, leaving relatively large spaces for papulae. Material and methods Data was entered us- ing Mesquite version 3.61 software (Maddison and Maddison 2019; SOM 1, Supplementary Online Material available at Terminology used for morphological characters fol- lows previous descriptions (Hayashi 1943, 1961; Blake and Zinsmeister 1979, 1988; Blake and Aronson 1998; Blake 1987; Blake and Hotchkiss 2004; Mah 2007) and other publi- cations about extant species (Mooi and David 2000; Sumida et al. 2001; Howell et al. 2004; Mah and Blake 2012; Fau and Villier 2018). Systematic classification follows Spencer and Wright (1966), Blake (1987) and Mah (2007). PALÓPOLO ET AL.—A NEW EOCENE ASTEROID FROM ANTARCTICA 305 Figs. 4–9, SOM. Figs. 4–9, SOM. Figs. 4–9, SOM. Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E Primary spines short and blunt (on carinals) and long and slender (on ac- tinolaterals). Secondary spines blunt on abactinal surface, Carinals large, subcircular to hexagonal, weakly lobate (Fig. 5C4), transversely elongated proximally, equidimen- sional or slightly longitudinally elongated distally. Each carinal overlaps adjacent adradials and proximal carinal (Figs. 5C1, 9A3, C2). One big, short and blunt spine to each carinal, in a central knob of the ossicle (Figs. 4A, 6D, 7C). Well-developed adradials in a single series along both sides of carinals, slightly depressed, covered by small sec- ondary spines. Adradial ossicles hexagonal, almost equi- dimensional, sometimes transversely elongated (Figs. 5C1, 7B–D, 9A3, B1, C2), overlapped by carinals and marginals. Marginals in a single series (Figs. 4, 9), hexagonal, twice as wide as long, proximally bearing one primary spine every two marginals, distally without spines (Fig. 4A). Marginal series abutting adradials, but not actinolaterals (Figs. 5C2, 7B–D, 9A3, B1, C2). Actinolaterals polygonal to subtriangular, arranged in 4–5 rows proximally, reduced to three on the distal half 306 ACTA PALAEONTOLOGICA POLONICA 66 (2), 2021 Table 1. Characters used in original descriptions to differenciate Zoroaster species. Abbreviations: ch., character; R, major radius (distance be- tween the disc center and the arm tip); r, minor radius (distance between the disc center to the edge of the disc in the middle of an interradius); “?”, not stated in the original description; “–”, absent. Characters Zoroaster actinocles Fisher, 1919 Zoroaster magnificus Ludwig, 1905 Zoroaster macracantha H.L. Clark, 1916 Zoroaster microporus Fisher, 1916 Zoroaster carinatus Alcock, 1893 Zoroaster fulgens Wyville Thomson, 1873 Zoroaster ophiactis Fisher, 1916 Zoroaster ophiurus Fisher, 1905 Zoroaster spinulosus Fisher, 1906 Zoroaster marambioensis sp. nov. R 161 295 160 205 194 152 282 140 118 150 r 11 14 14 12 13.5 31 15,5 10 11 18 R/r 14.6 21.1 11.5 17 14 4.9 17 14 10.7 8.3 Carinal primary spines number 1 1 1 0 1 or more 1 1 1 1–3 1 shape ? short, cil- indrical long (5 mm), sharp, pointed – similar to secondary spines short, blunt stout, cilin- drical, long thimble- shaped tubercle short, conical, blunt short, conical, blunt Shape of secondary spines on abactinal surface flesh, grooved sharp, curved, grooved short, blunt, grooved ? thorn-like thin, sharp small, ung- rooved short (1mm), sharp, slender short, delicate, papiliform short, straight, sharp Pedicellariae on abactinal surface (ch. 71–72) small, associated to popular pores big, straight big, straight ? Ambulacrals compressed, high, squarish-blocky in shape, Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E few, small, scattered ? small, asso- ciated with papulae 2–5, big, associated to carinals – small, associated to papulae Number of actinolateral rows (ch. 73) 3+1 4 3 ? ? 5 6 5 5 5 First row of actinolaterals different (ch. 74) yes yes yes ? ? yes yes yes no yes Actino- lateral primary spines shape slender long, blunt, cilindrical sharp, flattened long, central long, slender long, flattened, sharp sharp, flattened, long sharp, slender, long sharp, fine, delicate sharp, flattened adpressed (ch. 75) yes yes no ? ? yes yes no yes yes Actino lateral pedi cellariae number (ch. 76) 1 ? several ? ? ? ? 1 several 0 size (ch. 77) big ? small ? ? ? ? big small – Carinate adambula crals spine number 5 5 ? 2 2–3 3–5 4–5 4–5 4 3 pedicellariae above furrow (ch. 79) small big ? ? ? big big big big big pedicellariae on innermost spine (ch. 78) ? 2–5 ? several 1 or 2? 5–8 10 6–8 5–8 2–3 non-carinate ones (Figs. 4B1, D2, 6E, G, 9C3). Carinate adambulacrals bearing transverse series of stout cylindrical spines (Fig. 9A2, B2). Non-carinate adambulacrals at one side of the furrow is opposite to a carinate adambulacrals on the other side (Fig. 9A1, B2). One to three spines for each prominent adambulacral, preserved in (or near) life position. One or two big pedicellariae (or cup-shaped basal piece), two or three small pedicellariae, and one or two secondary spines (Figs. 6H1, H2, 9B2) associated to each prominent adambulacral. of the arm and becoming a single row near the arm tip (Figs. 5C2, C3, 7B–D, 9A1, A2). Upper row of actinolaterals smaller than marginals, equidimensional, alternated with marginals, without spines (Figs. 5C2, C3, 9A3). This series does not articulate with the terminal ossicle, although the last actinolateral ossicle is located near the last distal mar- ginal (Fig. 8A2). The three lower abactinal series bearing slender, elongated, usually flattened spines, directed up- ward and towards proximal part of arm, articulating with a central knob on each plate (Fig. 5C2, C3). Ambulacrals compressed, high, squarish-blocky in shape, Adambulacral plates alternating long carinate and short PALÓPOLO ET AL.—A NEW EOCENE ASTEROID FROM ANTARCTICA PALÓPOLO ET AL.—A NEW EOCENE ASTEROID FROM ANTARCTICA 30 directed towards the center of the ambulacral groove, with a long and well-developed furrow on actinal view (Figs. 4B1, H2, 6E, H1). Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E Four rows of podial pores on actinal surface in the proximal part of the arm, becoming reduced to two serie at the arm tip. Superambulacrals not observed, apparentl reduced or absent. Fig. 4. Zoroasterid asteroid Zoroaster marambioensis sp. nov., Eocene, Cucullaea I Allomember, La Meseta Formation of Seymour Island, Antarctica A. IAA-Pi-373-A, general view of abactinal surface. B–H. General appearance of each fragment in abactinal (B1–H1) and actinal (B2–H2) views. B. IAA Pi-373-B. C. IAA-Pi-373-C. D. IAA-Pi-373-D. E. IAA-Pi-373-E. F. IAA-Pi-373-F. G. IAA-Pi-373-G. H. IAA-Pi-373-H. 307 Fig. 4. Zoroasterid asteroid Zoroaster marambioensis sp. nov., Eocene, Cucullaea I Allomember, La Meseta Formation of Seymour Island, Antarctica. A. IAA-Pi-373-A, general view of abactinal surface. B–H. General appearance of each fragment in abactinal (B1–H1) and actinal (B2–H2) views. B. IAA- Pi-373-B. C. IAA-Pi-373-C. D. IAA-Pi-373-D. E. IAA-Pi-373-E. F. IAA-Pi-373-F. G. IAA-Pi-373-G. H. IAA-Pi-373-H. directed towards the center of the ambulacral groove, with a long and well-developed furrow on actinal view (Figs. 4B1, H2, 6E, H1). Four rows of podial pores on actinal surface in directed towards the center of the ambulacral groove, with a long and well-developed furrow on actinal view (Figs. 4B1, H2, 6E, H1). Four rows of podial pores on actinal surface in the proximal part of the arm, becoming reduced to two series at the arm tip. Superambulacrals not observed, apparently reduced or absent. 308 ACTA PALAEONTOLOGICA POLONICA 66 (2), 2021 Fig. 5. Zoroasterid asteroid Zoroaster marambioensis sp. nov., Eocene, Cucullaea I Allomember, La Meseta Formation of Seymour Island, Antarctica. A. IAA-Pi-373-B, actinal (A1) and abactinal (A2) views. Gastropod valve near peristome location, partially attached to orals in actinal side (arrow). B. IAA-Pi-373-M, actinal view, showing oral depression, inferred position of actinostome and orals. C. IAA-Pi-373-E, detail of arm structures; abactinal view, indicating ossicle rows (C1); lateral view of distal, denuded part of the arm (C2), note the insertion marks left by primary and secondary spines on marginals and actinolaterals (arrows); lateral view of proximal part of arm, primary and secondary spine number and arrangement (C3); carinal ossicle structure and position of papular orifices (C4). D. IAA-Pi-373-I, interbrachial zone of disc on abactinal view, modified triangular marginals (arrows). E. IAA-Pi-373-C; position of madreporic plate on fragmented disc (E1); detailed structure of madreporite (arrow) (E2). Abbreviations: Adr, adradial; Al, actinolateral; C, carinal; Ct, central; Ird, interradial; M, marginal; R, radial. 308 ACTA PALAEONTOLOGICA POLONICA 66 (2), 2021 Fig. 5. Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E Zoroasterid asteroid Zoroaster marambioensis sp. nov., Eocene, Cucullaea I Allomember, La Meseta Formation of Seymour Island, Antarctica. A. IAA-Pi-373-B, actinal (A1) and abactinal (A2) views. Gastropod valve near peristome location, partially attached to orals in actinal side (arrow). B. IAA-Pi-373-M, actinal view, showing oral depression, inferred position of actinostome and orals. C. IAA-Pi-373-E, detail of arm structures; abactinal view, indicating ossicle rows (C1); lateral view of distal, denuded part of the arm (C2), note the insertion marks left by primary and secondary spines on marginals and actinolaterals (arrows); lateral view of proximal part of arm, primary and secondary spine number and arrangement (C3); carinal ossicle structure and position of papular orifices (C4). D. IAA-Pi-373-I, interbrachial zone of disc on abactinal view, modified triangular marginals (arrows). E. IAA-Pi-373-C; position of madreporic plate on fragmented disc (E1); detailed structure of madreporite (arrow) (E2). Abbreviations: Adr, adradial; Al, actinolateral; C, carinal; Ct, central; Ird, interradial; M, marginal; R, radial. Terminals, when preserved, highly enlarged, crescent- shaped, wider than long (terminal length = 2/3 terminal width; Figs. 6B1, B2, 8A1, A2). Two lobes on abactinal sur- face of terminals articulated with last marginals, last carinal on prominent notch of terminals (Fig. 8A1). On actinal view, terminals have an oval depression, where the last pair of distal ambulacral and adambulacral ossicles are articulated (Fig. 8A2). Stereom of terminal plate well preserved on actinal side (with smooth and regular calcitic trabeculae), altered and pitted on abactinal side (Fig. 8A3, A4). Primary and secondary spines attached in life position in adambulacrals, actinolaterals, marginals, and carinal os- sicles, with massive spine bases. Secondary spines arranged in groups around the carinal spine bases, closely spaced in other plates of actinal and abactinal surface. When not preserved, secondary spine position is inferred by circular PALÓPOLO ET AL.—A NEW EOCENE ASTEROID FROM ANTARCTICA 309 Fig. 6. Zoroasterid asteroid Zoroaster marambioensis sp. nov., Eocene, Cucullaea I Allomember, La Meseta Formation of Seymour Island, Antarctica. A. IAA-Pi-373-K, abactinal (A1) and actinal (A2) views of a regenerating arm tip, note the size differences between ossicles and terminal small and inconspicuous. B. IAA-Pi-373-N, abactinal (B1) and actinal (B2) views of arm tip. C. IAA-Pi-373-L, regenerating arm tip on abactinal (C1) and actinal (C2) views, note small ossicles in chaotic arrangement on abactinal side. D. IAA-Pi-373-Q1, transverse section of a partially deformed ray. E. Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E IAA-Pi- 373-Q4, actinal view of a ray on the second third section, note that the third row of actinolaterals is reduced towards the arm tip (arrows). F. IAA-Pi-373-E, close-up of popular pore with small pedicellariae basal plate (arrow). G. IAA-Pi-373-E, small pedicellariae blades (arrow) on associated to a non-carinate adambulacral (arrow). H. IAA-Pi-373-R, inclined views of arms in life position; furrow with two big and three small pedicellariae (arrows) (H1); arm with three big pedicellariae (arrows) (H2). Fig. 6. Zoroasterid asteroid Zoroaster marambioensis sp. nov., Eocene, Cucullaea I Allomember, La Meseta Formation of Seymour Island, Antarctica. A. IAA-Pi-373-K, abactinal (A1) and actinal (A2) views of a regenerating arm tip, note the size differences between ossicles and terminal small and inconspicuous. B. IAA-Pi-373-N, abactinal (B1) and actinal (B2) views of arm tip. C. IAA-Pi-373-L, regenerating arm tip on abactinal (C1) and actinal (C2) views, note small ossicles in chaotic arrangement on abactinal side. D. IAA-Pi-373-Q1, transverse section of a partially deformed ray. E. IAA-Pi- 373-Q4, actinal view of a ray on the second third section, note that the third row of actinolaterals is reduced towards the arm tip (arrows). F. IAA-Pi-373-E, close-up of popular pore with small pedicellariae basal plate (arrow). G. IAA-Pi-373-E, small pedicellariae blades (arrow) on associated to a non-carinate adambulacral (arrow). H. IAA-Pi-373-R, inclined views of arms in life position; furrow with two big and three small pedicellariae (arrows) (H1); arm with three big pedicellariae (arrows) (H2). 5C, 9A1–A3), consistently sized marginals (Figs. 4A, 7D, 9B1), secondary spines widely spaced in actinal and abacti- nal surface (Figs. 5, 6H), carinate adambulacral plates alternating with non-carinate plates (Fig. 6E) and podial pores quadriserial proximally, becoming biserial distally. Within the imbricate Zoroasteridae, Bythiolopus Fisher, 1916, Doraster Downey, 1970, and Cnemidaster Sladen, 1889, have internal buttress and a ring of oral pedicellariae, while these structures are absent in Zoroaster and Pholi daster Sladen, 1889. Also, Bythiolopus and Pholidaster have alternated big and small marginal plates, while in Zoroaster marginals are consistently sized. Cnemidaster has enlarged, rounded and swollen disc plates, and discontinuous disc and arm plates. Doraster has a similar disc plate arrangement, but differs from Cnemidaster in having highly stellated disc plates. Zoroaster has weakly lobated disc plates, similar in size with the arm plates. Fig. 7. Diagrams of plate arrangement on disc and arms of zoroasterid aster- oid Zoroaster marambioensis sp. nov., Eocene, Cucullaea I Allomember, La Meseta Formation of Seymour Island, Antarctica. Note the presence of a single row of marginal ossicles. A. IAA-Pi-373-B, abactinal view of disc. B. IAA-Pi-373-H, transverse section of arm. C. IAA-Pi-373-Q1, transverse section of a partially deformed ray. D. Shape and arrangement of all the ossicle rows of an arm, projected on a plane. D not to scale. Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E The body wall in Cnemidaster is covered by membranous skin, while in Zoroaster is cov- ered by secondary spines, that are frequently absent in marks on the ossicles (Figs. 4D1, F2, G1, H1, 5C1, C2). There are two types of secondary spines; those on the actinal surface are slender and longer than those on the abactinal surface (Figs. 4F2, G1, 6G, H1, H2). Pedicellariae straight, 200–600 μm long, formed by two blades and a cup-shaped basal piece. Pedicellariae significantly more abundant on actinal surface than in abactinal surface, inferred by the presence of complete pedicellariae and basal pieces without attached blades on both actinal and abactinal surfaces. marks on the ossicles (Figs. 4D1, F2, G1, H1, 5C1, C2). There are two types of secondary spines; those on the actinal surface are slender and longer than those on the abactinal surface (Figs. 4F2, G1, 6G, H1, H2). Pedicellariae straight, 200–600 μm long, formed by two blades and a cup-shaped basal piece. Pedicellariae significantly more abundant on actinal surface than in abactinal surface, inferred by the presence of complete pedicellariae and basal pieces without attached blades on both actinal and abactinal surfaces. Actinal surface of disc preserved in detail. Actinostome deeply sunken, in a central depression (Figs. 5B2, 9B2). Oral area delimited by one pair of adambulacrals from each arm, articulated with small orals (Figs. 5A, B2, 9B2). Each oral ossicle bearing oral spines (Fig. 9A4). Remarks.—Studied specimens are assigned to Zoroaster because of disc characters (i.e., weakly lobate disc plates, disc and arm plates continuous, see Figs. 4A, 5A2, 7A, 8B2), imbricate ossicle arrangement (Figs. 4, 9), the presence of plates aligned in transverse and longitudinal series (Figs. 310 ACTA PALAEONTOLOGICA POLONICA 66 (2), 2021 Doraster and Pholidaster. Straight pedicellariae are absent in Pholidaster but present in Zoroaster. Doraster and Pholidaster. Straight pedicellariae are absent in Pholidaster but present in Zoroaster. Zoroaster marambioensis sp. nov. has the same number of actinolateral rows as Zoroaster microporus Fisher, 1916, but the former has spines only in the three or four actino- lateral lower rows while the latter has primary spines in all actinolaterals. Also, Z. microporus lacks primary spines on carinals and marginals, while Z. marambioensis sp. nov. has primary spines on each carinal, and on some proximal marginals. The carinal plates of Z. marambioensis sp. nov are weakly lobate, as in all the other species of the genus, ex- cept Z. Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E microporus, which has quadrate carinals. Zoroaster marambioensis sp. nov. has quadriserial arrangement of po- dial pores proximally, while Z. microporus and Zoroaster ophiactis Fisher, 1916, have a biserial tube feet arrangement. Zoroaster carinatus Alcock, 1893, differs from Antarctic species by showing a centrally domed disc, disc plates with- out secondary spines, lack of carinal and marginal spines along arms, and quadriserial tube feet along almost all of the rays. Zoroaster marambioensis sp. nov. is similar to Zoroaster variacanthus McKnight, 2006, in having four or five rows of actinolaterals at arm base reduced to three or two at prox- imal half of the arm (the lower three with a large and usually flattened spine), plates densely covered by secondary spines partially obscuring ossicle outlines, and rare pedicellariae in abactinal surface. Nevertheless, Z. variacanthus has longer than wide carinal plates, inconspicuous madreporite, more than one spine on disc plates, and spines in each marginal plate. Zoroaster marambioensis sp. nov. differs from Zoroaster fulgens Thomson, 1873, in having a flattened or slightly depressed central disc plate (Fig. 9B1, C2); weakly lobate radials overlapping polygonal interradials on the disc circlet (Figs. 4B2, 5A2, 9A3, B1, C2); marginal plates hexagonal or polygonal in shape lacking spines distally (Figs. 4A, D1, G1, 5C; 7B–C, 9B1); but with spines present on all carinals and radials (Figs. 4B2, H1, 6A2, D, 7C; note that in Z. fulgens primary carinal spines are absent and radials and interradial plates of disc have the same shape). Z. marambioensis sp. nov. has wider than long terminal ossicle, with a prominent notch (Figs. 6B1, B2, 8A1, A2), while terminal plates on Z. ful gens are longer than wide and have reduced notch. Also, their primary and secondary spine arrangement on the actinal sur- face is different (Z. fulgens has two primary spines and three secondary spines by each carinate adambulacral, while Z. marambioensis sp. nov. has one to three primary spines and one or two secondary spines by each adambulacral; see Figs. 6G, H1, H2, 8B1, B2). In Z. marambioensis sp. nov. the adradi- PALÓPOLO ET AL.—A NEW EOCENE ASTEROID FROM ANTARCTICA 311 als and the last row of actinolaterals extends until most distal part of the arm, near the terminal ossicle, separated from it only for the last marginal plate (Figs. 6A1–C2, 8A1, A2), while in Z. Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E fulgens the last adradial and actinolateral plates are aligned with the fourth or fifth marginal plate (countin from the arm tip). This actinolateral row arrangement i closer to those described by Ludwig (1905) for Zoroaste magnificus than the observed in Z. fulgens specimens. Fig. 8. Zoroasterid asteroid Zoroaster marambioensis sp. nov., Eocene, Cucullaea I Allomember, La Meseta Formation of Seymour Island, Antarctic A. IAA-Pi-373-N, arm tip, detailed abactinal (A1) and actinal (A2) views of distal and terminal ossicles; detailed stereom structure of terminal ossicle i abactinal (A3) and actinal (A4) surface, note that the fossil was preserved in life poisition, then the stereom in abactinal surface was unaltered. B. IAA Pi-373-G; position and arrangement of primary and secondary spines and pedicellariae in actinal inclined side of arm (B1); lateral view of arm, showin spines and pedicellariae associated to actinolaterals (B2). Abbreviations: 1Sp, primary spine; 2Sp, secondary spine. Fig. 8. Zoroasterid asteroid Zoroaster marambioensis sp. nov., Eocene, Cucullaea I Allomember, La Meseta Formation of Seymour Island, Antarctica. A. IAA-Pi-373-N, arm tip, detailed abactinal (A1) and actinal (A2) views of distal and terminal ossicles; detailed stereom structure of terminal ossicle in abactinal (A3) and actinal (A4) surface, note that the fossil was preserved in life poisition, then the stereom in abactinal surface was unaltered. B. IAA- Pi-373-G; position and arrangement of primary and secondary spines and pedicellariae in actinal inclined side of arm (B1); lateral view of arm, showing spines and pedicellariae associated to actinolaterals (B2). Abbreviations: 1Sp, primary spine; 2Sp, secondary spine. are aligned with the fourth or fifth marginal plate (counting from the arm tip). This actinolateral row arrangement is closer to those described by Ludwig (1905) for Zoroaster magnificus than the observed in Z. fulgens specimens. als and the last row of actinolaterals extends until most distal part of the arm, near the terminal ossicle, separated from it only for the last marginal plate (Figs. 6A1–C2, 8A1, A2), while in Z. fulgens the last adradial and actinolateral plates 312 ACTA PALAEONTOLOGICA POLONICA 66 (2), 2021 A d ib d b H ll t l (2004) R t Z f l b t h t f Z f l i h t t i ll Fig. 9. Zoroasterid asteroid Zoroaster marambioensis sp. nov., Eocene, Cucullaea I Allomember, La Meseta Formation of Seymour Island, Antarctica. Volume rendering captions from microCT (SOM 2). A. Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E fulgens Blake and Zinsmeister, 1979), with few to no character differences (Mah 2007). from New Zealand. Despite the poor preservation of Z. whan gareiensis, several differences with Z. marambioensis sp. nov. are recognizable. Z. whangareiensis has carinal spines placed proximally after the 4th or 5th carinal, and distally every second carinal; enlarged, central, single, conical spines every second marginal and 5–6 small spinules for each marginal. Z. marambioensis sp. nov. is larger than Z. whangareiensis and has a more robust armature. Madreporite, terminal ossicles, pedicellariae, secondary spines, ambulacral armature, spine number, size and arrangement on oral surface are unknown characters in Z. whangareiensis. According to the descriptions of species of Zoroaster, it seems likely that the diagnostic characters within the genus could be (i) type of primary and secondary spines (com- bining shape and size), (ii) actinal armature configuration (including number of primary and secondary spines asso- ciated with prominent adambulacral plates; number, shape and size of pedicellariae within the actinal area; number of spines that are directed into the furrow; etc.), (iii) number of actinolateral plate series, and (iv) spine distribution within abactinal and lateral plate series. Stratigraphic and geographic range.—Type locality and horizon only. Zoobank LSID: urn:lsid:zoobank.org:act:A9C9FB9D-A846-48CD- A6A3-FB3B8B65A00E IAA-Pi-373-E; general actinal surface reconstruction (A1); detailed arm structure on actinal side (A2), see primary and secondary spines preserved in detail and last ossicles of 4th actinolateral row (arrows). general abactinal surface reconstruction (A3) transverse view of disc structures, see orals and oral spines on disc center (A4). B. IAA-Pi-373-D, general reconstructions of abactinal (B1) and actinal (B2) surfaces. C. IAA-Pi-373-G; reconstructions of abactinal (C1) and actinal (C2) surfaces; structure detail on inclined side of arm (C3); transverse section of a ray (C4). Fig 9 Zoroasterid asteroid Zoroaster marambioensis sp nov Eocene Cucullaea I Allomember La Meseta Formation of Seymour Island Antarctica Fig. 9. Zoroasterid asteroid Zoroaster marambioensis sp. nov., Eocene, Cucullaea I Allomember, La Meseta Formation of Seymour Island, Antarctica. Volume rendering captions from microCT (SOM 2). A. IAA-Pi-373-E; general actinal surface reconstruction (A1); detailed arm structure on actinal side (A2), see primary and secondary spines preserved in detail and last ossicles of 4th actinolateral row (arrows). general abactinal surface reconstruction (A3) transverse view of disc structures, see orals and oral spines on disc center (A4). B. IAA-Pi-373-D, general reconstructions of abactinal (B1) and actinal (B2) surfaces. C. IAA-Pi-373-G; reconstructions of abactinal (C1) and actinal (C2) surfaces; structure detail on inclined side of arm (C3); transverse section of a ray (C4). As described by Howell et al. (2004), Recent Z. fulgens from the Atlantic Ocean appears as three morphotypes that could show reproductive isolation: the robust morphotype, the slender form and the long-armed one, which inhabit at depths of 975–1750 m, 1300–2200 m, and 3300–4020 m, respectively. This suggests that cryptic species are pres- ent across the bathymetric range of this species. Zoroaster marambioensis sp. nov. shows many similarities with the robust morphotype of Z. fulgens in characters typically as- sociated with shallower environments (i.e., solid skeletons, short arms and strong oral armature), although the former were found in beds deposited at 10–20 m depth and the latter inhabits (at least) at 200 m depth. In addition to Zoroaster marambioensis, described herein from Seymour Island, only one other Eocene fossil Zoroaster species is known, i.e., Zoroaster whangareiensis Eagle, 2006, PALÓPOLO ET AL.—A NEW EOCENE ASTEROID FROM ANTARCTICA 313 grouping the other seven species in a single clade (Zoroaster actinocles Fisher, 1919, Zoroaster macracantha Clark, 1916, Zoroaster magnificus Ludwig, 1905, Zoroaster ophiactis Fisher, 1916, Zoroaster ophiurus Fisher, 1905, Zoroaster spinulosus Fisher, 1906, and Zoroaster aff. Z. Systematic and phylogenetic analyses ophiurus is recovered in all trees, supported by characters 71 (pedicel- lariae associated to carinal ossicles), 72 (big pedicellariae on abactinal surface), 73 (five actinolateral rows) and 78 (5–8 pedicellariae on innermost spine of carinate adambu- lacrals). This consensus tree rendered a basal group within the class Asteroidea including Calliasterella, Trichasteropsis, and Ampheraster (node 26) and Neomorphaster. The lat- ter remains as sister group of all the Zoroasteridae clade. The group including species of Myxoderma and Sagenaster (node 30) is well separated from the rest of the zoroasterid species. Thus, “reticulated” and “imbricated” zoroasterids are clearly differentiated (as established by Mah 2007). A third well-supported group is the clade “Cnemidaster + Doraster + Bythiolopus” (node 33). It is supported by characters 19 (1.19), 44 (3.10), 66 (8.8) and 74. The genera Zoroaster and Pholidaster are grouped to- gether (node 43), with Pholidaster as sister group of Zoro aster. Zoroaster microporus remains basal to all species of Zoroaster (node 42). Within the other Zoroaster species, Z. actinocles, Z. fulgens, Z. macracantha, and Z. ophiurus (node 36) are recovered as sister group of Z. marambio ensis sp. nov., Eocene Zoroaster aff. Z. fulgens Blake and Zinsmeister, 1979, Z. magnificus, and Z. spinulosus (node 39). Node 36 is supported by characters 76 and 77, while node 39 is supported only by character 78. Within node 39, Z. marambioensis sp. nov. is supported by characters 16 (1.16), 17 (1.17), and 65 (8.7). Fifteen characters code differently for Z. fulgens, Eocene Zoroaster aff. Z. fulgens Blake and Zinsmeister, 1979, and Z. marambioensis sp. nov. Among these, seven characters cannot be observed in the studied specimens of Z. maram bioensis sp. nov. as they were all related to soft tissue or superambulacral features. The material studied herein are remarkably similar to those of the Eocene Z. aff. Z. fulgens from Seymour Island. A more detailed review of certain characters not recogniz- able in the specimens studied by Blake and Zinsmeister (1979) allowed improving the description of the species and clearly differentiate the specimens found in Seymour Island from Z. fulgens. The Bootstrap and Jackknife values show (within the Zoroaster species) a strongly supported group including Z. macracantha and Z. ophiurus. The clade Z. fulgens– Z. macracantha + Z. ophiurus is moderately to poorly sup- ported, but is recovered in all the most parsimonious trees as separated from the other Zoroaster species. Systematic and phylogenetic analyses fulgens Blake and Zinsmeister, 1979, but Blake and Zinsmeister (1979) did not mention the presence of soft parts and thus the states of these characters cannot be assumed as present or absent in fossils. It remains unclear then how these could be presence/ absence characters in the Eocene species. Despite these observations, Z. marambioensis sp. nov. characters were added to the previous matrix without previously modifying it, i.e., we used the original matrix published by Mah (2007). multaneously killed and buried by a rapid event. The fossil assemblage could be assigned to taphofacies IIA, as they are concentrated in a particular 3–5 cm thick bed, well-calci- fied, showing little or no breakage and minor to no abrasion, corrosion, or bioerosion (Brett et al. 1997). Zoroasterids colonized a distal part of an estuary under normal marine salinity and were killed by the input of freshwater carried by a hyperpycnal flow, and immediately buried by fine grained sand from suspension clouds related to the buoyant inver- sion of hyperpycnal flows at flow margin areas. Preservation of labile structures (i.e., primary and sec- ondary spines, and pedicellariae basal plates with or with- out articulated blades; Fig. 6I–L) helped to assess possible hypotheses regarding the studied material. Straight pedi- cellariae and cup-shaped basal plates were observed in our specimens, which are closely similar to those found in the extant species of the genus. Results seem to show that char- acters of pedicellariae did not change since the Eocene. Nine characters were added to the matrix of Mah (2007) (Appendix 1, SOM). A Heuristic Search with PAUP and TNT software returned 7 most parsimonious trees with a tree length of 172. Values of Consistency Index, Homoplasy Index and Retention Index were 0.6395, 0.3605, and 0.7989, respectively. Consensus tree is displayed in Fig 10. Phylogenetic results support the “reticulate” and “imbri cate” zoroasteridae identified by Mah (2007). The former includes Myxoderma and Sagenaster, while the latter com- prises Bythiolopus, Doraster, Cnemidaster, Pholidaster, and Zoroaster. The characters added to the matrix slightly changed the cladogram. Zoroaster microporus was retained as basal within the Zoroaster species, then Z. carinatus was identified as basal to a cluster of nine species. The cluster including Z. fulgens, Z. macracantha, and Z. Systematic and phylogenetic analyses The addition of characters to the matrix published by Mah (2007) reveals that a profound revision of Zoroaster is needed to clearly establish which are the key characters to identify species within this genus. Future research should include morphological characters related to the “ambulacral armature” and the “disc plate arrangement” as well as mo- lecular data (for extant species). Molecular characters could prove a higher resolution than morphological characters, considering that Howell et al. (2004) stated that the gene flux and reproductive isolation of the morphotypes of Z. fulgens could indicate a depth-controlled speciation. Systematic and phylogenetic analyses We carefully re-evaluated the matrix published by Mah (2007) and found out that some characters in this matrix seem to be inconsistent. Character 1.21 is a presence-ab- sence character (0–1), but it is coded “2” for C. wyvillei and C. sigsbeii. Character Group 3 has some problems too, i.e., Calliasterella species do not have actinal or actinolateral plates, as stated by Mah (2007) in character 3.1. Characters Mah (2007) published an extensive revision of the family Zoroasteridae. Although the author did not attempt a re- view of the genus Zoroaster, he found that resolution within the Zoroaster clade is poor, only moderately supporting the clades Zoroaster carinatus and Zoroaster fulgens, and Fig. 10. Most parsimonious (A) and consensus (B) tree from the Zoroasterida italics to the left above the node numbers in the cladogram. Fig. 10. Most parsimonious (A) and consensus (B) tree from the Zoroasteridae. Bootstrap values are in boldface below and Jackknife values are shown in italics to the left above the node numbers in the cladogram. Fig. 10. Most parsimonious (A) and consensus (B) tree from the Zoroasteridae. Bootstrap values are in boldface below and Jackknife values are shown in italics to the left above the node numbers in the cladogram. 314 ACTA PALAEONTOLOGICA POLONICA 66 (2), 2021 3.2–3.6 refer to distribution, orientation, and spinulation of such ossicle type and are coded in Calliasterella as absent (3.1, 3.2, 3.3, and 3.5) and imbricate (3.4), perhaps meaning lack of orientation, spines, and density instead of ossicle ab- sence. Finally, soft tissue characters (5.1 and 5.2) are coded as absent for Eocene Zoroaster aff. Z. fulgens Blake and Zinsmeister, 1979, but Blake and Zinsmeister (1979) did not mention the presence of soft parts and thus the states of these characters cannot be assumed as present or absent in fossils. It remains unclear then how these could be presence/ absence characters in the Eocene species. Despite these observations, Z. marambioensis sp. nov. characters were added to the previous matrix without previously modifying it, i.e., we used the original matrix published by Mah (2007). 3.2–3.6 refer to distribution, orientation, and spinulation of such ossicle type and are coded in Calliasterella as absent (3.1, 3.2, 3.3, and 3.5) and imbricate (3.4), perhaps meaning lack of orientation, spines, and density instead of ossicle ab- sence. Finally, soft tissue characters (5.1 and 5.2) are coded as absent for Eocene Zoroaster aff. Z. PALÓPOLO ET AL.—A NEW EOCENE ASTEROID FROM ANTARCTICA Some fossil zoroasterids appear in locations and envi- ronments different from those where extant members of the family live, although this could be due to an artefact of depositional environment. Eocene zoroasterids were epi- faunal, inhabited shallow environments in the proximal platform, and coexisted with several groups of predatory and scavenger organisms (Blake and Zinsmeister 1979). Recent species of Zoroaster live in deep water, where pred- ator pressure is lower than in shallow marine environments (Downey 1970; Howell et al. 2004; Mah, 2007; Aronson et al. 2009). there was an increase in high latitude surface sea tempera- ture during the early Eocene, with a climatic optimum in the middle Eocene. Surface sea temperatures at the Middle Eocene Climate Optimum were estimated in 10–17°C using δ18O (Douglas et al. 2014) and in 24°C with TEXL86 calibra- tion (Bijl et al. 2013). After that there was a sharp cooling caused by the opening of the Drake Passage and the devel- opment of the Antarctic Circumpolar Current, with a 7–9°C drop in sea water temperatures at the Eocene/Oligocene boundary (Aronson et al. 1997; Aronson and Blake 2001; Ivany et al. 2008; Casadío et al. 2010). As sedimentological data suggest that Z. marambioensis sp. nov. and Z. whanga reirensis lived in shallow-water environments, it seems pos- sible that they were able to tolerate a broader temperature range than other Recent species of the genus, which inhabit cold, deep marine environments. ) Meyer and Oji (1993) suggested that predation pressure and temperature are very important factors that could con- trol the presence of echinoderms in Eocene nearshore envi- ronments in the Antarctic continent. Teleosts and other pred- ators (including sea urchins) generated selection pressure on the crinoids causing their migration into deeper waters (Aronson et al. 1997, 2009). In fact, Gorzelak et al. (2012) concluded that benthic predation by sea urchins was an im- portant, if not the main, causal driver of biological change throughout the Mesozoic, and that it may have set the stage for the recent pattern in which motile crinoids greatly pre- dominate over sessile forms that live only at great depths. Like crinoids, stelleroids and the studied material shows no significant signals of damage caused by predation (Blake and Zinsmeister 1979, 1988; Baumiller and Gaździcki 1996; Aronson et al. 1997; this publication), except for two frag- ments of possible regenerated arms of Zoroaster marambi oensis sp. nov. (Fig. PALÓPOLO ET AL.—A NEW EOCENE ASTEROID FROM ANTARCTICA 6A, B, E, F). Also, teleost fishes, which prey upon asteroids, are poorly represented in La Meseta Formation (Clarke and Johnston 2003). Therefore, it could be possible that these factors aided in the survival of those echinoderm groups in shallow water environments until the late Eocene. The discovery of new strata with echi- noderm concentrations in the La Meseta Formation could reinforce the anomaly defined by Aronson et al. (1997) as “anachronistic, Paleozoic-type, low-predation communi- ties”, where echinoderms were dominant and show almost non-existent damage and regeneration rates. Whittle et al. (2018), however, argued that benthic marine faunas from South America, Antarctica, Australia and New Zealand had the same community structure with a continuous record of shallow marine stalked crinoids from the Cretaceous to the Paleogene, without signs of reversions. They also stated that subsequent changes in benthic faunal composition could have been driven either by increasing predation pressure or, more likely, by competition with other echinoderm groups (such as comatulid crinoids). Regarding changes in depth of habitat, we agree that, considering the current knowledge on the group and the fossil material available, the onshore-offshore theory could apply in this case. Migration to deeper habitats could prob- ably have been related to climate changes occurring in Antarctica during the Oligocene–Miocene. Unfortunately, Oligocene marine rocks preserved in Patagonia (i.e., the San Julián Formation) are not widespread and in all cases do not record deep environments. This kind of environment is not recorded in other post-Eocene deposits either in areas of the southern Atlantic Ocean or areas surrounding Seymour Island, thus limiting our chances of recovering material of Zoroaster that could prove such a migration of species of this genus into deeper waters or, conversely, its migration onto shallow water in Antarctica during the Eocene. Acknowledgements Authors acknowledge Carolina Acosta Hospitaleche and Javier Gelfo (both Universidad Nacional de La Plata, Argentina) for helping with the specimens collection in the field; Sergio Marenssi (Universidad de Buenos Aires, Argentina) and Carlos Zavala (Universidad Nacional del Sur, Bahía Blanca, Argentina) for the comments on the stratigraphy and sedimentology; and Carolina Martín Cao-Romero (Universidad Nacional Autónoma de México, Ciudad de México, México) for the relevant comments about important characters to differentiate species of the genus. We also thank the very useful comments and suggestions made by the reviewers, Christopher Mah (National Museum of Natural History, Washington, USA), Daniel Blake (University of Illinois, Springfield, USA), and an anonymous. This publication was supported by CONICET and Instituto Antártico Argentino. Funding to this project was provided by Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (PICT 2018-917). The Antarctic continent experimented great climatic changes since the middle Eocene. During the Paleogene, surface water temperature was stable between 10–15°C (Zinsmeister 1982; Meyer and Oji 1993). Several studies using δ18O (Gaździcki et al. 1992; Aronson and Blake 2001; Dutton et al. 2002 and references therein; Ivany et al. 2008) and, recently, multiproxy data including TEXL86 calibration of surface sea temperatures (Douglas et al. 2014) agree that Concluding remarks All evidence suggests that the layer yielding the fossil ma- terial studied represents an autochthonous simple episodic deposition event (Kidwell 1991), where starfishes were si- 315 Alcock, A. 1893. Natural History notes from the HM Indian Marine Survey Steamer Investigator. An account of the collection of deep-sea Aster- oidea. 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Montes, M., Nozal, F., Olivero, E., Gallastegui, G., Santillana, S., Maestro, ACTA PALAEONTOLOGICA POLONICA 66 (2), 2021 318 Ch. 71: Distribution of pedicellariae on abactinal surface. 0: asso- ciated to popular pores, 1: associated to carinals. Ch. 72: Size of pedicellariae on abactinal surface. 0: small, 1: big. Ch. 73: Number of actinolateral rows. 0: three, 1: four, 2: five, 3: six. Ch. 74: First actinolateral row differentiated from others. 0: absent, 1: present. Ch. 75: Actinolateral primary spines adpressed. 0: absent, 1: pres- ent. Ch. 76: Number of actinolateral pedicellariae. 0: none, 1: one, 2: more than one. Ch. 77: Size of actinolateral pedicellariae. 0: small, 1: big, 2: ped- icellariae absent. Ch. 78: Number of pedicellariae on carinate adambulacral inner most spine. 0: 0-2, 1: 2-5, 2: 5-8, 3: more than 8. Ch. 79: Both small and big pedicellariae associated to carinate adambulacrals. 0: absent, 1: present. Characters added to the zoroasterid matrix of Mah (2007). Ch. 76: Number of actinolateral pedicellariae. 0: none, 1: one, 2: more than one. Ch. 71: Distribution of pedicellariae on abactinal surface. 0: asso- ciated to popular pores, 1: associated to carinals. Ch. 72: Size of pedicellariae on abactinal surface. 0: small, 1: big. Ch. 73: Number of actinolateral rows. 0: three, 1: four, 2: five, 3: six. Ch. 74: First actinolateral row differentiated from others. 0: absent, 1: present. Ch. 75: Actinolateral primary spines adpressed. 0: absent, 1: pres- ent. Ch. 77: Size of actinolateral pedicellariae. 0: small, 1: big, 2: ped- icellariae absent. Ch. 78: Number of pedicellariae on carinate adambulacral inner most spine. 0: 0-2, 1: 2-5, 2: 5-8, 3: more than 8. Ch. 79: Both small and big pedicellariae associated to carinate adambulacrals. 0: absent, 1: present.
https://openalex.org/W2022526689	https://potravinarstvo.com/journal1/index.php/potravinarstvo/article/download/158/158	Slovak	null	Sensory evaluation of meat chickens Ross 308 after application of propolis in their nutrition	Potravinárstvo	2,012	cc-by	5,940	ÚVOD Ľudská populácia vytvára tlak na potrebu a tvorbu vysoko kvalitných univerzálnych potravín, ktoré sú zdrojom najmä bielkovín a preto sa v osttanom období neustále zvyšuje dopyt po hydinových výrobkoch (FAO, 2002). Pre tvorbu výrobkov z hydiny sú najpočetnejším chovaným živočíšnym druhom na svete brojlerové kurčatá (Perry et al., 2002; Moreki et al., 2010). Ľudská populácia vytvára tlak na potrebu a tvorbu vysoko kvalitných univerzálnych potravín, ktoré sú zdrojom najmä bielkovín a preto sa v osttanom období neustále zvyšuje dopyt po hydinových výrobkoch (FAO, 2002). Pre tvorbu výrobkov z hydiny sú najpočetnejším chovaným živočíšnym druhom na svete brojlerové kurčatá (Perry et al., 2002; Moreki et al., 2010). Racionálna výživa obyvateľstva sa v súčasnosti zameriava na vysoko stráviteľné živočíšne produkty. Z nich je za najvýznamnejší zdroj považované hydinové mäso, ktoré má vysokú nutričnú a biologickú hodnotu a jeho skladbu ovplyvňuje genotyp, výživa, vek, chovateľské prostredie a rôzne ďalšie extravitálne a intravitálne činitele (Jedlička, 1988; Karas, 1998; Holoubek, 2001; Haščík et al., 2005a). Mnoho autorov okrem selekcie a tvorby nových hybridných kombinácií kurčiat uskutočnilo experimenty aj za účelom návrhu zloženia nových kŕmnych zmesí a vytvorili rôzne modely pre dosiahnutie maximálnej úžitkovosti kurčiat (McDonald and Evans, 1977; Greig et al., 1977; Allison et al., 1978; Pesti et al., 1986; Gonzalez-Alcorta et al., 1994). Základom návrhu na tvorbu a zloženie používaných kŕmnych zmesí s ich správnym obsahom a pomerom živín a energie je dosiahnutie maximálnej úžitkovosti vyjadrenej prírastkom telesnej hmotnosti pri najekonomickejšom využití krmiva a dosiahnutom čo najvyššom zisku, pričom súbor požiadaviek na živiny a ich obmedzenia vytváraných špeciálne pre jednotlivé hybridné kombinácie kurčiat a kvalita ich jatočného tela sú ovplyvnené cenou surovín tvoriacich kŕmnu zmes a taktiež požiadavkami na nutričné zloženie mäsa kurčiat (Donaldson et al., 1957; Combs a Nicholson, 1964; Saleh et al., 2004; Cerrate a Waldroup, 2009). Benková et al. (2005) označujú hydinové mäso ako vhodnú komoditu pre tvorbu tzv. funkčných potravín pre ľudskú výživu, čo je v súčasnosti v centre záujmu humánneho, poľnohospodárskeho ako aj potravinárskeho výskumu. Produkcia hydinového mäsa pre ľudskú populáciu predstavuje dôležitý systém dodávky vysoko kvalitných proteínov, ktoré sú najdôležitejšou zložkou hydinového mäsa s vysokým obsahom esenciálnych aminokyselín (Straková et al., 2003; Gueye, 2009). potravinárstvo potravinárstvo ABSTRACT The objective of the experiment was to verify the effect of propolis extract in Ross 308 broiler on the sensory quality of breast and thigh muscle modified by baking at temperature 200 ºC for 60 minutes and finish baking for a period of 10-15 minutes. In the experiment were used 180 chickens divided into 2 groups (control and experimental group) with 90 chickens (45 ♂ and 45 ♀). Fattening lasted 40 days. The chickens were fed ad libitum with the same starter feed mixtures to 21 days and from 22 days of age through 40 days of age with the grower feed mixtures in the both followed groups. Feed mixtures were made without antibiotics and coccidiostatics. The feed mixtures used in experimental group were enriched with the feed extract of propolis in a dose of 0.2 g.kg-1. After heat treatment of breast and thigh muscle 60 pieces chickens (30 pieces ♀, ♂ 30 pieces) of each group samples were sensory analyzed (smell, taste, juiciness, softness). Statistically significant differences were found by sex (P≤0.05 to 0.001) in aroma and taste of cocks in the thigh muscle (+0.290 points, +0.300 points) and hens (P≤0.01) in flavor (+0.250 points) and softness (+0.372 points) in breast muscle. Sensory assessment of the individual characters of either gender had significant differences (P≤0.05 to 0.001) in favor of the experimental group achieved only in the evaluation of the smell in the breast (+0.207 points) as well as thigh muscle (+0.207 points). In the final evaluation the most valuable parts of Ross 308 chickens carcass were found a positive effect of propolis extract on their sensory properties, but significant differences (P≤0.01) were observed only in chickens in the breast muscle (+0.917 points) compared with control group. The results have confirmed that propolis extract in a dose of 0.2 g.kg-1 feed mixture can be applied in the diet of chickens, as it positively affects the sensory quality of Ross 308 chickens meat, which is one of the most important parts of chicken meat for use in human food chain. Keywords: Ross 308 chicken, propolis extract, sensory evaluation, breast and thigh muscle Potravinarstvo, vol. 6, 2012, no. 1, p. 14-20 doi:10.5219/158 Received: 9. January 2012. Accepted: 12. January 2012. Available online 15. February 2012 at www.potravinarstvo.com © 2011 Potravinarstvo. All rights reserved. ISSN 1337-0960 online Received: 9. January 2012. Accepted: 12. January 2012. Available online 15. February 2012 at www.potravinarstvo.com © 2011 Potravinarstvo. All rights reserved. ISSN 1337-0960 online SENSORY EVALUATION OF MEAT CHICKENS ROSS 308 AFTER APPLICATION OF PROPOLIS IN THEIR NUTRITION Jozef Garlík ml., Miroslava Kačániová, Juraj Čuboň, Martin Mellen, Michal Mihok, Ibrahim Omer Eliman Elimam Peter Haščík, Jozef Garlík ml., Miroslava Kačániová, Juraj Čuboň, Martin Mellen, M Ibrahim Omer Eliman Elimam MATERIÁL A METÓDY Experiment bol realizovaný v testovacej stanici hydiny Katedry hydinárstva a malých hospodárskych zvierat pri FAPZ SPU v Nitre na výkrmových kurčatách hybridnej kombinácie Ross 308. Do pokusu bolo zaradených 180 ks jednodňových kurčiat, z ktorých boli vytvorené 2 skupiny: kontrolná (K) a pokusná (P) po 90 ks kurčiat (45 ♂ a 45 ♀). Vlastný výkrm trval 40 dní. Kurčatá boli kŕmené systémom ad libitum rovnakou štartérovou kŕmnou zmesou HYD-01 (sypká štruktúra) do 21. dňa veku a od 22. dňa do 40. dňa rastovou kŕmnou zmesou HYD-02 (sypká štruktúra) v oboch sledovaných skupinách. Skrmované kŕmne zmesi HYD-01 a HYD-02 boli vyrobené bez antibiotických preparátov a kokcidiostatík. Priemerná výživná hodnota podávaných kŕmnych zmesí počas experimentu bola rovnaká v oboch skupinách, ale v pokusnej skupine bol navyše do kŕmnych zmesí HYD-01 a HYD-02 pridávaný extrakt propolisu v dávke 0,2 g.kg-1. Propolisový extrakt bol pripravený z rozomletého propolisu. Navážka propolisu bola 150 g a objem použitého 80 %-ného etanolu 500 cm3. Extrakcia prebiehala vo vodnom kúpeli pri 80 °C pod spätným chladičom po dobu 1 hodiny. Zmes bola po extrakcii a ochladení centrifugovaná. Získaný supernatant bol odparený na rotačnej vákuovej odparke pri teplote kúpeľa 40-50 °C a následne odvážený. Odparok v množstve 20 g bol rozpustený v 1000 cm3 80 %-ného etanolu a aplikovaný do 100 kg kŕmnej zmesi. Neoddeliteľnou súčasťou hodnotenia hydinového mäsa je posudzovanie jeho senzorickej kvality, ktorá patrí medzi najstaršie, aj keď menej objektívne metódy (Jedlička, 1988). Naopak Guárdia et al. (2010) považujú senzorickú analýzu za vedeckú disciplínu, ktorá nám umožňuje stanovovať charakteristiky výrobku objektívne a reprodukovateľne prostredníctvom zmyslov človeka, ale variabilitu medzi posudzovateľmi a rozdiely medzi vzorkami môžu značne zvýšiť aspekty ako je nejednotnosť v teplote vzoriek počas degustácie alebo poradie pri hodnotení. Neoddeliteľnou súčasťou hodnotenia hydinového mäsa je posudzovanie jeho senzorickej kvality, ktorá patrí medzi najstaršie, aj keď menej objektívne metódy (Jedlička, 1988). Naopak Guárdia et al. (2010) považujú senzorickú analýzu za vedeckú disciplínu, ktorá nám umožňuje stanovovať charakteristiky výrobku objektívne a reprodukovateľne prostredníctvom zmyslov človeka, ale variabilitu medzi posudzovateľmi a rozdiely medzi vzorkami môžu značne zvýšiť aspekty ako je nejednotnosť v teplote vzoriek počas degustácie alebo poradie pri hodnotení. Jedlička (1988), Uhrín et al. (1993) a Haščík et al. (2004) charakterizujú zmyslové posúdenie kvality hydinového mäsa ako subjektívne hodnotenie, pretože schopnosť vnímania predovšetkým chute a vône u ľudí je značne variabilná, ale chuť a čuch ako najdôležitejšie zmysly človeka zatiaľ nie je možné nahradiť žiadnou aparatívnou metódou. potravinárstvo potravinárstvo Nové legislatívne obmedzenia a zákazy EÚ pri využití živočíšnych múčok, klasických antibiotických stimulátorov rastu a antimikrobiálnych látok v krmivárstve pre výživu polygastrických a monogastrických zvierat vedú tak vo vede ako aj praxi k alternatíve aplikovania nových biotechnologických možných doplnkov a produktov (Haščík et al., 2006, 2007; Bobko et al., 2009). Vo výžive kurčiat sa už bežne používajú kompletné kŕmne zmesi, ktoré sú často obohacované v ostatnom období o prídavok rôznych doplnkov vrátane rastlinných silíc, probiotických, prebiotických a enzymatických preparátov (Berri, 2000; Lee et al., 2003, 2004; Angelovičová et al., 2006, 2008; Khojasteh a Shivazad, 2006; Haščík et al., 2006, 2007; Angelovičová a Angelovič, 2009). neškodnosťou a cenou sú rozhodujúcimi kritériami pre jeho úspešnosť na trhu. Civille a Szczesniack (1973) ako jednu z najdôležitejších častí metodického postupu senzorickej analýzy považujú výber kandidátov v závislosti od ich psychologických alebo fyziologických schopností. Odborník na senzorickú analýzu musí mať predovšetkým správne zmyslové schopnosti, má určenie zmyslovej citlivosti, vnímania a komunikáciu (Meilgaard et al., 1987). Na základe vyššie uvedených skutočností bolo cieľom našej práce preveriť využitie bežne vyrábaných komerčných kŕmnych zmesí s doplnkom propolisu na senzorické hodnotenie prsnej a stehennej časti jatočne opracovaného tela kurčiat Ross 308 po ich tepelnej úprave pečením. Ako alternatívy sa využívajú aj včelie produkty (peľ, propolis, resp. ich extrakty), ktoré v konečnom dôsledku môžu mať tiež pozitívny vplyv na zdravotný stav, hospodárske využitie krmiva, nutričnú a senzorickú kvalitu produktu, ako aj ekonomiku výroby v hydinárskom priemysle (Chrappa et al., 1991; Kováč et al., 1993; Výmola et al., 1995; Kimoto et al., 1999; Mojto a Zaujec 2001; Prytzyk et al., 2003; Haščík et al. 2004; Wang et al., 2004; Haščík et al., 2005ab, 2007; Shalmany a Shivazad, 2006; Seven et al., 2008; a i.). Zloženie včelích produktov často závisí od ich času získavania a rastlinného zdroja (Greenaway et al., 1991; Markham et al., 1996). ÚVOD Bielkoviny kuracieho a morčacieho mäsa majú v porovnaní s bravčovým a hovädzím mäsom viac a v priaznivejšom pomere esenciálnych aminokyselín, najmä arginínu, leucínu, izoleucínu, metionínu a valínu, pričom hydinové mäso je aj zdrojom lipidov, ktoré sú rezervoárom energie, vitamínov rozpustných v tuku a dodávateľom esenciálnych mastných kyselín (Benková et al., 2005). Volume 6 14 No. 1/2012 MATERIÁL A METÓDY Ďalšie senzorické vlastnosti mäsa, ku ktorým patrí aj jemnosť (textúra, tuhosť, tvrdosť, mäkkosť) mäsa, resp. šťavnatosť mäsa, je možné okrem senzorického posúdenia vyhodnotiť aj napríklad za pomoci penetrometra, konzistometra, resp. stanovením obsahu vody vo vzorke mäsa. Senzorické hodnotenie sa najčastejšie vykonáva po tepelnej úprave, pričom pre každú sledovanú vlastnosť, t.j. vôňu, chuť, šťavnatosť a jemnosť sa používa 5-bodová stupnica, t.j. za komplexné posúdenie kvality mäsa je maximálny počet 20 bodov. Na konci experimentu (40. deň výkrmu) bolo z každej skupiny vybratých po 60 ks kurčiat na jatočný rozbor (30 ks sliepočiek a 30 ks kohútikov), ktorý sa uskutočnil na Katedre hodnotenia a spracovania živočíšnych produktov pri FBP SPU Nitra s následným zhodnotením senzorických (kulinárskych) vlastností prsnej a stehennej časti jatočného tela kurčiat po tepelnej úprave pri 200 °C počas doby 60 minút a dopečení v trvaní 10 až 15 minút. Senzorické posúdenie anonymných vzoriek bolo uskutočnené 6-člennou komisiou, kde pre vlastné vyhodnotenie sa použila metóda hodnotenia 5-bodovou stupnicou. Z hľadiska senzorickej analýzy sme sledovali vôňu, chuť, šťavnatosť a jemnosť mäsa. Sledované senzorické vlastnosti závisia podľa Augustina a Fischera (1999), Brestenského (2002), Mojta a Zaujeca (2003), Haščíka et al. (2004) od druhu použitého krmiva, intramuskulárneho tuku, množstva extraktívnych látok, spôsobu prípravy, výživy, genetiky a ďalších intravitálnych a extravitálnych činiteľov. Steinhauser et al. (1995) zároveň konštatujú, že senzorické posúdenie mäsa patrí do komplexu hodnôt, ktoré spolu so zdravotnou Výsledky experimentu (aritmetický priemer, smerodajná odchýlka, minimum, maximum, variačný koeficient) sme spracovali v štatistickom programe Statgraphics 5.0 a na určenie preukaznosti rozdielov medzi skupinami experimentu bol použitý F-test s následným t-testom. Volume 6 No. 1/2012 15 potravinárstvo (tabuľka 1-6), ako aj celkové senzorické hodnotenie prsnej a stehennej svaloviny (tabuľka 7-8) bez a po aplikácii propolisového extraktu v kŕmnych zmesiach výkrmových kurčiat Ross 308 sú uvedené v tabuľke 1-8. Výsledky Výsledky zmyslového posúdenia jednotlivých senzorických vlastností cenných častí jatočného tela kurčiat (prsná a stehenná svalovina) po úprave pečením Tabuľka 1 Senzorické hodnotenie stehien (sliepky) Tabuľka 1 Senzorické hodnotenie stehien (sliepky) Ukazovateľ Vôňa Chuť Šťavnatosť Jemnosť kontrola pokus kontrola pokus kontrola pokus kontrola pokus n 30 30 30 30 30 30 30 30 (body) 4,25 4,375 4,293 4,25 4,378 4,375 4,503 4,583 sx 0,0913 0,0913 0,1463 0,1581 0,001 0,0791 0,078 0,0697 min. 4,0 4,125 4,0 3,75 4,375 4,25 4,38 4,375 max. MATERIÁL A METÓDY 4,5 4,625 4,75 4,5 4,38 4,625 4,78 4,75 vk 5,26 5,11 8,35 9,11 0,06 4,42 4,24 3,73 t-test (P value) 0,356 0,845 0,967 0,462 Poznámka: n – počet vzoriek, – aritmetický priemer, sx – stredná chyba aritmetického priemeru, min. – minimum, max. – maximum, vk – variačný koeficient, t-test = P≥0,05 – štatisticky nepreukazná hodnota, P≤0,05+ štatisticky preukazná hodnota, P≤0,01++ štatisticky stredne preukazná hodnota, P≤0,001+++ štatisticky vysoko preukazná hodnota Poznámka: n – počet vzoriek, – aritmetický priemer, sx – stredná chyba aritmetického priemeru, min. – minimum, max. – maximum, vk – variačný koeficient, t-test = P≥0,05 – štatisticky nepreukazná hodnota, P≤0,05+ štatisticky preukazná hodnota, P≤0,01++ štatisticky stredne preukazná hodnota, P≤0,001+++ štatisticky vysoko preukazná hodnota Tabuľka 2 Senzorické hodnotenie stehien (kohúty) Tabuľka 2 Senzorické hodnotenie stehien (kohúty) Ukazovateľ Vôňa Chuť Šťavnatosť Jemnosť kontrola pokus kontrola pokus kontrola pokus kontrola pokus n 30 30 30 30 30 30 30 30 (body) 4,002 4,292 4,17 4,417 4,377 4,208 4,5 4,542 sx 0,0466 0,0264 0,0705 0,0527 0,078 0,0697 0,0913 0,095 min. 3,875 4,25 4,0 4,25 4,13 4,0 4,25 4,25 max. 4,13 4,375 4,38 4,5 4,5 4,375 4,75 4,75 vk 2,85 1,50 4,14 2,92 4,36 4,06 4,97 5,12 t-test (P value) 0,0003 0,019 0,139 0,758 Tabuľka 3 Senzorické hodnotenie pŕs (sliepky) Ukazovateľ Vôňa Chuť Šťavnatosť Jemnosť kontrola pokus kontrola pokus kontrola pokus kontrola pokus n 30 30 30 30 30 30 30 30 (body) 4,167 4,417 4,167 4,25 3,792 3,958 4,045 4,417 sx 0,0527 0,0264 0,0527 0 0,095 0,0697 0,1044 0,0264 min. 4,0 4,375 4,0 4,25 3,5 3,75 3,88 4,375 max. 4,25 4,5 4,25 4,25 4,0 4,125 4,375 4,5 vk 3,10 1,46 3,10 0 6,14 4,31 6,32 1,46 t-test (P value) 0,002 0,999 0,188 0,006 Tabuľka 4 Senzorické hodnotenie pŕs (kohúty) Ukazovateľ Vôňa Chuť Šťavnatosť Jemnosť kontrola pokus kontrola pokus kontrola pokus kontrola pokus n 30 30 30 30 30 30 30 30 (body) 4,127 4,292 4,21 4,25 3,958 3,958 4,293 4,25 sx 0,0457 0,0697 0,0253 0,0456 0,0697 0,095 0,0689 0,1208 min. 4,0 4,125 4,13 4,125 3,75 3,75 4,13 4,0 max. MATERIÁL A METÓDY 4,25 4,5 4,25 4,375 4,125 4,25 4,5 4,625 vk 2,71 3,98 1,47 2,63 4,31 5,88 3,93 6,96 t-test (P value) 0,076 0,461 1,00 0,762 Tabuľka 3 Senzorické hodnotenie pŕs (sliepky) Ukazovateľ Vôňa Chuť Šťavnatosť Jemnosť kontrola pokus kontrola pokus kontrola pokus kontrola pokus n 30 30 30 30 30 30 30 30 (body) 4,167 4,417 4,167 4,25 3,792 3,958 4,045 4,417 sx 0,0527 0,0264 0,0527 0 0,095 0,0697 0,1044 0,0264 min. 4,0 4,375 4,0 4,25 3,5 3,75 3,88 4,375 max. 4,25 4,5 4,25 4,25 4,0 4,125 4,375 4,5 vk 3,10 1,46 3,10 0 6,14 4,31 6,32 1,46 t-test (P value) 0,002 0,999 0,188 0,006 Tabuľka 4 Senzorické hodnotenie pŕs (kohúty) Ukazovateľ Vôňa Chuť Šťavnatosť Jemnosť kontrola pokus kontrola pokus kontrola pokus kontrola pokus n 30 30 30 30 30 30 30 30 (body) 4,127 4,292 4,21 4,25 3,958 3,958 4,293 4,25 sx 0,0457 0,0697 0,0253 0,0456 0,0697 0,095 0,0689 0,1208 min. 4,0 4,125 4,13 4,125 3,75 3,75 4,13 4,0 max. 4,25 4,5 4,25 4,375 4,125 4,25 4,5 4,625 vk 2,71 3,98 1,47 2,63 4,31 5,88 3,93 6,96 t-test (P value) 0,076 0,461 1,00 0,762 Volume 6 No. 1/2012 16 potravinárstvo Tabuľka 5 Senzorické hodnotenie pŕs (bez ohľadu na pohlavie) Ukazovateľ Vôňa Chuť Šťavnatosť Jemnosť kontrola pokus kontrola pokus kontrola pokus kontrola pokus n 60 60 60 60 60 60 60 60 (body) 4,147 4,354 4,188 4,25 3,875 3,958 4,169 4,333 sx 0,0338 0,0402 0,0286 0,0218 0,0615 0,0562 0,0704 0,0641 min. 4,0 4,125 4,0 4,125 3,5 3,75 3,88 4,0 max. 4,25 4,5 4,25 4,375 4,125 4,25 4,5 4,625 2,82 3,20 2,37 1,77 5,50 4,92 5,85 5,12 t-test (P value) 0,0007 0,100 1,328 0,099 Tabuľka 6 Senzorické hodnotenie stehien (bez ohľadu na pohlavie) Ukazovateľ Vôňa Chuť Šťavnatosť Jemnosť kontrola pokus kontrola pokus kontrola pokus kontrola pokus n 60 60 60 60 60 60 60 60 (body) 4,126 4,333 4,232 4,333 4,377 4,297 4,502 4,563 sx 0,0615 0,047 0,0796 0,0833 0,0372 0,0562 0,0572 0,0565 min. 3,875 4,125 4,0 3,75 4,13 4,0 4,25 4,25 max. 4,5 4,625 4,75 4,5 4,5 4,625 4,75 4,75 vk 5,17 3,76 6,52 6,66 2,94 4,53 4,41 4,29 t-test (P value) 0,014 0,387 0,216 0,458 Tabuľka 6 Senzorické hodnotenie stehien (bez ohľadu na pohlavie) Tabuľka 7 Celkové hodnotenie najcennejších častí jatočne opracovaného tela (sliepky) Ukazovateľ Stehná Prsia kontrola pokus kontrola Pokus n 30 30 30 30 (body) 17,333 17,583 16,125 17,042 sx 0,2157 0,3206 0,2415 0,0697 min. LITERATÚRA ALLISON, J. R., ELY, L. O., AMATO, S. V. 1978. Broiler profit maximizing models. In Poult. Sci., vol. 57, 1978, p. 845-853. ANGELOVIČOVÁ, M., MELLEN, M., ANGELOVIČ, M. 2006. Uplatnenie biotechnologického postupu náhrady kŕmneho antibiotika premixom škoricovej silice vo výžive výkrmových kurčiat. In Biotechnológie, JU: České Budĕjovice, 2006, p. 134-136, ISBN 8085- 645-53-X. Dosiahnuté pozitívne výsledky senzorického hodnotenia najcennejších častí JOT kurčiat Ross 308 v preverovanom experimente s aplikáciou propolisového extraktu v kŕmnych zmesiach sú v súlade s hodnotami a tendenciami, ktoré vo svojich pokusoch zistili pri aplikácii iných kŕmnych aditív vo forme rôznych probiotických preparátov vo výžive kurčiat Mudřík et al. (1990), Haščík et al. (2004, 2007) a Mihok et al. (2010). Pozitívne ovplyvnenie senzorických ako aj technologických vlastností mäsa vplyvom probiotických preparátov, ale aj rastlinných silíc a iných prirodzených aditív vo výžive kurčiat deklarujú aj Urminská a Michalík (1991), Brož (1991), Haščík et al. (2004, 2007), resp. Bobko et al. (2006, 2009). ANGELOVIČOVÁ, M., LADYKOVÁ, M., LIPTAIOVÁ, D., MOČÁR, K., ŠTOFAN, D. 2008. Riešenie náhrady kŕmnych antibiotík rastlinnými silicami pri výrobe kuracieho mäsa. In Otvorené fórum o stave bezpečnosti, kvality a kontroly potravín, Bratislava, 2008, p. 41–45. ANGELOVIČOVÁ, M., LADYKOVÁ, M., LIPTAIOVÁ, D., MOČÁR, K., ŠTOFAN, D. 2008. Riešenie náhrady kŕmnych antibiotík rastlinnými silicami pri výrobe kuracieho mäsa. In Otvorené fórum o stave bezpečnosti, kvality a kontroly potravín, Bratislava, 2008, p. 41–45. ANGELOVIČOVÁ, M., ANGELOVIČ, M. 2009. Zhodnotenie efektivity výkrmu kurčiat vo vzťahu k ich produkcii. In Bezpečnosť a kontrola potravín, SPU Nitra, 2009, p. 199-203, ISBN 978-80-552-0193-1. ANGELOVIČOVÁ, M., ANGELOVIČ, M. 2009. Zhodnotenie efektivity výkrmu kurčiat vo vzťahu k ich produkcii. In Bezpečnosť a kontrola potravín, SPU Nitra, 2009, p. 199-203, ISBN 978-80-552-0193-1. p AUGUSTIN, CH., FISCHER, K. 1999. Fleischreifung und sensorische Qulität. In Fleischwirtschaft, vol. 79, 1999, no. 12, p. 96-98. Zlepšené organoleptické (zmyslové) vlastnosti mäsa kurčiat potvrdili vo svojich štúdiách pri aplikácii tuku a olejnatých semien aj Połtowicz (2000), Osek et al. (2001), Barteczko et al. (2003), Marcinčák et al. (2009) a pri aplikácii cesnaku aj Kim et al. (2009). Autori zároveň konštatujú, že požadovanú a správnu technologickú, nutričnú a taktiež senzorickú kvalitu mäsa kurčiat je možné dosiahnuť len preverenými a otestovanými kŕmnymi doplnkami, čo potvrdzujú aj výsledky Bobka et al. (2006, 2009), ktorí upozorňujú, že nie všetky aditívne látky, resp. LITERATÚRA možné doplnky a ich množstvo aplikované vo výžive kurčiat má priaznivý vplyv na senzorické vlastnosti mäsa, pretože zistili pri aplikácii rôznych rastlinných silíc vo väčšom množstve vo výžive kurčiat aj opačnú tendenciu, t.j. mierne zhoršenú senzorickú kvalitu mäsa. BARTECZKO, J., BOROWIEC, F., WĘGLARZ, A. 2003. Chemical composition and sensory traits of meat of broiler chickens fed probiotic supplemented diets. In Ann. Anim. Sci., vol. 2, 2003, p. 169-173. BENKOVÁ, J., BAUMGARTNER, J., HETÉNYI, L. 2005. Hydinové mäso – významná zložka racionálne výživy obyvateľstva. In Realizácia komplexného programu ozdravenia výživy obyvateľstva SR – využitie nutričných poznatkov v primárnej a sekundárnej prevencii neinfekčných chorôb, no. 49, SAPV, Nitra, 2005, p. 31-32, ISBN 80-89162- 18-5. BERRI, C., BESNARD, J., RELANDEAU, C. 2008. Increasing dietary lysine increases final pH and decreases driploss of broiler breast meat. In Poult. Sci., vol. 87, 2008, no. 3, p. 480-484. BOBKO, M., LAGIN, L., KROČKO, M. 2006. Zmeny senzorických vlastností hydinového mäsa po nahradení antibiotík rastlinnými silicami. In Zborník z mezinárodní konference: „Drůbež a mléko ve výživě člověka“, ČZU Praha, 2006, p. 88-91, ISBN 80-213-1548-2. Č Á MATERIÁL A METÓDY 16,875 16,75 15,375 16,875 max. 18,0 18,5 16,625 17,25 vk 3,05 4,47 3,67 1,00 t-test (P value) 0,532 0,004 Tabuľka 8 Celkové hodnotenie najcennejších častí jatočne opracovaného tela (kohúty) Ukazovateľ Stehná Prsia kontrola pokus kontrola Pokus n 30 30 30 30 (body) 17,0417 17,458 16,583 16,833 sx 0,2342 0,2342 0,2058 0,2157 min. 16,5 16,75 16,0 16,375 max. 17,75 18,0 17,125 17,5 vk 3,37 3,29 3,04 3,14 t-test (P value) 0,237 0,421 Tabuľka 8 Celkové hodnotenie najcennejších častí jatočne opracovaného tela (kohúty Uk t ľ St h á Organoleptickým hodnotením stehennej svaloviny sliepok medzi kontrolnou a pokusnou skupinou neboli zistené štatisticky významné rozdiely (P≥0,05), ale u kohútov boli významné rozdiely (P≤0,001; P≤0,05) v prospech pokusnej skupiny v hodnotení vône (+0,290 bodu) a chuti (+0,300 bodu). V prsnej svalovine u sliepok boli dosiahnuté vyššie hodnoty u všetkých sledovaných zmyslových vlastností v pokusnej skupine oproti kontrole, ale významné rozdiely (P≤0,01) boli zistené len v hodnotení vône (+0,250 bodu) a jemnosti (+0,372 bodu). Kohúty v hodnotení prsnej svaloviny nedosiahli medzi kontrolnou a pokusnou Volume 6 No. 1/2012 No. 1/2012 17 potravinárstvo skupinou významné štatistické rozdiely (P≥0,05) podobne ako sliepky pri hodnotení stehennej svaloviny. skupinou významné štatistické rozdiely (P≥0,05) podobne ako sliepky pri hodnotení stehennej svaloviny. sliepok v prsnej svalovine. Výsledky experimentu potvrdili, že propolisový extrakt v dávke 0,2 g.kg-1 KKZ môžeme aplikovať vo výžive bojlerových kurčiat, nakoľko pozitívne ovplyvňuje senzorickú kvalitu mäsa kurčiat, ktorá je jedným z najdôležitejších článkov pre využitie kuracieho mäsa v potravinovom reťazci človeka. Hodnotením senzorických vlastností bez rozdielu pohlavia u kurčiat Ross 308 boli významné rozdiely (P≤0,001) dosiahnuté v prsnej svalovine v hodnotení vône v pokusnej skupine (+0,207 bodu) a podobne v stehennej svalovine (P≤0,05) pri hodnotení vône v prospech pokusnej skupiny (+0,207 bodu) oproti kontrole. Acknowledgments: Acknowledgments: This work was supported by grant VEGA No. 1/0360/09. Acknowledgments: This work was supported by grant VEGA No. 1/0360/09. Celkovým senzorickým zhodnotením stehennej a prsnej svaloviny podľa pohlavia sme zaznamenali vyššie hodnoty v pokusnej skupine kŕmenej doplnkom extraktu propolisu oproti kontrole, ale preukazné rozdiely (P≤0,01) boli zistené len v prsnej svalovine sliepok, ktorá dosiahla v pokusnej skupine o 0,917 bodu vyššiu hodnotu ako kontrola. This work was supported by grant VEGA No. 1/0360/09. potravinárstvo kŕmnych zmesí a kŕmnych aditív v podmienkach trhovej ekonomiky, VÚK, Ivánka pri Dunaji, 1991, p. 39-43. 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ZÁVER V experimente bol preverovaný vplyv extraktu propolisu aplikovaného v kŕmnych zmesiach kurčiat Ross 308 v dávke 0,2 g.kg-1 KKZ na senzorické hodnotenie prsnej a stehennej svaloviny po tepelnej úprave pečením. Na základe dosiahnutých výsledkov podľa pohlavia boli dosiahnuté štatisticky významné rozdiely (P≤0,05 až 0,001) v hodnotení vône a chuti u kohútov v stehennej svalovine a u sliepok (P≤0,01) vo vôni a jemnosti v prsnej svalovine. Senzorickým posudzovaním jednotlivých znakov bez rozdielu pohlavia boli významné rozdiely (P≤0,05 až 0,001) v prospech pokusnej skupiny dosiahnuté len v hodnotení vône tak v prsnej ako aj stehennej svalovine. Celkovým zhodnotením najcennejších častí jatočného tela kurčiat Ross 308 sme zistili pozitívny vplyv extraktu propolisu na ich senzorické vlastnosti, ale preukazné rozdiely (P≤0,01) boli zaznamenané len u BOBKO, M., LAGIN, L., ANGELOVIČOVÁ, M., BOBKOVÁ, A., HAŠČÍK, P. 2009. Vplyv prídavku fytoaditív na kvalitu kuracieho mäsa. 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ŠČÍ Č Ň ČÁ Á ÍŠ MIHOK, M., HAŠČÍK, P., ČUBOŇ, J., KAČÁNIOVÁ, M., BOBKO, M., HLEBA, L, PRÍVARA, Š., VAVRIŠINOVÁ, K., ARPÁŠOVÁ, H. 2010. Aplikácia probiotického preparátu vo výžive kurčiat Hybro na senzorické vlastnosti mäsa. In Potravinarstvo, vol. 4, special issue, 2010, p. 466-473. p p MOJTO, J., ZAUJEC, K. 2001. Aktuálne údaje o chemickom zložení a nutričnej hodnote mäsa hospodárskych a divých zvierat. In Maso, vol. 13, 2001, no. 4, p. 39-41. MOJTO, J., ZAUJEC, K. 2003. Analýza krehkosti (strižnej sily) hovädzieho mäsa v jatočnej populácii. In Maso, vol. 15, 2003, no. 1, p. 25-27. HAŠČÍK, P., ČUBOŇ, J., KAČÁNIOVÁ, M., KULÍŠEK, V. 2006. Vplyv probiotického preparátu na zloženie mäsa kurčiat. In Maso, vol. 17, 2006, no. 5, p. 13-15. ŠČÍ ČÁ Á Č Ň MOJTO, J., ZAUJEC, K. 2001. Aktuálne údaje o chemickom zložení a nutričnej hodnote mäsa hospodárskych a divých zvierat. In Maso, vol. 13, 2001, no. 4, p. 39-41. HAŠČÍK, P., BOBKO, M., KAČÁNIOVÁ, M., ČUBOŇ, J., KULÍŠEK, V., PAVLIČOVÁ, S. 2007. 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The effect of Turkish propolis on growth and carcass characteristics in broilers under heat stress. In Anim. Feed Sci. and Techn., vol. 146, 2008, p. 137-148. Ing. Mgr. Martin Mellen, PhD., Agrokonzult s.r.o., Branovo, 941 31, email: martin.mellen@gmail.com SHALMANY, S. K., SHIVAZAD, M. 2006. The effect of diet propolis supplementation on ross broiler chicks performance. In J. Poult. Sci. vol. 5, 2006, p. 84-88. Ing. Michal Mihok, Department of Evaluation and Processing of Animal Products, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Trieda A. Hlinku 2, 949 76 Nitra, E-mail: mihok.michal@gmail.com STEINHAUSER, L., BENEŠ, J., BUDIG, J., GOLA, J., HOFMAN, I., INGR, I., KAMENIK, J. 1995. Hygiena a technológie mäsa. In Last Brno, 1st ed., 1995, p. 664, ISBN 80-9000260-4-4. Msc. Ibrahim Omer Eliman Elimam, Department of Evaluation and Processing of Animal Products, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Trieda A. Hlinku 2, 949 76 Nitra, E-mail: alkrshola@yahoo.com STRAKOVÁ, E., VEČEREK, V., SUCHÝ, P., VITULA, F. 2003. The comparison of carcass quality in fattening chicks and pheasants. In Současnost a perspektivy chovu drůbeže, Praha, 2003, p. 83-87, ISBN 80-213-1037-5. Volume 6 No. 1/2012 20
https://openalex.org/W2734497636	https://bmcpalliatcare.biomedcentral.com/track/pdf/10.1186/s12904-017-0221-0	English	null	Better palliative care for people with a dementia: summary of interdisciplinary workshop highlighting current gaps and recommendations for future research	BMC palliative care	2,017	cc-by	9,744	* Correspondence: s.fox@ucc.ie 1Centre for Gerontology and Rehabilitation, School of Medicine, University College Cork, The Bungalow, Block 13, St Finbarr’s Hospital, Douglas road, Cork T21XH60, Republic of Ireland Full list of author information is available at the end of the article Better palliative care for people with a dementia: summary of interdisciplinary workshop highlighting current gaps and recommendations for future research Siobhán Fox1*, Carol FitzGerald1, Karen Harrison Dening2, Kate Irving3, W. George Kernohan4, Adrian Treloar5, David Oliver6,7, Suzanne Guerin8 and Suzanne Timmons1 © The Author(s). 2017 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: Dementia is the most common neurological disorder worldwide and is a life-limiting condition, but very often is not recognised as such. People with dementia, and their carers, have been shown to have palliative care needs equal in extent to those of cancer patients. However, many people with advanced dementia are not routinely being assessed to determine their palliative care needs, and it is not clear why this is so. Main body: An interdisciplinary workshop on “Palliative Care in Neurodegeneration, with a focus on Dementia”, was held in Cork, Ireland, in May 2016. The key aim of this workshop was to discuss the evidence base for palliative care for people with dementia, to identify ‘gaps’ for clinical research, and to make recommendations for interdisciplinary research practice. To lead the discussion throughout the day a multidisciplinary panel of expert speakers were brought together, including both researchers and clinicians from across Ireland and the UK. Targeted invitations were sent to attendees ensuring all key stakeholders were present to contribute to discussions. In total, 49 experts representing 17 different academic and practice settings, attended. Key topics for discussion were pre-selected based on previously identified research priorities (e.g. James Lind Alliance) and stakeholder input. Key discussion topics included: i. Advance Care Planning for people with Dementia; ii. Personhood in End-of-life Dementia care; iii. Topics in the care of advanced dementia at home. These topics were used as a starting point, and the ethos of the workshop was that the attendees could stimulate discussion and debate in any relevant area, not just the key topics, summarised under iv. Other priorities. Conclusions: The care experienced by people with dementia and their families has the potential to be improved; palliative care frameworks may have much to offer in this endeavour. However, a solid evidence base is required to translate palliative care into practice in the context of dementia. This paper presents suggested research priorities as a starting point to build this evidence base. An interdisciplinary approach to research and priority setting is essential to develop actionable knowledge in this area. Keywords: Dementia, Neurodegenerative diseases, Interdisciplinary research, Research priorities, Advance care planning, Personhood, Care at home © The Author(s). Fox et al. BMC Palliative Care (2018) 17:9 DOI 10.1186/s12904-017-0221-0 Fox et al. BMC Palliative Care (2018) 17:9 DOI 10.1186/s12904-017-0221-0 Background difficult to identify [16]. Research is also hindered by the lack of agreed outcome measures, and the challenge of adapting existing tools for use with someone with ad- vanced dementia who is verbally non-communicative [17]. Assessment of symptoms can be further confounded by the presence of concurrent illnesses. Dementia causes impairment of memory, problem-solving and communication, and in advanced disease, the ability to perform everyday tasks [1]. Dementia is one of the major causes of disability and dependency among older people and it is not a normal part of ageing. In the United Kingdom and Wales, dementia is the leading cause of death [2]. Worldwide, 47.5 million people have dementia and there are 7.7 million new cases every year [3]. There are at least 48,000 people in the Republic of Ireland living with dementia; given the ageing population, this number is expected to increase to about 150,000 by 2046 [4]. While recent population-based research suggests that the prevalence rate of dementia in older people may actually be declining [5], due partly to improved healthcare, the number of people affected by dementia directly or indir- ectly continues to rise as the population ages and the number at risk rises. In recognition of the importance of this challenge, the international research community has called for more clinically-relevant, collaborative, and strategic approaches to palliative care research [18–22]. While many disciplines have recognised the importance of research in palliative care for neurodegeneration individually, a problem is that researchers are tackling the problem from different per- spectives, theoretical frameworks, and using diverse meth- odologies; these are complementary but require a platform for discussion, debate and collaboration. Further- more, this discussion needs to be interdisciplinary, and in- clude academics, practitioners and service-users, as one discipline alone cannot manage the complex physical, psy- chological, social, and ethical problems in palliative care for people with dementia. It is important that priorities for future research are set so that questions pertinent to de- mentia and palliative care in Ireland could be addressed effectively by researchers of all relevant disciplines, to en- able a strong evidence base to be developed. There is a significant need to increase and expand the re- search base for palliative and end-of-life care, in recognition of emerging global priorities [6], including moving beyond cancer to examine other chronic diseases such as dementia [7]. Abstract 2017 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 11 Fox et al. BMC Palliative Care (2018) 17:9 Background Dementia is a life-limiting condition, but very often is not recognised as a terminal illness. People with dementia, and their carers, have been shown to have palliative care needs equal to those of cancer patients [8]. A palliative care approach is also favoured by informal caregivers [9]. Main text Palliative care can be defined as: “an approach to care that improves the quality-of-life of patients and their fam- ilies facing problems associated with life-threatening ill- ness, through the prevention and relief of suffering by means of early identification and impeccable assessment and treatment of pain and other problems, physical, psy- chosocial and spiritual” [10]. This broad definition covers both i) generalist palliative care (approach which involves all healthcare workers practicing palliative care principles as a core skill, supplemented by some healthcare workers who are not engaged full time in palliative care, but have had additional training and experience in palliative care); and ii) Specialist Palliative Care services whose core activ- ity is the provision of palliative care to individuals with more complex and demanding care needs [11]. Overview of research in neurodegenerative disease There is an imperative for the development of research into the care of people with neurodegenerative disease, as at present there are no curative treatments, and the aim of care is to provide the best supportive and palliative care for these patients and their families. There have been sev- eral documents and discussions about the future of this research including the Priority Partnership Project in 2015, which was based on a wide consultation on the fu- ture priorities for research in palliative care, initiated by Marie Curie and facilitated by the James Lind Alliance [25]. Ten areas were prioritised, and of these, the follow- ing four have particular relevance to neurodegenerative disease: access to palliative care; Advance Care Planning; determination of patient needs; assessment and treatment of pain when communication is complex (see Table 2). Table 1 Details of professional backgrounds of workshop delegates Table 1 Details of professional backgrounds of workshop delegates Discipline n Nursing 11 Consultant Physician 8 Palliative Medicine 4 Geriatrician 2 Neurologist 1 Old Age Psychiatrist 1 Psychology 7 Voluntary Sector 7 Medical Researchers 4 Law 3 Family Carers 2 Pharmacy 1 General Practitioner 1 Neuroscience 1 Microsystems 1 Physical Sciences 1 ICT For Healthcare 1 Speech And Language Therapist 1 The longer list of suggested research topics is also rele- vant, specifically: the best way of providing palliative care to people with dementia; swallowing problems at end-of- life; drooling, which often accompanies reduced swallow- ing; assessment of distress in dementia; carer support and training for carers; continuity of care; understanding the person’s needs in neurological disease and dementia. Within Europe, the Joint Programme - Neurodegenera- tive Disease Research (JPND) has been considering the re- search priorities and suggested the following: needs assessment, the identification of transitions along the pathway (such as the move to institutional care), and con- sideration of effective models across Europe [26]. Sug- gested priorities include quality improvement and research funding to establish effective strategies to achieve them. Specific priorities within these two related domains have been identified (Table 3). These areas may now be considered in greater depth and it is hoped that there will be opportunities for funding to look at these areas. A recent Consensus document on neurological palliative care has been produced and endorsed by the European Academy of Neurology and the European Association for Palliative Care [14]. Planning the workshop A consortium was established, representing two uni- versities and five non-profit organisations for demen- tia and palliative care. The goal of the consortium was to plan an interdisciplinary workshop to explore the theme: “Palliative Care in Neurodegeneration with a focus on Dementia: Addressing complex questions through interdisciplinary research and reflection.” The aim of the workshop was to bring experts together from different disciplines to discuss this theme, to en- hance cross-discipline learning, and to identify and discuss research gaps, priorities and methodologies in palliative care in neurodegeneration. There are other examples of using a similar approach to identify re- search priorities in palliative care (e.g. Stevinson, Preston, & Todd [23]; Jones et al. [24]). Recent international reviews have highlighted the im- portance of palliative care in neurodegeneration [12–14]. The Irish National Dementia Strategy placed a particular focus on palliative care [15]. However, it is difficult to enact policy as the evidence base for the value of palliative care for people with dementia is still lacking and many people with advanced dementia are still not routinely being pro- vided with palliative care in practice. Providing high quality palliative care for people with dementia presents unique challenges, for example the person’s inability to verbally ex- press preferences for their care as the illness progresses, and the fact that the end-of-life phase may be long and The consortium members identified a long-list of key priority areas for the workshop through review of exist- ing priority setting exercises. Members then conferred within their own organisations (this included input from a wider stakeholder network of academics and re- searchers, clinicians, and people affected by dementia) and a final short-list with particular relevance to the Irish context was agreed by the consortium. Next, ex- perts in the chosen priorities were identified by the con- sortium and invited to the workshop. Five invited speakers presented at the workshop. Page 3 of 11 Fox et al. BMC Palliative Care (2018) 17:9 Outcomes In total, 49 experts attended the workshop, representing academics, researchers, and clinicians, from a range of relevant disciplines (see Table 1). All attendees were identified and targeted as leading experts in Ireland in either palliative care, neurodegeneration, or both, and at- tendance was on an invite only basis. There was also substantial Patient and Public Involvement in both the organisation and attendance at the event, including fam- ily carers of people with dementia and representatives from national voluntary and charitable organisations. The purpose of this paper is to summarise some of the key research priorities and suggestions for future re- search in dementia palliative care, based on core discus- sion points which arose during the workshop. We have provided a general overview of a selection of these key topics against a brief background literature. We con- clude with specific priorities for future research which are taken directly from discussions during the workshop. This paper is not intended as an exact summary of the proceedings on the day, however video recordings of the workshop presentations are available online. y g The workshop was highly participatory, and scheduled such that all delegates had ample opportunity to partake in discussions throughout the day. The workshop in- cluded five facilitated discussions. In these sessions, in- vited speakers gave a brief introduction to one of the pre-identified key themes, and then an independent, sec- ond expert facilitated the consequent discussion with the floor. A longer keynote presentation was delivered by a leading international expert. Two workshop consor- tium members independently recorded the core discus- sion points as they arose, and also gathered and collated anonymous written comments (each attendee received blank comment cards for each session). The discussion points and the comments were synopsised by an expert in an afternoon session with further brief discussion to clarify content and fidelity. The workshop closed with a facilitated question and answer session with a panel of six experts, three of whom had presented earlier. Priority research questions identified in May 2016 workshop 4.1 . Multi-method 4.2 . Interdisciplinary What are the limits and potential of proxy, i.e. family carers, decision making? What are the limits and potential of proxy, i.e. family carers, decision making? optimal treatment of symptoms and providing comfort, setting care goals and advance planning. How best to include people with dementia in research studies, to achieve useful and actionable outcomes? How best to include people with dementia in research studies, to achieve useful and actionable outcomes? What is the economic benefit, if any, of care at home services for dementia, and other neurodegenerative disease? What is the economic benefit, if any, of care at home services for dementia, and other neurodegenerative disease? Together, these documents suggest that the palliative care needs of people with neurodegenerative diseases, including dementia, requires more research and there needs to be a unified approach, linked to existing evi- dence, and at all levels – locally, nationally, across Eur- ope, and across the world. Such an approach should be informed by regional priorities and may be guided using specific frameworks and models of care. What are the factors that contribute to and build carer resilience in advanced dementia care? What are the factors that contribute to and build carer resilience in advanced dementia care? How can assessment and support through video technology be utilised? How can assessment and support through video technology be utilised? What are the most appropriate outcome measures to explore benefit (if any) of palliative care? These need to be validated in dementia, Parkinson’s disease, motor neuron disease, etc. Table 3 JPND Palliative and End-Of-Life Care Research in Neurodegenerative Diseases Suggested Priorities Priority research questions identified by James Lind Alliance and All Ireland Institute of Hospice and Palliative Care (2015) Priority research questions identified by James Lind Alliance and All Ireland Institute of Hospice and Palliative Care (2015) Improvement of Quality 1. Support for transnational networking, aiming for multi-professional engagement in palliative care research across EU How can access to palliative care services be improved for everyone regardless of where they are in the UK? James Lind Alliance #2 2. Co-ordination of best practices across EU member states 2. Co-ordination of best practices across EU member states 2.1.Working groups looking at developing evidence a. Advance Care Planning b. Cognitive impairment and challenges c. Effectiveness of education d. Primary care involvement in planning for palliative care e. Engagement with voluntary groups What are the benefits of Advance Care Planning and other approaches to listening to and incorporating patients’ preferences? Who should implement this and when? James Lind Alliance #3 What are the best ways to begin and deliver palliative care for patients with non-cancer diseases (such as COPD, heart failure, MND, AIDS, multiple sclerosis, Crohn’s disease and stroke)? James Lind Alliance #6 / AIIHPC #9 d. Primary care involvement in planning for palliative care e Engagement with voluntary groups d. Primary care involvement in planning for palliative care e. Engagement with voluntary groups Research Funding What are the best ways to assess and treat pain and discomfort in people at the end of life with communication and/or cognitive difficulties, perhaps due to motor neurone disease (MND), dementia, Parkinson’s disease, brain tumour (including glioblastoma) or head and neck cancer, for example? James Lind Alliance #10 3. Collaborative research, especially enhancing and using existing population and disease based longitudinal cohort studies 3.1 . Looking at triggers / transitions leading to changes in care 4. Support of research into identification of best practices for needs assessment Priority research questions identified in May 2016 workshop Overview of research in neurodegenerative disease This Consensus has suggested areas for development in the palliative care for all patients with Page 4 of 11 Fox et al. BMC Palliative Care (2018) 17:9 Table 3 JPND Palliative and End-Of-Life Care Research in Neurodegenerative Diseases Suggested Priorities The following areas are suggested priorities in two related domains: Table 2 Selection of research priorities set through the James Lind Alliance and revised for Ireland by All Ireland Institute of Hospice and Palliative Care (2015) deterioration such as in feeding or breathing, but at other times the disease progression is slow. four domains: sciences; political; sociology; and interper- sonal, and challenges us to consider the potential to in- fluence health outcomes from a range of viewpoints. From the mechanistic side, science and politics attempt to deal with cause and effect, costs and benefits, trade- offs and “hard” evidence to shape services. From the humanistic side come psychology, ethics, culture and sociology to address fear and stigma of illness, death and dying; addressing our relationships in support of one an- other. Hodges recognises the complexity of disease and, through his model, challenges modern thinking about how we address these challenging and interrelated symp- toms of a complex disease. Interested readers are re- ferred to this blog [29] for further reading. This model may provide a useful theoretical and conceptual frame- work for researching dementia and palliative care. Within the holistic remit of palliative care lie four pri- mary components: the physical, social, spiritual and psy- chological. These frameworks aim to inform thinking, and highlight gaps in our knowledge where multi-professional, inter-disciplinary views, expertise and effort can be brought together to help make sense of complex issues in dementia. Specific research priorities have been identified (Table 2) and go some way to highlight the current un- answered questions. Hodges Health Career Model and the Palliative Care Continuum can help to ensure that the journey ahead is well-travelled. Future research could use- fully explore the intersection of these two models. Frameworks for planning and conducting research in palliative care and dementia Dementia is a devastating illness which can affect every one of us in some way. Most widely, we all know some- one with dementia and its symptoms and might all aim to achieve prevention in our own lives; a smaller number of us provide support and care for those so affected (and ourselves may need support); and an even smaller num- ber attempt to address these needs through research and practice development. What is the effect on quality of death and dying, of being transferred from an acute hospital to die at home? How can recognition of need be improved among primary care and other healthcare workers of palliative care needs in their patients with dementia, and other neurodegenerative disease? chronic and progressive neurological disease, considering in particular: ensuring palliative care approach is included in overall care, communication and Advance Care Plan- ning, symptom management, multidisciplinary team ap- proach, family support, carer support, bereavement care, discussion of end-of-life care and the recognition of end- of-life care and the identification and use of triggers for palliative care [14]. Research into these areas would help to facilitate these developments and provide the evidence base that is so often missing. A Delphi Study on palliative care for people with dementia, produced as a White Paper from the EAPC [27], found that the areas for research that received the highest importance ratings were person- centred care, communication and shared decision making; The life-long journey is fraught with difficulty: those affected by dementia experience pain, loss of appetite, poor swallow, general fear and agitation, relationship problems and mental illness, infections, pressure ulcers and communication difficulties. If so affected, we need substantial help with activities of daily living and we might suffer social stigma and even the side-effects of treatments. The journey is at once unique to each of us, yet we must navigate it together and make decisions at all levels about where to place our emphasis. Two frameworks are offered to guide our thinking. First is the Health Career model devised by Hodges [28] which can be seen in Fig. 1. The model distinguishes Page 5 of 11 Fox et al. BMC Palliative Care (2018) 17:9 Fig. 1 Showing the four quadrants of Hodges’ Health Career Model (1989) that provide a unique systematic way to think about research to inform holistic care Research priorities in Advance Care Planning for people with dementia The second framework is the more familiar schematic timeline, see Fig. 2. The palliative care continuum offers a somewhat more one-dimensional or simpler view of the journey from screening for disease in an otherwise healthy population, through diagnosis into a zone where elements of curative and palliative care combine to achieve quality-of-life, right up to (and including) death and (for those close by) bereavement support. The long course of the illness allows some potential to navigate the journey, address secondary prevention and consider rehabilitation models in order to achieve as good a quality-of-life as possible. There are other models of pal- liative care involvement, including a varying involve- ment, according to need, throughout the disease progression – as shown in Fig. 3. This model is of par- ticular relevance in progressive neurological disease, such as dementia, where there are times of specific In the United Kingdom and Ireland, various policy docu- ments have called for improvements in care for people with dementia towards the end-of-life by promoting the use of ‘Advance Care Planning’ [30–33]. In Ireland, pio- neering legislation was introduced in 2015 in the form of the Assisted Decision Making (Capacity) Act [34], which provides the legal guidance to uphold the auton- omy and dignity of the person with dementia, and may be an exemplar for other countries. It has been sug- gested that everyone should be encouraged to identify their needs, priorities and preferences for end-of-life care [30]. This may seem to be a challenge for those with mental capacity, but will be especially challenging for people with cognitive impairment and language defi- cits which reduces their ability to express their prefer- ences. Autonomy in decision making depends upon Page 6 of 11 Fox et al. BMC Palliative Care (2018) 17:9 Fig. 2 Showing the Palliative Care Continuum as one-dimensional journey from screening and diagnosis to end-of-life care. Evidence is required to inform practice in all segments (coloured) Future research priorities for Advance Care Planning in Dementia. Overall there is little evidence to support Advance Care Planning in dementia as a specific interven- tion. We need to test a feasible and acceptable Advance Care Planning intervention for families affected by de- mentia [43] and to test it over time. However, given the average life span of a person with dementia [44], this pre- sents the researcher with considerable challenges. Research priorities in Advance Care Planning for people with dementia Funding for such a study that would recruit people with dementia from an early stage, when they are more likely to have the capacity to develop an Advance Care Plan; through to end- of-life, to be able to measure its effectiveness, may render it unfeasible in respect of normal funding time scales. consciousness of our past and future thoughts and ac- tions in the same way as we are conscious of our present thoughts and actions [35]. However, as dementia pro- gresses, in particular, the ability to consider future thoughts [36] and actions become compromised and this affects the capacity to make decisions [37]. Proxy decision making. Older people often trust loved ones to make healthcare decisions on their behalf [38] and want those decisions to be in keeping with their own wishes and preferences [39]. Family carers are assumed to know what these wishes and preferences would have been had the person with dementia not lost capacity [40] and profes- sionals often rely on family members to predict and articu- late these preferences with assumed accuracy [41]. However, research shows this assumption to be misplaced [37], with family carers often not able to accurately reflect the preferences of a person with dementia in the absence of prior discussions or a documented advance care plan [42]. Proxy decision making can be confounded as such deci- sions may be impossible to separate from the family carers’ own views and furthermore, where the family carer has supportive (or other) care needs of their own. Accordingly, the limits and potential of proxy decision making in the Irish context, require further clarification and research. These long time scales assume that the only evidence for practice comes from long term prospective trails. Other forms of ethical decision-making can be informed by pro- fessional and personal experience of patients and family members. However, as noted above, there is scant evidence on the compatibility of the priorities and wishes of the fam- ily carer and the person with dementia, and if these change over time, converging or diverging, and if it is influenced by the progression of the disease or by transitions in care. Such evidence as exists suggests their perspectives differ greatly at the outset [42, 45] but, could an intervention be Fig. Research priorities in Advance Care Planning for people with dementia 3 The model of dynamic involvement of palliative services based on trigger points (adapted from NHS England, End of life in long term neurological conditions: A framework for Implementation, pg.11) Fig. 3 The model of dynamic involvement of palliative services based on trigger points (adapted from NHS England, End of life in long term neurological conditions: A framework for Implementation, pg.11) Page 7 of 11 Fox et al. BMC Palliative Care (2018) 17:9 Page 7 of 11 developed that works systemically with the whole family to develop a realistic, shared decision making approach? We know that families affected by dementia do benefit from early and ongoing practical and emotional support [46], but can this be extended to prepare them for potential changes and aid decision making in the context of the realities of care towards the end-of-life [47]? To do this, we need to develop a greater understanding of what factors influence the agreement or divergence of views, or how these issues are handled in skilled practice. [49], a position which has led to a silence of the voice of people with dementia for too long. This position serves to reinforce the idea that people with dementia may not have a worthwhile contribution to make or that they are too vulnerable to require anything of them. Of course these concerns are to be taken seriously but the larger danger may well be the resulting lack of voice. Assuming ethical permissions, there is an emerging but neophyte literature on the methods required to elicit useful data when people with dementia are taking part in research studies. As people with dementia are not in any way homogenous, the skills required are hugely var- ied not just from person to person, but from day to day and week to week, depending on context and many other factors we are yet to fully understand. We also lack knowledge as to whether an Advance Care Planning intervention is a viable option for people in dif- ferent stages of dementia. Often capacity assessments are not always carried out to consider specific decisions in re- spect of end-of-life care preferences so further study is warranted on how we can ensure people with dementia in the moderate to advanced stages of the illness are sup- ported to engage in the decision making processes for their end-of-life care. Research priorities in Advance Care Planning for people with dementia We also need to establish the stabil- ity of these views over the dementia journey. One example that explores the uniqueness of human response at the later stages of dementia is the Aware- Care study [50]. They proposed that if care staff can be trained to identify signs of awareness this should support greater responsiveness and facilitate the expression of awareness. They found seven spontaneously occurring stimuli (e.g. someone nearby) and three introduced stim- uli (e.g. call by name), with 14 response categories sub- divided into movement (eyes, face, head, arm and body) and sounds. Importantly, use of the tool led to relatives rating improvements in wellbeing and quality-of-life of the person with dementia. Research priorities in personhood in end-of-life dementia care ‘Person-centred care,' since its rise in popularity in the 1980’s, has become a catchphrase for good dementia care. However, while the phrase is central to policy and education on dementia, many people with dementia have not experienced improvements in care. The pri- mary proponent of person-centred care in dementia, Tom Kitwood [48], made a very insightful statement in his book, Dementia Reconsidered: There is a great need for creativity in research to gen- erate knowledge that supports the translation of person- centred care not just as a watch-word for good care but as an illumination of how that may be practiced. Research topics in the care of advanced dementia at home and in 24-h care “It is conceivable that most of the advances that have been made in recent years might be obliterated, and that the state of affairs in 2010 might be as bad as it was in 1970, except that it would be varnished by eloquent mis- sion statements, and masked by fine buildings and glossy brochures” p.133. In Ireland and the United Kingdom, acute hospital care is under huge pressure with large overspends on un- planned emergency admissions. Older people occupy in- creasing numbers of acute care beds, and most people with dementia present to the Emergency Department or and/or acute medical assessment unit in the last six months of life [51]. Good care at home may help avoid this, and the associated costs, as well as supporting good outcomes. Advanced dementia care at home has been piloted by Treloar et al. [52] and further described by the Kings Fund [53]. Data from studies have indicated substantial savings as a result of advanced dementia care at home. Sampson et at [51] found that care costs over the six months before death were higher in care homes or continuing care (£37,029) than for those living at home (£19,854). The Housing 21 Dementia Voice pro- ject in Westminster [54] reported that “over a 24-month period, it is estimated that the Dementia Voice Nurse service wholly or partly contributed to savings of £314,440 through the avoidance of hospital, nursing and If we are to ensure that person-centred care is more than a name-check in a mission statement, it is essential that we explore the meaning of personhood right along the spectrum of dementia to end-of-life care. Personhood is a standing or status that is bestowed on one human be- ing, by others, in the context of relationship and social be- ing. It implies recognition, respect and trust. It is a commitment on behalf of one to recognize the unique contribution of all human beings: primarily the person liv- ing with dementia, but also the family carer, the volunteer, the unqualified assistant and healthcare professionals [48]. For the research community there are many hurdles to surpass before we can realize this challenge. It can be difficult for ethics committees to accept the necessity of involving vulnerable people in research as co-researchers For the research community there are many hurdles to surpass before we can realize this challenge. Research topics in the care of advanced dementia at home and in 24-h care There is a real need for better quality economic data to comple- ment patient-focused outcome data. Table 4 Principles of care of the Oxleas Advanced Dementia Service Table 4 Principles of care of the Oxleas Advanced Dementia Service A core belief of the Oxleas Advanced Dementia Service is that anyone cared for at home with advanced dementia deserves care co-ordination and on-going support. The service combines mental and physical health expertise, to look competently after patients with advanced dementia living at home and to: • Comprehensively assess and plan ahead; • Co-ordinate care; • Respond quickly when needs are changing; • Establish a palliative care framework with a focus on maximising quality-of-life, helping to avoid or shorten unnecessary and traumatic hospital admissions, treatments and investigations, and replace them with home care whenever possible; • Offer excellent care towards the end-of-life; • Relieve the carer from having to navigate alone within a complex care system while grieving. • Relieve the carer from having to navigate alone within a complex care system while grieving. any) of palliative care. It was agreed that this still- emerging research area would benefit from smaller scale studies in the short-term, including: quality im- provement studies, smaller pilot studies, and observa- tional studies to better inform interventions in future trials. This aligns with the recommendations of the Medical Research Council (MRC) framework for the evaluation of complex health interventions [56]. The MRC framework was developed in light of the limita- tions of randomised control trials, mainly limited con- textual data, and outlines the steps for process evaluation, i.e. methods to assess fidelity and quality of implementation, clarify causal mechanisms and iden- tify contextual factors associated with variation in outcome. Despite the possible economic savings, supporting the care of people with advanced dementia at home is poorly understood and rarely prioritised by statutory ser- vices. Central to enabling care at home for a person with advanced dementia, is carer resilience. The START (STrAtegies for RelaTives) trial implemented a manua- lised intervention programme and aimed to improve carer coping strategies. The trial demonstrated reduced depression and anxiety in family carers of people with Dementia at 8 months and 2 years post intervention and also suggested savings [55]. ii. Palliative care of a person with dementia at home also depends upon skilled healthcare, and expertise that en- ables competent professional advice to support carers in what they are doing. Research topics in the care of advanced dementia at home and in 24-h care The principles of care of the Oxleas Advanced Dementia Service are good guiding principles, these are outlined in Table 4. ii. There is a research gap concerning our understanding of the lived experience of the person with advanced dementia. In this context Public and Patient Involvement (PPI) in research is critical. However, it is important that PPI is not incorporated as a token exercise, but rather researchers must aim to achieve useful and actionable outcomes and goals through patient and public participation in research. It is essential that people with advanced dementia are also included in research. For this to happen, innovative research methods must be utilised, as many people living with dementia at this advanced stage will be verbally non-communicative. ii. There is a research gap concerning our understanding of the lived experience of the person with advanced dementia. In this context Public and Patient Involvement (PPI) in research is critical. However, it is important that PPI is not incorporated as a token exercise, but rather researchers must aim to achieve useful and actionable outcomes and goals through patient and public participation in research. It is essential that people with advanced dementia are also included in research. For this to happen, innovative research methods must be utilised, as many people living with dementia at this advanced stage will be verbally non-communicative. Research topics in the care of advanced dementia at home and in 24-h care It can be difficult for ethics committees to accept the necessity of involving vulnerable people in research as co-researchers Fox et al. BMC Palliative Care (2018) 17:9 Page 8 of 11 Page 8 of 11 residential home admission and the use of ambulance services”. Results from the Hope for Home study [52] in- dicated that total savings of home care compared with nursing home care for 14 patients was approximately £700,000 and that 57% of participants died in their own home. An audit of 23 patients cared for by the Green- wich Advanced Dementia service in 2009 found that, in total, these patients were cared for at home for 6205 days or approximately 886 weeks. Savings to local health and social care commissioners from these patients were esti- mated at between £200 and £350 per week, saving up- wards of £177,200 to £310,100 for these patients. These savings are notional as the numbers of people using the service are too small to enable commissioners to release money from closing beds [53]. Using similar assump- tions, the Greenwich Advanced Dementia Project esti- mates that it saved over £2 million caring for 100 patients. However, this data is “soft” and formal eco- nomic analysis of such services is very difficult. There is a real need for better quality economic data to comple- ment patient-focused outcome data. residential home admission and the use of ambulance services”. Results from the Hope for Home study [52] in- dicated that total savings of home care compared with nursing home care for 14 patients was approximately £700,000 and that 57% of participants died in their own home. An audit of 23 patients cared for by the Green- wich Advanced Dementia service in 2009 found that, in total, these patients were cared for at home for 6205 days or approximately 886 weeks. Savings to local health and social care commissioners from these patients were esti- mated at between £200 and £350 per week, saving up- wards of £177,200 to £310,100 for these patients. These savings are notional as the numbers of people using the service are too small to enable commissioners to release money from closing beds [53]. Using similar assump- tions, the Greenwich Advanced Dementia Project esti- mates that it saved over £2 million caring for 100 patients. However, this data is “soft” and formal eco- nomic analysis of such services is very difficult. Conclusions some may be ethically questionable, such as financially incentivising nurses and other healthcare staff. A better course may be to look at implementing education programmes, and critically assess the sustainability of change following an education intervention. These programmes might include methods to help staff to get to know the person with dementia better, to improve quality of care, etc. Overall, research is needed to investigate which methods are the best way to sustain positive changes in staff behaviours for the long-term. The care experienced by people with dementia and their families has the potential to be improved through using palliative care frameworks. However, a solid evidence base is required to inform how to achieve such improvements. As a relatively new field, there are significant methodo- logical and content areas where research is needed. An ex- pert consortium has highlighted priorities for future research (Table 2). Integrated care may improve out- comes, notably quality-of-life, for people with dementia [57], hence an interdisciplinary approach to research and priority setting is essential to further actionable knowledge in this area. It is also imperative that there needs to be a unified approach at all levels – nationally, across Europe, and across the world. g g v. Another priority is to develop useful and transferrable models of best care. In developing these, the key questions are: how to best integrate palliative care and dementia care, and identification of the facilitators and barriers to such integration; how to integrate care not only across disciplines but also sectors, including acute, community, residential care; and determining the existing access to specialist services for people with dementia. A small number of existing clinics have pioneered models of palliative care for dementia or other neurodegenerative illnesses, and these can serve as exemplary models of excellence. Learning from existing models that are performing well may be done through a cross-case analysis to identify the core principles and practices that are happening at each site, mapping across the models to look at the commonalities and differences and build a taxonomy of that model. Thus (as above) more conceptual re- search is needed, in addition to large scale trials and studies. In any model, cost effectiveness is critical, but it is impossible to accurately measure cost effect- iveness unless the model of care is properly de- scribed. Other research priorities In addition to the aforementioned themes, there were a number of recurring issues raised during discussion ses- sions during the workshop; these are discussed briefly in the following paragraphs and summarised in Table 2. i. Research design, including the choice of appropriate methodologies, can be challenging in palliative care and dementia. By nature, large scale trials and longitudinal studies will be difficult and may not always be feasible. It is also critical to identify the best ways to capture the potential benefit of Advance Care Planning in palliative care and dementia. A research priority must be the identification and validation of appropriate outcome measures to explore benefit (if i. Research design, including the choice of appropriate methodologies, can be challenging in palliative care and dementia. By nature, large scale trials and longitudinal studies will be difficult and may not always be feasible. It is also critical to identify the best ways to capture the potential benefit of Advance Care Planning in palliative care and dementia. A research priority must be the identification and validation of appropriate outcome measures to explore benefit (if iii. Palliative care for dementia, and neurodegeneration has been supported in policy for some years, however in practice this is a new area for many healthcare staff and there is a need for it to be actioned in routine practice across disciplines. Therefore, research needs to investigate the optimal methods to change healthcare workers’ behaviours concerning palliative care for their patients with dementia. There are various recognised methods, iii. Palliative care for dementia, and neurodegeneration has been supported in policy for some years, however in practice this is a new area for many healthcare staff and there is a need for it to be actioned in routine practice across disciplines. Therefore, research needs to investigate the optimal methods to change healthcare workers’ behaviours concerning palliative care for their patients with dementia. There are various recognised methods, Page 9 of 11 Page 9 of 11 Fox et al. BMC Palliative Care (2018) 17:9 Conclusions The development of these frameworks would be highly useful as they could be subsequently replicated in multiple sites. This paper summarises key topics in dementia palliative care, based in part on a consensus workshop, and the re- search priorities discussed here were not identified through systematic or empirical research studies. Further, the priorities were discussed primarily with relevance to the Irish context, and while most are common to inter- national dementia research, there may be country-specific priorities owing to unique cultures, different healthcare systems, different state of current research, etc. However notable strengths of this paper, and the workshop which stimulated its development, are that the consensus group included targeted national and international experts from a variety of academic and professional disciplines, and had substantial Patient and Public Involvement. A literature review was also performed to place the research priorities discussed into context of international research literature. We have highlighted some of the research priorities for palliative care and neurodegeneration, as discussed by a consortium of multidisciplinary experts. We have also suggested two models or frameworks that may be useful in mapping out topics to guide research in pallia- tive care for people with dementia and continue to prompt further questions. v. Other topics that arose at this workshop included “chemical restraint” and the issue of inappropriate antipsychotic prescribing; dying at home, particularly transferring people at end-of-life from an acute hos- pital setting to die at home, and the effect of this on quality of death and dying; palliative care in primary care; improving staff and carers’ recognition of need (i.e. if a need is not recognised by others, it will never be addressed); the potential use of technology to assist in assessment where communication is limited, and in supporting care provision; exploration of potential conflicts in the views of the person with dementia, their family and healthcare workers towards end-of- life. The considered application of frameworks (such as Hodges Model) may provide a useful mapping framework for priority setting and enable other areas requiring attention to be highlighted. Abbreviations EAPC E Abbreviations EAPC: European Association of Palliative Care; JPND: Joint Programme - Neurodegenerative Disease Research; MRC: Medical Research Council; PPI: Patient and Public Involvement Acknowledgements The authors would like to thank all of the workshop participants who engaged in the discussions summarised here. Availability of data and materials Not applicable. Competing interests 21. Murray S, Barclay S, Bennett MI, Kendall M, Amir Z, Lloyd-Williams M. Editorial: palliative care research in the community: it is time to progress this emerging field. Palliat Med. 2008;22(5):609–11. 22. Sigurdardottir KR, Haugen DF, van der Rijt CC, Sjøgren P, Harding R, Higginson IJ, Kaasa S. Clinical priorities, barriers and solutions in end-of-life cancer care research across Europe. Report from a workshop. Eur J Cancer. 2010;46(10):1815–22. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 23. Stevinson C, Preston N, Todd C. Defining priorities in prognostication research: results of a consensus workshop. Palliat Med. 2010;24(5): 462–8. Funding g This research was supported by a grant from the Irish Research Council. The purpose of the ‘Creative Connections’ grant award is to fund workshops to cultivate interdisciplinary research in Ireland focussing on key national and international research priorities. The Council had no significant role in the design of the workshop or in writing the manuscript. Availability of data and materials Not applicable. Page 10 of 11 Page 10 of 11 Page 10 of 11 Fox et al. BMC Palliative Care (2018) 17:9 Author details 1 1Centre for Gerontology and Rehabilitation, School of Medicine, University College Cork, The Bungalow, Block 13, St Finbarr’s Hospital, Douglas road, Cork T21XH60, Republic of Ireland. 2Research & Evaluation Dementia UK, London, UK. 3Nursing and Human Sciences, Dublin City University, Dublin, Ireland. 4Institute of Nursing and Health Research, Ulster University, Co., Antrim, UK. 5Old Age Psychiatry, Oxleas NHS Trust, London, UK. 6Tizard Centre, University of Kent, Canterbury, UK. 7EAPC Board Member, European Association of Palliative Care, Vilvoorde, Belgium. 8Research Design & Analysis, School of Psychology, University College Dublin, Dublin, Ireland. 1Centre for Gerontology and Rehabilitation, School of Medicine, University College Cork, The Bungalow, Block 13, St Finbarr’s Hospital, Douglas road, Cork T21XH60, Republic of Ireland. 2Research & Evaluation Dementia UK, London, UK. 3Nursing and Human Sciences, Dublin City University, Dublin, Ireland. 4Institute of Nursing and Health Research, Ulster University, Co., Antrim, UK. 5Old Age Psychiatry, Oxleas NHS Trust, London, UK. 6Tizard C U i i f K C b UK 7EAPC B d M b E 24. Jones L, Candy B, Davis S, Elliott M, Gola A, Harrington J, Kupeli N, Lord K, Moore K, Scott S. Development of a model for integrated care at the end of life in advanced dementia: a whole systems UK-wide approach. Palliat Med. 2016;30(3):279–95. 25. Palliative and end-of-life care Priority Setting Partnership [https://www. mariecurie.org.uk/globalassets/media/documents/research/PeolcPSP_ ExecSummary_English.pdf]. Accessed 5 July 2017. Association of Palliative Care, Vilvoorde, Belgium. 8Research Design & Analysis, School of Psychology, University College Dublin, Dublin, Ireland. 26. Palliative and end-of-life care research in neurodegenerative disease. Report of the JPND Action Group. [http://www.neurodegenerationresearch.eu/ initiatives/jpnd-alignment-actions/palliative-care/ ]. Accessed 5 July 2017. Received: 30 January 2017 Accepted: 29 June 2017 27. van der Steen JT, Radbruch L, Hertogh CM, de Boer ME, Hughes JC, Larkin P, Francke AL, Jünger S, Gove D, Firth P. 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Current opinion in supportive and palliative care. 2014;8(3):303–7. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries
W4378229232.txt	https://sosains.greenvest.co.id/index.php/sosains/article/download/702/1250	id		Pengaruh Gaya Kepemimpinan dan Motivasi Kerja terhadap Kinerja Pegawai Badan Penghubung Pemerintah Provinsi Papua di Jakarta	Jurnal sosial dan sains/Jurnal Sosial dan Sains	2,023	cc-by-sa	3,971	JURNAL SOSAINS JURNAL SOSIAL DAN SAINS VOLUME 3 NOMOR 3 2023 P-ISSN 2774-7018, E-ISSN 2774-700X PENGARUH GAYA KEPEMIMPINAN DAN MOTIVASI KERJA TERHADAP KINERJA PEGAWAI BADAN PENGHUBUNG PEMERINTAH PROVINSI PAPUA DI JAKARTA Ayu Adriani Metalia, Syofiah Aryani Magister Manajemen Fakultas Ekonomi dan Bisnis, Universitas Budi Luhur Email : ayuadriani@gmail.com, syofiah.aryani@outlook.com ABSTRAK Latar Belakang : Salah satu hal yang dapat ditempuh perusahaan agar mampu bertahan dalam persaingan yang ketat yaitu dengan meningkatkan kinerja perusahaan dengan mempertahankan gaya kepemimpinan terhadap perusahaan. Kata kunci: Gaya Kepemimpinan, Motivasi Kerja, Tujuan : Penelitian ini bertujuan untuk mengetahui gaya kepemimpinan dan motivasi Kinerja Karyawan. kerja berpengaruh positif dan signifikan terhadap kinerja karyawan secara parsial dan simultan. Metode : Metode penelitian ini dilakukan di Instansi Penghubung Pemerintah Provinsi Papua di Jakarta, dengan populasi seluruh karyawan dan sampel yang digunakan dalam penelitian ini adalah 56 pegawai di Badan Penghubung Pemerintah Provinsi Papua di Jakarta.Teknik penelitian menggunakan penelitian kausal adalah untuk mengidentifikasi hubungan kausal antara variabel yang berfungsi sebagai sebab dan variabel mana yang berfungsi sebagai variabel efek. Metode pengumpulan menggunakan kuesioner. Alat analisis yang digunakan: (1) Uji Instrumen (uji validitas dan reliabilitas), (2) Uji Klasikal (Uji Normalitas, Uji Multikolinearitas dan Uji Heteroskedastisitas), (3) Uji Hipotesis (Analisis Linear Berganda, Uji t, Uji F dan Penentuan Koefisien) Hasil : Hasil analisis data menunjukkan bahwa: (1) hasil analisis uji-t menunjukkan bahwa gaya kepemimpinan dan motivasi kerja berpengaruh sebagian terhadap Kinerja Pegawai Badan Penghubung Pemerintah Provinsi Papua di Jakarta dan (2) hasil uji FAnalysis menunjukkan bahwa Gaya Kepemimpinan dan Motivasi Kerja berpengaruh terhadap Kinerja Pegawai Instansi Penghubung Pemerintah Provinsi Papua di Jakarta secara bersamaan. Kesimpulan: Variabel gaya kepemimpinan berpengaruh terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua di Jakarta. Variabel motivasi kerja berpengaruh terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua di Jakarta. Variabel gaya kepemimpinan dan motivasi kerja secara simultan berpengaruh terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua di Jakarta. ABSTRACT Background: One of the things that companies can take to be able to survive in fierce Keywords: Leadership Style, competition is to improve company performance by maintaining a leadership style Work Motivation, towards the company. Employee Performance. 248 Purpose: This study aims to determine the leadership style and work motivation have a positive and significant effect on employee performance partially and simultaneously http://sosains.greenvest.co.id Volume 3, Nomor 3, Maret 2023 p-ISSN 2774-7018 ; e-ISSN 2774-700X Method: This research method was carried out at the Papua Provincial Government Liaison Agency in Jakarta, with a population of all employees and the sample used in this study was 56 employees at the Papua Provincial Government Liaison Agency in Jakarta.The research technique using causal research is to identify the causal relationship between variables that function as causes and which variables function as effect variables. The method of collection using questionnaires. Analysis tools used: (1) Instrument Test (validity and reliability test), (2) Classical Test (Normality Test, Multicholinearity Test and Heteroskedasticity Test), (3) Hypothesis Test (Multiple Linear Analysis, t Test, F Test and Coefficient Determination). Results: The results of the data analysis show that: (1) the results of the t-test analysis show that leadership style and work motivation have a partial effect on the Performance of Employees of the Papua Provincial Government Liaison Agency in Jakarta and (2) the results of the F-Analysis test show that Leadership Style and Work Motivation affect the Performance of Employees of the Papua Provincial Government Liaison Agency in Jakarta simultaneously. Conclusion: Leadership style variables affect the performance of employees of the Papua Provincial Government Liaison Agency in Jakarta. The variable of work motivation affects the performance of employees of the Papua Provincial Government Liaison Agency in Jakarta. The variables of leadership style and work motivation simultaneously affect the performance of employees of the Papua Provincial Government Liaison Agency in Jakarta. PENDAHULUAN Dalam dunia bisnis yang semakin berkembang dari waktu ke waktu, menurut (Winarso, 2019). Setiap organisasi harus mampu mengelola dan memaksimalkan sumber daya manusianya. Faktor karyawan yang diharapkan dapat mencapai kinerja terbaik untuk memenuhi tujuan perusahaan, tidak dapat dipisahkan dari pengelolaan SDM (Sinambela, 2021). Sistem model hubungan saat ini, termasuk hubungan dengan rekan kerja dan atasan atau manajer, berdampak pada operasi bisnis. Sumber daya manusia harus dikelola secara efektif dan efisien karena merupakan salah satu variabel internal yang mempengaruhi keberhasilan atau kegagalan suatu perusahaan. Dalam pasar yang sangat kompetitif, perusahaan yang meningkatkan kinerja mereka sambil mempertahankan gaya manajemen mereka dapat berkembang (Ningrum et al., 2022). Gaya manajemen bisnis memainkan peran yang memotivasi dan mendukung dalam mendorong orang untuk meningkatkan tingkat kinerja mereka. Pegawai yang dapat diandalkan dan terampil sangat penting, seperti halnya pendekatan manajemen kinerja manajer, yang harus sempurna dan berkualitas tinggi untuk meningkatkan kinerja pegawai. Organisasi selain dipandang sebagai wadah kegiatan orang juga dipandang sebagai proses, yaitu menyoroti interaksi diantara orang-orang yang menjadi anggota organisasi. Keberhasilan suatu organisasi ditentukan oleh kualitas sumber daya manusia yang saling berinteraksi dan mengembangkan organisasi yang bersangkutan (Djuwita, 2011). Organisasi dalam meningkatkan Sumber Daya Manusia dalam rangka mengoptimalkan kinerja pegawai tidak terlepas dari gaya kepemimpinan yang ada (Tampi, 2014). Perusahaan diharapkan memiliki pemimpin yang mampu mendorong motivasi para pegawainya untuk dapat bekerja dengan baik dan optimal. Salah satu hal yang dapat ditempuh perusahaan agar mampu bertahan dalam persaingan yang ketat yaitu dengan meningkatkan kinerja perusahaan dengan mempertahankan gaya kepemimpinan terhadap perusahaan. Karena kinerja suatu perusahaan merupakan modal, untuk mengerjakan produksi barang dan jasa pada perusahaan dan gaya kepemimpinan dari suatu perusahaan menjadi faktor pendorong dan 249 http://sosains.greenvest.co.id pendukung dalam memimpin pegawai dalam meningkatkan kinerjanya (Heriyanti & Putri, 2021). Segala usaha dilakukan untuk mencapai tujuan di antaranya dengan menggunakan sumber daya manusia yaitu tenaga kerja yang handal dan professional dan gaya kepemimpinan dari kinerja seorang pemimpin, dimana harus memiliki sifat yang ideal dan baik sehingga bisa meningkatkan kinerja pegawainya. Gaya kepemimpinan seorang dalam organisasi sangat penting dalam organisasi sangat penting dalam kemajuan sebuah perusahaan. Dari gaya kepemimpinan seorang baik dalam proses mengarahkan dan memberikan pengaruh terhadap pegawai atau bawahan penting agar tujuan perusahaan tercapai (Hanafi, Almy, & Siregar, 2018). Kata pimpin mengandung pengertian mengarahkan, membina atau mengatur, menuntun dan juga menunjukkan ataupun mempengaruhi. Secara teori perilaku karywan bergantung pada tipe kepemimpinan dan sulit untuk di identifikasi (Heriyanti & Putri, 2021). Gaya kepemimpinan dapat dijelaskan dalam skala mulai dari otokratis melalui demokrasi hingga partisipatif untuk menunjukan tingkat kewenangan dan keputusan yang membuat kekuatan pemimpin dan karyawan (Iqbal, Anwar, & Haider, 2015). Pemimpin mempunyai tanggung jawab baik secara fisik maupun spiritual terhadap keberhasilan aktivitas kerja dari yang dipimpin, sehingga menjadi pemimpin itu tidak mudah dan tidak setiap orang mempunyai kesamaan di dalam menjalankan ke-pemimpinannya. Untuk menunjukkan betapa pentingnya kepemimpinan dan betapa manusia membutuhkannya pemimpin yang baik ada pendapat yang keras yang mengatakan bahwa dunia atau umat manusia pada hakekatnya hanya ditentukan oleh beberapa orang saja, yakni yang berstatus sebagai pemimpin. Demikian juga dalam sebuah organisasi atau perusahaan. Oleh karena itu, kepemimpian sangat diperlukan bila suatu organisasi ingin sukses. Terlebih lagi pekerja-pekerja yang baik selalu ingin tahu bagaimana mereka dapat menyumbang dalam pencapaian tujuan organisasi, dan paling tidak, gairah pekerja memerlukan kepemimpinan sebagai dasar motivasi eksternal untuk menjaga tujuantujuan mereka tetap harmonis dengan tujuan organisasi, maka dari itu suatu organisasi akan berhasil atau bahkan gagal sebagian besar ditentukan oleh kepemimpinan tersebut. Disinilah diperlukan figur kepemiminan yang mampu berkomunikasi yang baik dan benar pada bawahannya, agar tujuan organisasi tetap terarah sesuai dengan perencanaan. Kepemimpinan merupakan suatu kegiatan untuk dapat mempengaruhi perilaku orang lain, atau seni yang akan memengaruhi perilaku manusia baik perorangan maupun kelompok. Kepemimpinan merupakan salah satu faktor yang sangat penting dalam suatu organisai karena berpengaruh akan keberhasilan ataupun kegagalan suatu organisai yang akan ditentukan oleh kepemimpinan tersebut. Adapun mengenai lingkungan kerja, seperti adanya pembagian mengenai tugas di lingkungan kerja tersebut tidak dapat dipisahkan satu sama lain. Antara keduanya harus saling seimbang, karena baik lingkungan kerja fisik maupun nonfisik samasama memengaruhi kinerja pegawai (Putra, 2013). Untuk menyeimbangkan keduanya diperlukan kesadaran pihak manajemen dari perusahaan tersebut. Oleh sebab itu, diperlukan lingkungan kerja yang kondusif untuk menunjang kinerja pegawai dalam melaksanakan pekerjaannya, agar hasil kerja yang diperoleh dapat tercapai secara optimal. Sumber daya manusia yang kompeten dengan kinerja yang baik, dapat menunjang keberhasilan bisnis sebaliknya sumber daya manusia yang tidak kompeten dan kinerjanya buruk merupakan masalah kompetitif yang dapat menempatkan perusahaan dalam kondisi yang merugi (Kadarisman, 2012). Peran dan Fungsi sumber daya manusia tidak bisa digantikan oleh sumber daya lainya dikarenakan sumber daya manusia merupakan asset organisasi yang sangat vital. Dalam mencapai tujuann ya suatu organisasi memerlukan sumber daya manusia sebagai pengelola sistem. Agar Sistem ini berjalan tentu dalam pengelolaannya harus memperhatikan beberapa aspek penting seperti kepemimpinan, motivasi, disiplin kerja, kinerja dan aspek-aspek lainya. Ayu Adriani Metalia, Syofiah Aryani 250 Volume 3, Nomor 3, Maret 2023 p-ISSN 2774-7018 ; e-ISSN 2774-700X Hal ini akan menjadikan sumber daya manusia sebagai salah satu indikator penting pencapaian tujuan organisasi secara efektif dan efisien. Sumber daya manusia dalam pengertian sederhana dan terbatas adalah “Personil, pekerja tenaga kerja, karyawan atau pegawai” (Tamarindang, Mananeke, & Pandowo, 2017). Hal ini membuktikan bahwa sumber ada manusia adalah suatu lembaga atau departemen di dalam masyarakat yang memerlukan peningkatan atau pengembangan agar dicapai suatu hasil kerja yang optimal. Kinerja dalam organsasi merupakan jawaban dari berhasil atau tidaknya tujuan organisasi yang telah ditetapkan. Kinerja merupakan sebuah aksi, bukan kejadian. Aksi kinerja itu sendiri terdiri dari banyak komponen dan bukan merupakan hasil yang dapat dilihat pada saat itu juga. Pada dasarnya kinerja merupakan hal yang bersifat individual, karena setiap pegawai memiliki tingkat kemampuan yang berbeda dalam mengerjakan tugasnya. Kinerja tergantung pada kombinasi antara kemampuan, usaha dan kesempatan yang diperoleh. Kinerja seorang pegawai merupakan hal yang bersifat individual, karena setiap pegawai mempunyai tingkat kemampuan yang berbeda-beda dalam mengerjakan tugasnya. Kinerja (performance) adalah gambaran mengenai tingkat pencapain pelaksanaan suatu kegiatan/program/kebijakan dalam mewujudkan sasaran, tujuan, misi dan visi organisasi yang tertuang dalam strategic planning suatu organisasi (Suwandi, 2013). Faktor Pertama yang mempengaruhi kinerja adalah Kepemimpinan. Kepemimpinan merupakan suatu aktivitas untuk mempengaruhi perilaku orang lain agar mereka mau diarahkan untuk mencapai tujuan tertentu. Kepemimpinan juga merupakan proses mempengaruhi atau memberi contoh oleh pemimpin kepada bawahannya dalam upaya mencapai tujuan organisasi. Sebagai proses, Kepemimpinan difokuskan kepada apa yang dilakukan oleh para pemipin, yaitu proses dimana para pemimpin menggunakan pengaruhnya untuk memperjelas tujuan organisasi bagi para karyawan, bawahan, atau yang dimpimpinnya, memotivasi mereka untuk mencapai Tujuan tersebut, serta membantu menciptakan budaya prouktif dalam organisasi. Thoha (2012:259) merumuskan bahwa kepemimpinan itu adalah aktivitas untuk mempengaruhi orang-orang agar di arahkan mencapai tujuan organisasi. Fungsi Kepemimpinan dalam Organisasi. Merupakan elemen yang sangat penting dalam pengelolaan sumber daya manusia. Selain memberikan pengarahan juga memberikan motivasi upaya peningkatan kinerja pegawai. Untuk mengembangkan kemajuan dan perkembangan kinerja pegawai sangat tergantung kepada sumber daya manusia sebagai pengelola langsung. Oleh sebab itu kepemimpinan mempunyai peran besar dalam meningkatkan kinerja pegawai. Sikap dan gaya serta perilaku kepemimpinan seorang pemimpin sangat besar pengaruhnya terhadap organisasi yang dipimpin bahkan sangat berpengaruh terhadap kinerja pegawai dalam organisasi tersebut. Kelangsungan hidup perusahaan juga tergantung pada motivasi kerja para pegawai. Motivasi merupakan hal yang sangat penting untuk diperhatikan oleh pihak manajemen dan perusahaan lainnya, jika mereka menginginkan setiap karyawan dapat mendapatkan kontribusi positif terhadap pencapain tujuan visi dan misi perusahaan dalam meningkatkan kinerja karyawan. Demikian pula setiap pekerjaan pegawai juga mempunyai suatu motivasi misalnya dia mengharapkan pengahasilan atau gaji dan peningkatan status. Motivasi yaitu sebuah dorongan atau alasan yang mendasari semangat dalam melakukan sesuatu Motivasi adalah hal-hal yang menimbulkan dorongan, motivasi kerja yang merupakan pendorong semangat sehingga menimbulkan suatu dorongan. Mengacu pada latar belakang yang telah disampaikan, maka perumusah masalah dalam penelitian ini dapat ditentukan sebagai berikut: 1. Apakah terdapat pengaruh gaya kepemimpinan terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua Di Jakarta? 251 http://sosains.greenvest.co.id 2. Apakah terdapat pengaruh motivasi kerja terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua Di Jakarta? 3. Apakah gaya kepemimpinan dan motivasi kerja terdapat pengaruh terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua Di Jakarta? 4. Dengan demikian, tujuan penelitian ini yakni: 5. Untuk mengetahui dan membuktikan pengaruh gaya kepemimpinan terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua Di Jakarta. 6. Untuk mengetahui dan membuktikan pengaruh motivasi kerja terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua Di Jakarta. 7. Untuk mengetahu dan membuktikan pengarub gaya kepemimpinan dan motivasi kerja terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua Di Jakarta. METODE PENELITIAN Pendekatan penelitian yang digunakan adalah penelitian asosiatif atau hubungan. (Umdiana & Claudia, 2020) mengemukakan pendekatan penelitian asosiatif adalah pendekatan penelitian yang bertujuan untuk mengetahui hubungan antara dua variabel atau lebih. Melalui penelitian ini diharapkan dapat diketahui pengaruh Gaya Kepemimpinan dan Motivasi Kerja Terhadap Kinerja Pegawai Badan Penghubung Pemerintah Provinsi Papua Di Jakarta. Populasi dalam penelitian ini merupakan Pegawai Badan Penghubung Pemerintah Provinsi Papua Di Jakarta yang berjumlah 56 orang. Sampel yang diambil adalah Pegawai Badan Penghubung Pemerintah Provinsi Papua Di Jakarta sehingga jumlah sampel yang diperoleh berjumlah 56 orang. Sumber data primer yang diperoleh dengan membagikan kuesioner (angket penilaian) kepada objek yang akan diteliti dalam hal ini Pegawai Badan Penghubung Pemerintah Provinsi Papua Di Jakarta. Data yang diperoleh dari arsip perusahaan mengenai produktivitas kerja karyawan dari tahun-tahun sebelumnya, selain itu juga dapat diperoleh dari penelitian terdahulu, literature, dan jurnal yang berhubungan dengan permasalahan dalam penelitian ini. Dalam penelitian ini penulis menggunakan beberapa metode dalam pengumpulan data yaitu: teknik kuesioner, teknik observasi dan teknik wawancara. Analisis data yang digunakan adalah data kuantitatif. Data penelitian diolah menggunakan program Statistical Package for the Social Science (SPSS) versi 25 dan data dianalisis dengan menggunakan metode regresi linear berganda (multiple linier regression) melalui uji data dan hipotesis. Uji asumsi klasik dapat digunakan untuk mengetahui apakah residual berdistribusi normal atau tidak, multikolinearitas, dan heteroskedastisitas pada model regresi. Model regresi linear dapat disebut juga dengan model yang baik jika model tersebut memenuhi beberapa asumsi klasik, yaitu data residual terdistribusi normal, tidak adanya multikolinearitas dan heteroskedastisitas. HASIL DAN PEMBAHASAN A. Hasil Penelitian 1. Hasil Uji Validitas Berdasarkan hasil uji validitas, semua pernyataan dari variabel gaya kepemimpinan, motivasi kerja dan kinerja pegawai seluruhnya valid, karena nilai r hitung pada tabel Corrected Item-Total Correlation lebih besar dari nilai r tabel. Nilai r tabel adalah sebesar 0,2632. 2. Uji Reliabilitas Berdasarkan hasil uji reliabilitas, semua pernyataan dari variabel gaya kepemimpinan, motivasi kerja dan kinerja pegawai seluruhnya dikatakan reliable, karena seluruh nilai Cronbach’s alpha dari seluruh varibel lebih besar dari 0,6. Ayu Adriani Metalia, Syofiah Aryani 252 Volume 3, Nomor 3, Maret 2023 p-ISSN 2774-7018 ; e-ISSN 2774-700X 3. Uji Asumsi Klasik a. Uji Normalitas Dari graifk P-P Plot diketahui semua data yang ada berdistribusi normal, karena semua data menyebar dan membentuk garis lurus searah dengan garis diagonal maka data tersebut memenuhi asumsi normal atau mengikuti garis normalitas. Gambar 1. Uji Normalitas b. Uji Multikolinearitas Nilai tolerance variabel gaya kepemimpinan (0,423 > 0.05), dan variabel motivasi kerja (0,423 > 0,05), sedangkan nilai VIF variabel kepemimpinan (2,367 < 10), dan variabel motivasi kerja (2,367 < 10). Maka dengan demikian dapat disimpulkan bahwa pada model regresi tidak terdapat gejala multikolinearitas. c. Uji Heteroskedastisitas Hasil pengujian dengan menggunakan metode scatterplots, hasil uji ini menunjukkan bahwa titik-titik menyebar secara acak serta tersebar baik di atas maupun di bawah angka 0 pada sumbu Y, maka dapat disimpulkan dalam model regresi ini kedua persamaan tersebut tidak terjadi masalah heteroskedastisitas. Gambar 2. Uji heteroskedatisitas 4. Analisis Regresi Linier Berganda Tabel 1 Regresi Linier Berganda Model 253 Coefficientsa Unstandardized Standardized Coefficients Coefficients t Sig. Collinearity Statistics http://sosains.greenvest.co.id Std. B Error 1 (Constant) 4.462 3.475 Gaya_Kepemimpinan .449 .160 Motivasi_Kerja .424 .184 a. Dependent Variable: Kinerja_Pegawai Beta Tolerance VIF 1.284 .205 .416 2.801 .007 .342 2.301 .025 .423 2.367 .423 2.367 Sumber: Hasil Pengolahan SPSS Berdasarkan Tabel 1 di atas, diketahui persamaan regresi linear berganda sebagai berikut: Y = 4,462 + 0,449 Gaya Kepemimpinan + 0,424 Motivasi Kerja + e Persamaan regresi tersebut dapat diinterpretasikan sebagai berikut: 1. Nilai 4,462 adalah nilai konstana (α) yang artinya ketika variabel Gaya Kepemimpinan (X1), dan Motivasi Kerja (X2) adalah 0 (nol), maka besarnya nilai untuk variabel Kinerja Pegawai (Y) adalah sebesar 4,462. 2. Nilai 0,449 adalah nilai koefisien variabel gaya kepemimpinan (X1), artinya jika variabel gaya kepemimpinan (X1) mengalami kenaikan 1 persen, maka tingkat pengaruh terhadap variabel kinerja pegawai (Y) akan mengalami peningkatan 44,9%. Nilai 0,424 adalah nilai koefisien variabel motivasi kerja (X2), artinya jika variabel motivasi kerja (X2) mengalami kenaikan 1 persen, maka tingkat pengaruh terhadap variabel kinerja pegawai (Y) akan mengalami peningkatan sebesar 42,47%. 5. Koefisien Determinasi (X2) Tabel 2 Koefisien Determinasi (R2) Model Summaryb Change Statistics Std. Error R Adjusted of the R Square F Sig. F Model R Square R Square Estimate Change Change df1 df2 Change 1 .711a .506 .487 4.38629 .506 27.145 2 53 .000 a. Predictors: (Constant), Motivasi_Kerja, Gaya_Kepemimpinan b. Dependent Variable: Kinerja_Pegawai Sumber: Hasil Pengolahan SPSS Dari tabel 2 diatas menunjukkan nilai Adjusted R Square sebesar 0,487 atau 48,7% yang berarti penerapan variabel kinerja pegawai yang ada pada penelitian ini dijelaskan oleh variabel independen sebesar 48,7% dan sisanya 51,3% dijelaskan oleh variabel lain diluar penelitian ini. a. Uji F Tabel 3 Uji F ANOVAa Sum of Model Squares df Mean Square 1 Regression 1044.501 2 522.250 Residual 1019.697 53 19.240 Total 2064.197 55 a. Dependent Variable: Kinerja_Pegawai b. Predictors: (Constant), Motivasi_Kerja, Gaya_Kepemimpinan F 27.145 Sig. .000b Sumber: Hasil Pengolahan SPSS Ayu Adriani Metalia, Syofiah Aryani 254 Volume 3, Nomor 3, Maret 2023 p-ISSN 2774-7018 ; e-ISSN 2774-700X Dari tabel 3 diatas di peroleh nilai F hitung sebesar 27,145 dengan probabilitas 0,000 karena probabilitas jauh lebih kecil dari 0,05 maka dapat di katakan bahwa tingkat pendidikan, usia dan pengalaman kerja berpengaruh terhadap produktivitas kerja karyawan, sedangkan nilai F tabel sebesar 3,17 dengan angka df1 = 2 dan df2 = 56-2-1, sehingga F hitung (29,886) > F tabel (2,66) dan nilai signifikansi sebesar 0,000 menunjukkan bahwa signifikansi lebih kecil dari 0,05 (0,000 < 0,05), menunjukkan bahwa model regresi dapat digunakan dalam penelitian ini. Artinya secara simultan variabel gaya kepemimpinan dan motivasi kerja mampu mempengaruhi variabel kinerja pegawai. b. Uji Hipotesis (Uji t) Tabel 4 Uji t Coefficientsa Unstandardized Standardized Coefficients Coefficients B Std. Error Beta 4.462 3.475 .449 .160 .416 t 1.284 2.801 Sig. .205 .007 .342 2.301 .025 Model 1 (Constant) Gaya_Kepemim pinan Motivasi_Kerja .424 .184 a. Dependent Variable: Kinerja_Pegawai Collinearity Statistics Tolerance VIF .423 2.367 .423 2.367 Sumber: Hasil Pengolahan SPSS Hasil perhitungan variabel gaya kepemimpinan menunjukkan bahwa nilai signifikan kurang dari 0,05 (0,007 < 0,05) dan nilai t hitung lebih besar dari t tabel (2,801 > 2,005), jadi dapat disimpulkan hipotesis H1 diterima, artinya gaya kepemimpinan secara parsial berpengaruh signifikan terhadap variabel kinerja pegawai. Hasil perhitungan variabel motivasi kerja menunjukkan bahwa nilai signifikan kurang dari 0,05 (0,025 > 0.05) dan nilai t hitung lebih besar dari t tabel (2,301 > 2,005), jadi dapat disimpulkan hipotesis H 2 diterima. Artinya motivasi kerja secara parsial berpengaruh signifikan terhadap variabel kinerja pegawai. B. Pembahasan 1. H1 = Terdapat Pengaruh antara Gaya Kepemimpinan (X1) Terhadap Kinerja Pegawai (Y) Badan Penghubung Pemerintah Provinsi Papua Di Jakarta Hasil penelitian menunjukkan bahwa gaya kepemimpinan berpengaruh terhadap kinerja pegawai, dengan nilai signifikan kurang dari 0,05 (0,007 < 0,05) dan nilai t hitung lebih besar dari t tabel (2,801 > 2,005). Variabel gaya kepemimpinan mempunyai nilai koefisien regresi sebesar 0,4429 (bernilai positif). Hal in berarti semakin baik sikap seorang pemimpin dalam sebuah perusahaan maka akan memberikan kenyamanan kerja bagi bawahannya, sehingga akan meningkatkan kinerja para pegawai. Hasil penelitian sesuai dengan penndapat yang dikemukakan oleh Herawati (2015), bahwa kepemimpinan adalah seseorang yang mempengaruhi dan memotivasi orang lain untuk melakukan sesuatu sesuaoi tujuan bersama. Kepemimpinan adalah proses yang mempengaruhi dalam menentukan tujuan organisasi, memotivasi perilaku pengikut untuk mencapai tujuan, mempengaruhi untuk memperbaiki kinerja pegawai. Selain itu, hasil penelitian ini sejalan dengan penelitian yang dilakkan oleh Bryan Johannes Tampi (2018) yang mengungkapkan bahwa Gaya Kepemimpinan dan Motivasi Kerja berpengaruh positif dan signifikan terhadap Kinerja Pegawai. 255 http://sosains.greenvest.co.id 2. H2 = Terdapat Pengaruh antara Motivasi Kerja (X2) Terhadap Kinerja Pegawai (Y) Badan Penghubung Pemerintah Provinsi Papua Di Jakarta Hasil penelitian menunjukkan bahwa motivasi kerja berpengaruh terhadap kinerja pegawai, dengan nilai signifikan kurang dari 0,05 (0,025 > 0.05) dan nilai t hitung lebih besar dari t tabel (5,311 > 1,974). Variabel motivasi kerja mempunyai nilai koefisien regresi sebesar 0,424 (bernilai positif). Hal ini menunjukkan bahwa motivasi sangat penting bagi pegawai, karena semakin tinggi motivasi yang dimiliki oleh seorang pegawai maka akan memiliki prestasi kerja yang baik dan kinerja yang baik (Abdilah & Djastuti, 2011). Hasil penelitian sesuai dengan pendapat, yang menyatakan juka karyawan memiliki motivasi yang rendah cenderung tidak memiliki prestasi kerja yang baik, daripada pegawai yang memiliki motivasi yang tinggi (Murty, 2012). Sebaliknya apabila terdapat motivasi yang tinggi dari para pegawai, maka hal ini merupakan suatu jaminan atas keberhasilan suatu perusahaan dalam mencapai tujuannya. Selain itu penelitian ini sejalan dengan hasil penelitian yang dilakukan oleh Aries Susanty dan Sigit Wahyu Baskoro (2021) yang menyatakan bahwa walaupun tidak signifikan, motivasi kerja dari karyawan PT. PLN (Persero) APD Senmarang memiliki pengaruh positif terhadap kinerja karyawan. Hal ini dapat terjadi karena seorang karyawan yang memiliki motivasi kerja tinggi belum tentu dapat menghasilkan output kerja yang baik atau memuaskan (FACHRIZA, 2022). KESIMPULAN Berdasarkan hasil analisis data dan pembahasan mengenai pengaruh gaya kepemimpinan dan motivasi terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua Di Jakarta dengan metode analisis yang digunakan yaitu metode regresi berganda, maka dapat ditarik kesimpulan bahwa Variabel gaya kepemimpinan berpengaruh terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua di Jakarta. Variabel motivasi kerja berpengaruh terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua di Jakarta. Variabel gaya kepemimpinan dan motivasi kerja secara simultan berpengaruh terhadap kinerja pegawai Badan Penghubung Pemerintah Provinsi Papua di Jakarta. DAFTAR PUSTAKA Abdilah, Rokhmaloka Habsoro, & Djastuti, Indi. (2011). Analisis Pengaruh Gaya Kepemimpinan Dan Motivasi Kerja terhadap Kinerja Pegawai (Studi pada Pegawai Badan Kesatuan Bangsa Politik dan Perlindungan Masyarakat Provinsi Jawa Tengah). Universitas Diponegoro. Djuwita, Tita Meirina. (2011). Pengembangan sumber daya manusia dan produktivitas kerja pegawai. Manajerial: Jurnal Manajemen Dan Sistem Informasi, 10(2), 15– 21. FACHRIZA, MUHAMMAD BAIHAQI. (2022). PENGARUH MOTIVASI DAN DISIPLIN KERJA TERHADAP KINERJA KARYAWAN STUDI PADA PT SINAR SOSRO DELI SERDANG BRANCH. Hanafi, Andhi Sukma, Almy, Chairil, & Siregar, M. Tirtana. (2018). Pengaruh gaya kepemimpinan dan motivasi kerja terhadap kinerja pegawai. Jurnal Manajemen Industri Dan Logistik, 2(1), 52–61. Heriyanti, Sinta Sundari, & Putri, Rahma. (2021). Pengaruh Gaya Kepemimpinan, Lingkungan Kerja dan Stres Kerja terhadap Kinerja Karyawan PT NT Cikarang. Jesya (Jurnal Ekonomi Dan Ekonomi Syariah), 4(2), 915–925. Iqbal, N., Anwar, Sanusi, & Haider, N. (2015). Effect of leadership style on employee performance. Arabian Journal of Business and Management Review, 5(5), 1–6. Ayu Adriani Metalia, Syofiah Aryani 256 Volume 3, Nomor 3, Maret 2023 p-ISSN 2774-7018 ; e-ISSN 2774-700X Kadarisman, Muhammad. (2012). Manajemen pengembangan sumber daya manusia. Jakarta: Rajawali Pers, 2, 13. Murty, Windy Aprilia. (2012). Pengaruh kompensasi, motivasi dan komitmen organisasional terhadap kinerja karyawan bagian akuntansi (studi kasus pada perusahaan manufaktur di Surabaya). STIE Perbanas Surabaya. Ningrum, Dilla Agista, Fauzi, Achmad, Supu, Amelya Lestin Anggraeni, Agustin, Primasari, Afriliani, Silvia Nurul Izati, & Mahardhika, Wahyu Tia. (2022). Pengaruh Gaya Kepemimpinan, Lingkungan Kerja Dan Stres Kerja Terhadap Kinerja Karyawan (Studi Pustaka Manajemen Kinerja). Jurnal Ekonomi Manajemen Sistem Informasi, 4(2), 224–233. Putra, Fariz Ramanda. (2013). Pengaruh Lingkungan Kerja Terhadap Kinerja (Studi Pada Karyawan PT. Naraya Telematika Malang). Brawijaya University. Sinambela, Lijan Poltak. (2021). Manajemen Sumber Daya Manusia: Membangun tim kerja yang solid untuk meningkatkan kinerja. Bumi Aksara. Suwandi, Annisa Pratiwy. (2013). Pengaruh kejelasan sasaran anggaran dan desentralisasi terhadap kinerja pemerintah daerah (Studi empiris pada SKPD Pemerintah Kota Padang). Jurnal Akuntansi, 1(2). Tamarindang, Billy, Mananeke, Lisbeth, & Pandowo, Merinda. (2017). Pengaruh Gaya Kepemimpinan, Motivasi Dan Disiplin Kerja Terhadap Kinerja Karyawan Di Bank Bni Cabang Manado. Jurnal Emba: Jurnal Riset Ekonomi, Manajemen, Bisnis Dan Akuntansi, 5(2). Tampi, Bryan Johannes. (2014). Pengaruh Gaya Kepemimpinan dan Motivasi terrhadap Kinerja karyawan pada PT. Bank Negara Indonesia, tbk (regional sales manado). Acta Diurna Komunikasi, 3(4). Umdiana, Nana, & Claudia, Hashifah. (2020). Analisis Struktur Modal Berdasarkan Trade off Theory. Jurnal Akuntansi: Kajian Ilmiah Akuntansi, 7(1), 52–70. Winarso, Widi. (2019). Pengaruh Gaya Kepemimpinan Terhadap Kinerja Karyawan (Studi Kasus: PT Agung Citra Tranformasi). Jurnal Ilmiah Akuntansi Dan Manajemen (JIAM), 15(2), 38–49. This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. 257 http://sosains.greenvest.co.id
https://openalex.org/W3043678472	https://iris.unive.it/bitstream/10278/3728352/1/fevo-08-00235.pdf	English	null	Bioaccumulation of Polychlorinated Dibenzo-p-Dioxins (PCDDs) and Dibenzofurans (PCDFs) in Hediste diversicolor (Polychaeta: Nereididae)	Frontiers in ecology and evolution	2,020	cc-by	11,161	ORIGINAL RESEARCH published: 17 July 2020 doi: 10.3389/fevo.2020.00235 Edited by: Alice Newton, University of Algarve, Portugal Reviewed by: Paolo Magni, National Research Council (CNR), Italy Michael Joseph Ahrens, Universidad de Bogotá Jorge Tadeo Lozano, Colombia Correspondence: Annamaria Volpi Ghirardini voghi@unive.it †††ORCID: Annamaria Volpi Ghirardini orcid.org/0000-0002-8834-2595 Annamaria Volpi Ghirardini orcid.org/0000-0002-8834-2595 Specialty section: This article was submitted to Conservation, a section of the journal Frontiers in Ecology and Evolution Received: 30 September 2019 Accepted: 26 June 2020 Published: 17 July 2020 Specialty section: This article was submitted to Conservation, a section of the journal Frontiers in Ecology and Evolution Keywords: Venice Lagoon, TCDD, TCDF, benthos, bioaccumulators Received: 30 September 2019 Accepted: 26 June 2020 Published: 17 July 2020 Bioaccumulation of Polychlorinated Dibenzo-p-Dioxins (PCDDs) and Dibenzofurans (PCDFs) in Hediste diversicolor (Polychaeta: Nereididae) Marco Picone1, Eugenia Delaney1, Davide Tagliapietra2, Irene Guarneri2 and Annamaria Volpi Ghirardini1† 1 Dipartimento di Scienze Ambientali, Informatica e Statistica, Università Ca’ Foscari, Venezia, Italy, 2 Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine, Venezia, Italy The effectiveness and reliability of the polychaete Hediste diversicolor (O.F. Müller, 1776) to bioaccumulate polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) was assessed in an in-situ passive biomonitoring study. Field collected specimens were sampled in ﬁve sites within the Venice Lagoon (Italy), selected along a PCDD/F contamination gradient. The homolog proﬁles in the tissues of the common ragworm were considerably different from those observed in the sediments, independent of sediment contamination. Moreover, H. diversicolor accumulated preferentially the less chlorinated 2,3,7,8-TCDD, 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF compared to the more chlorinated and hydrophobic hexa-, hepta- and octa- substituted congeners, as evidenced by the signiﬁcant and linearly decreasing trend of the Biota-to-Sediment Accumulation Factor (BSAF) with the increasing lipophilicity of the congeners, expressed as the logarithmic form of the octanol/water partition coefﬁcient (logKOW). The BSAFs for dioxins and furans were generally low compared to other organochlorine compounds such as polychlorinated biphenyls and organochlorine pesticides, suggesting that H. diversicolor may eliminate both dioxins and furans efﬁciently. Keywords: Venice Lagoon, TCDD, TCDF, benthos, bioaccumulators INTRODUCTION Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are highly persistent, bioaccumulative and toxic contaminants not intentionally produced, but occurring in the environment as byproducts of chemical processes involving the use of chlorine (i.e., wood pulp and magnesium industries), as a result of combustion processes from both natural (forest ﬁres) and anthropogenic sources (smoke from incinerators, car and boat exhaust fumes) or as impurities in chemicals (i.e., pesticides and herbicides) (Swerev and Ballschmiter, 1989). Citation: Picone M, Delaney E, Tagliapietra D, Guarneri I and Volpi Ghirardini A (2020) Bioaccumulation of Polychlorinated Dibenzo-p-Dioxins (PCDDs) and Dibenzofurans (PCDFs) in Hediste diversicolor (Polychaeta: Nereididae). Front. Ecol. Evol. 8:235. doi: 10.3389/fevo.2020.00235 Study Area The Venice Lagoon is one of the largest and relevant Coastal Transitional Ecosystems of the Mediterranean (Tagliapietra et al., 2009); it is about 50 km long and 10 km wide, accounting for a surface of about 550 km2. Out of them, 36 km2 are salt marshes, 30 km2 islands (excluding the barrier islands) and the rest is covered by water. The mean depth of the water column is about 1.2 m, with only 5% of the lagoon deeper than 5 m (Molinaroli et al., 2009). The volume of water contained in the lagoon is about 628 million m3, and according to Kjerfve (1994), the Venice Lagoon can be deﬁned as a “restricted” coastal lagoon. The basin is connected to the Adriatic Sea through three inlets (Lido, Malamocco, and Chioggia) which allow tidal ﬂushing twice a day (microtidal and predominantly semidiurnal tides) (Tagliapietra and Volpi Ghirardini, 2006). Every day the Venice Lagoon exchanges with the Adriatic Sea about 400 million m3 of water while the inﬂow from the inland through the rivers and subsoil averages 3.7 million m3 (Bernstein and Montobbio, 2011). The drainage basin is about 1850 km2, 40% of which is reclaimed land lying under the sea level. q ) A commonly used polychaete species in ecotoxicological research is the common ragworm Hediste diversicolor (O.F. Müller, 1776). This burrowing polychaete is omnivorous, but also behaves as a deposit feeder, by collecting detritus near the opening of its burrows, and is widespread in the shallow marine and estuarine waters of the European coasts of the Atlantic, the Mediterranean Sea, the Black Sea and the Caspian Sea (Scaps, 2002). Its large dispersion into brackish waters has favored its frequent use as a biological indicator for assessing exposure and eﬀects of sediment-bound contaminants in estuaries and coastal lagoons aﬀected by pollution due to anthropogenic sources, including metals (Volpi Ghirardini et al., 1999; Berthet et al., 2003; Frangipane et al., 2005) and organic contaminants, such as polynuclear aromatic hydrocarbons (PAHs) – although the ability of polychaetes to biotransform PAHs was demonstrated (Jørgensen et al., 2008) – and polychlorinated biphenyls (PCBs) (Gunnarsson et al., 1999; Cornelissen et al., 2006; Langston et al., 2012). Nevertheless, the ability of H. Citation: Citation: Picone M, Delaney E, Tagliapietra D, Guarneri I and Volpi Ghirardini A (2020) Bioaccumulation of Polychlorinated Dibenzo-p-Dioxins (PCDDs) and Dibenzofurans (PCDFs) in Hediste diversicolor (Polychaeta: Nereididae). Front. Ecol. Evol. 8:235. doi: 10.3389/fevo.2020.00235 Citation: Picone M, Delaney E, Tagliapietra D, Guarneri I and Volpi Ghirardini A (2020) Bioaccumulation of Polychlorinated Dibenzo-p-Dioxins (PCDDs) and Dibenzofurans (PCDFs) in Hediste diversicolor (Polychaeta: Nereididae). Front. Ecol. Evol. 8:235. doi: 10.3389/fevo.2020.00235 Once released, PCDD/F enter the marine and coastal environments via atmospheric deposition, riverine inputs from the inland, and direct discharges of urban and industrial wastewaters into the coastal waters (Armitage et al., 2009). July 2020 \| Volume 8 \| Article 235 Frontiers in Ecology and Evolution \| www.frontiersin.org 1 Accumulation of PCDD/F in Ragworms Picone et al. polychaetes. The Venice Lagoon was chosen as a case study since sources, inputs and sediment concentration of PCDD/F have been extensively studied in the past decades (Fattore et al., 1997; Marcomini et al., 1997; Jimenez et al., 1998; Bellucci et al., 2000; Frignani et al., 2001a). The aims of the study were the assessment of possible bioaccumulation in tissues of H. diversicolor, the identiﬁcation of the congeners with higher ability to accumulate into polychaete tissues, and the analysis of the possible relationships between accumulation and the lipophilicity (expressed as logKOW) of the 2,3,7,8 chlorinated congeners. In the marine environment PCDD/F are quickly adsorbed onto suspended matter, due to their high hydrophobicity, and then deposited onto the sediment where they accumulate over time, due to their persistence. As a consequence, sediment may act both as a secondary source of PCDD/F pollution for the water column (Khairy et al., 2016) and as a primary source of contamination for the benthic species living and/or feeding on sediment-bound contaminants (Pruell et al., 1993, 2000). Polychaetes are a major component of the coastal and estuarine systems, where they may represent up to 40% of the taxa constituting the communities (Amiard-Triquet et al., 2013). Many species living in soft-bottom habitats are endobenthic and occupy several ecological niches, serving as relevant vectors for the recycling of the detritus, as primary consumers of benthic algae and predators of other invertebrates, and as food for the organisms at the top of the benthic and aquatic trophic webs (Scaps, 2002; Dean, 2008; Amiard-Triquet et al., 2013). Citation: The results of this study, performed in 1998, are presented only now because we found that in the last 20 years no signiﬁcant progress has been made in the ﬁeld of bioaccumulation of organochlorine compounds in estuarine environments. Although they play a relevant role in the trophic webs of marine and estuarine environments, benthic infaunal organisms such as polychaetes, have been too often disregarded as bioindicators for bioavailability/bioaccessibility of PCDD/Fs, in favor of other species of greater commercial interest (ﬁsh and molluscs). The focal purpose of this paper is thus to underline how these organisms are suitable for studying bioaccumulation of organic contaminants (other than PAHs), as well as to promote a more widespread exploitation of polychaetes in monitoring programs. Close contact with the sediment as well as detritivorous habits of most species make polychaetes vulnerable to the uptake of contaminants through both contact with dissolved chemicals and ingestion of pollutants bound to sediment particles or adsorbed onto detritus particles (Dean, 2008). Consequently, contaminants accumulated into the tissues of the polychaetes may then be transferred to higher levels of the trophic web, leading to possible biomagniﬁcation phenomena (Ruus et al., 2012; Sizmur et al., 2013). For these reasons, polychaetes are often used in ecotoxicological studies to assess both early warning signals of chemical stress, by using relevant biomarkers, and the bioavailability of the contaminants (Durou et al., 2007a,b; Nesto et al., 2010; Ruus et al., 2012; Amiard-Triquet et al., 2013). Frontiers in Ecology and Evolution \| www.frontiersin.org Polychaete Holding and Analysis Once in the laboratory, the polychaetes were depurated for 4 days in small glass aquaria (20 × 20 × 15 cm) containing acid-washed quartz sand and artiﬁcial seawater (1:1 v/v sand/water ratio), to allow the elimination of the gut’s content. Then, they were transferred for 1 day in aquaria containing only artiﬁcial seawater, to remove also the quartz sand accumulated during depuration phase (Lobel et al., 1991; Volpi Ghirardini et al., 1999). Artiﬁcial seawater was prepared by dissolving an artiﬁcial sea salt mixture (Ocean Fish R⃝, PRODAC International, Cittadella, Italy) in Milli- Q R⃝puriﬁed water. Sediment sampling was performed following the integrated design and Quality Assurance/Quality Control (QA/QC) procedures reported in Volpi Ghirardini et al. (2005). Brieﬂy, in each site, the sampling area was deﬁned as a circle with a diameter of 30 m. Within this area, eight sediment cores (depth 0–20 cm, diameter 5 cm) were collected with a Plexiglas R⃝corer and then pooled to obtain an integrated sample. This procedure was performed in triplicate in each sampling area. Sediment samples were stored in 2 l glass containers and kept refrigerated until their arrival in the laboratory. Samples were processed within 2 weeks, according to ASTM guidelines (ASTM, 2014). During the last day of depuration in seawater, glass tubes were introduced into the aquaria to allow polychaetes to individually settle in there, to avoid aggressive behavior, cannibalism and also damages due to the lack of substrate (e.g., autotomy). At the end of the depuration phase, animals were anaesthetized using a 10% ethanol solution in seawater 20% and individually checked for species conﬁrmation under a dissecting microscope. After taxonomic conﬁrmation and determination of biometric parameters (in order to select polychaetes of comparable age), pooled samples of 20–30 specimens per sampling site were frozen until the analysis. Taxonomic identiﬁcation was performed in order to avoid mixing with specimens belonging to other species, such as Alitta succinea (Leuckhart, 1847) and Perinereis cultrifera (Grube, 1840), that may share the habitat with H. diversicolor (Volpi Ghirardini et al., 1999). PCDD/F in the tissues were analyzed using the same method reported for sediments; prior to analysis, the frozen polychaete samples were freeze-dried. One composite sample (n = 1) per site was analyzed. g g Polychaetes were collected within sediment sampling areas using a box corer (14×14×16 cm). Study Area diversicolor to also accumulate other persistent and bioaccumulative toxicants, as PCDD/F, has been rarely explored, although these polychlorinated compounds may accumulate in ﬁsh – and also humans (Raccanelli et al., 2007) – through the marine and estuarine trophic web, where H. diversicolor represents a relevant trophic link (Nunes et al., 2011). The primary source of pollution is the Porto Marghera industrial district (Bellucci et al., 2000; Frignani et al., 2001a,b; Zonta et al., 2007). Other point and non-point sources of pollution ﬂowing into the lagoon include also treated and untreated municipal wastewaters, streams, agricultural runoﬀ, boat traﬃc and atmospheric deposition (Guerzoni et al., 2004; Secco et al., 2005; Volpi Ghirardini et al., 2005; Gambaro et al., 2009). The recorded pattern of pollution follows the urban and industrial development: evidence exists that The present study examined the eﬀectiveness and reliability of H. diversicolor as an indicator of exposure for PCDD/F in an in-situ passive biomonitoring study, using ﬁeld-collected July 2020 \| Volume 8 \| Article 235 2 Accumulation of PCDD/F in Ragworms Picone et al. contamination of waters and sediments started in 1920 with the development of the ﬁrst part of the industrial area, then accelerated after 1933 and again after World War II (Frignani et al., 2001a,b). As a consequence, the contamination gradient was largely superimposed on the inland-sea transect (Picone et al., 2016, 2018). as a consequence, it was necessary to pool organisms collected in each site in order to have enough biological material for performing chemical analysis with an adequate limit of detection (0.25 pg g−1 dw). Sediment Analysis The atmospheric and riverine inputs of PCDD/F into the Venice Lagoon has been estimated in the order of 2.2–195.7 g y−1 (Guerzoni et al., 2007) and 6.9 g y−1 (Bettiol et al., 2005), respectively; the contribution of treated and untreated discharges from the industrial district of Porto Marghera has been estimated in 0.10–0.26 g y−1 as toxicity equivalents (MAG. ACQUE-Uta, 2011). Total organic carbon (TOC) analyses were performed using a CHNS-O analyzer on aliquots of 10–20 mg of dry sediment acidiﬁed with 20-µL of 1N HCl solution and dried at 105◦C for 15 min. Sediment grain-size was determined following a gravimetric procedure and was subsequently classiﬁed according to Shepard (1954). PCDD/F in the sediments were analyzed according to the procedure reported in Bellucci et al. (2000), based on the United States. EPA method 1613/94 for the determination of 17 congeners 2,3,7,8 substituted of dioxins and furans using gas chromatography-mass spectrometry (GC-MS). One composite sample (n = 1) per site was analyzed. Sediment and Polychaete Sampling Sediment and polychaete samples were collected during summer 1998 from 5 sub-tidal shallows, selected along a gradient of chemical contamination and aﬀected by diﬀerent sources of pollution (Figure 1). Palude della Centrega (CE) is an inter-tidal mudﬂat in the northern basin of the Lagoon, chosen as possible reference site due to the low concentrations of contaminants characterizing the area (Volpi Ghirardini et al., 2005; Picone et al., 2008). Dese river (DE) and Osellino canal (OS) sites are two estuaries inﬂuenced by agricultural runoﬀ; OS is also characterized by multi-sources pollution being aﬀected by both urban and industrial discharges, due to the proximity to the urban area of Mestre and the illegal landﬁll of Campalto (Critto et al., 2003). Canale Industriale Sud (SA) and Canale Lusore- Brentelle (BR) are two industrial canals located within the Porto Marghera district; in particular, site BR is a polluted site both as concern metals and organic micropollutants (Volpi Ghirardini et al., 2005; Bellucci et al., 2009; Picone et al., 2018) catching also freshwater from the Naviglio di Brenta canal. Frontiers in Ecology and Evolution \| www.frontiersin.org Polychaete Holding and Analysis Sediments collected with the box corer were washed into polyethylene tanks and then the sediment slurry was poured into a 1 mm mesh size sieve and thoroughly washed in situ with seawater to separate the worms from the substrate. Once collected, the polychaetes were placed in 1 liter polyethylene containers ﬁlled with a layer of clean quartz sand (grain size approx. 60–100 µm) and natural seawater, in order to minimize the stress during the transport in the laboratory. A variable number of polychaetes was retrieved in each site, from 30 up to 80 depending on the population density in the area. Only for sites BR and OS it was possible to obtain three replicates with suﬃcient biological material for analysis; Lipids were extracted by Accelerated Solvent Extraction (ASE) with 50 ml of n-hexane/dichloromethane 50/05 at 150◦C, 1500 psi 7-min heat-up and two cycles of 5 min static time. The extracts July 2020 \| Volume 8 \| Article 235 3 Accumulation of PCDD/F in Ragworms Picone et al. FIGURE 1 \| Map of the sampling sites within the Venice Lagoon. CE = Palude della Centrega; DE = Dese river; OS = Osellino canal; SA = Canale Industriale Sud; BR = Canale Lusore-Brentelle. FIGURE 1 \| Map of the sampling sites within the Venice Lagoon. CE = Palude della Centrega; DE = Dese river; OS = Osellino canal; SA = Canale Industriale Sud; BR = Canale Lusore-Brentelle. g sites within the Venice Lagoon. CE = Palude della Centrega; DE = Dese river; OS = Osellino canal; SA = Canale Industriale Sud; GURE 1 \| Map of the sampling sites within the Venice Lagoon. CE = Palude della Centrega; DE = Dese river; OS = Osellino canal; R = Canale Lusore-Brentelle. were desiccated using an evaporator equipped with a vacuum controller. The lipid content was then determined gravimetrically using an analytical balance. concentration in sediments (pg g−1 dw), normalized to the lipid content and to the organic carbon content, respectively (Burkhard, 2009). For the calculation of BSAFs, concentrations below detection limits were assumed to equal a value half of the detection limit used. Welch’s ANOVA and Games-Howell test were applied to the pooled BSAF data in order to check for the overall signiﬁcant diﬀerence in the accumulation of the congeners of PCDD/F. Polychaete Holding and Analysis Welch’s ANOVA was chosen as a parametric method for the analysis of variance since homogeneity of variances condition was not met also after logarithmic and square root transformations. Data Elaboration and Analysis Concentrations of PCDD/F in sediments and tissues were reported on dry weight (dw) basis. The dry weight concentrations were then also converted in 2,3,7,8-TCDD equivalents by multiplying the sediment concentrations by the toxic equivalency factors (TEFs) proposed by the World Health Organization (Van den Berg et al., 2006). Cluster analysis was used to categorize the homolog proﬁle of PCDD/F in sediments and tissues; Euclidean distances and complete linkage were used as distance metric and joining rule, respectively. Before cluster analysis, sediment and tissue concentrations were transformed to per mil 2,3,7,8 chlorosubstituted homologs, by normalizing the sum of the isomers with the same degree of chlorination (i.e., PeCDD or HxCDD) to the sum of all dioxins and furans congeners with a concentration above the detection limit (Marcomini et al., 1997). All the statistical analysis were performed using the StatSoft Statistica v8.0 (Spearman’s non-parametric correlation, linear regression) and IBM SPSS Statistics v.25 software (Welch’s ANOVA and Games-Howell post hoc test). Sediment Chemistry Total organic carbon in the sediments ranged from 1.88% (CE) up to 4.67% (OS); grain size was characterized by a prevailing fraction of silt, ranging from 52.9% (BR) up to 69.6% (SA). According to the ternary classiﬁcation of Shepard (1954) samples CE, DE, OS, and SA were classiﬁed as clayey silts, whilst sample BR was classiﬁed sandy silt. Spearman’s non-parametric correlation was applied to test for signiﬁcant correlations between sediment and tissue concentrations of the 17 congeners of PCDD/F, whilst linear regression was used to verify the linkage between BSAFs and the octanol-water partition coeﬃcients (KOW), after logarithmic transformation. LogKOW values were taken from Chen et al. (2001). Sediment PCDD/F concentrations at the ﬁve study sites ranged between 73 and 6,621 pg g−1 dw. The sediments collected at site SA (6,621 pg g−1 dw) were the most contaminated being characterized by total concentrations (6PCDD/F) of 1 or 2 order Biota-Sediment Accumulation Factors (BSAFs) were calculated for each sampling site as the ratio of contaminant concentration in tissues (pg g−1 dw) on the contaminant July 2020 \| Volume 8 \| Article 235 Frontiers in Ecology and Evolution \| www.frontiersin.org 4 Accumulation of PCDD/F in Ragworms Picone et al. of magnitude higher than the other samples (Table 1). As concern dioxins, the total sediment concentrations ranged from 24 pg g−1 dw (CE) to 677 pg g−1 dw (OS), with OCDD accounting for more than 75% of total dioxins in all samples; 2,3,7,8-TCDD occurred at concentrations above the detection limit (0.5 pg g−1 dw) only in sample SA (1 pg g−1 dw). With regard to furans, the highest concentration was measured in sample SA (6061 pg g−1 dw) whilst the reference sample collected in site CE was, as expected, the least contaminated one (47 pg g−1 dw); the congeners detected at highest concentrations were 1,2,3,4,6,7,8- HpCDF and 1,2,3,4,6,7,8,9-OCDDF. Cluster analysis on homolog proﬁles evidenced the occurrence of distinct patterns of accumulation in the tissue (Figure 3). At site BR the polychaetes accumulated mostly OCDF and secondarily OCDD and TCDF, whereas the polychaetes collected at sites SA and OS accumulated primarily furans (contributing up to 87% and 94% to 6PCDD/F, respectively), with a prevalence of HxCDF, TCDF and PeCDF, over the other furans and OCDD. Comparison Between Sediments and Tissues Homolog proﬁles in sediment showed diﬀerent patterns among sites, especially as concern the contribution of OCDD and OCDF (Figure 2). In sample SA, the proﬁle was dominated by OCDF and characterized by negligible contributions of the other homologs except for OCDD and HpCDF; in contrast, in samples BR and OS the proﬁles showed a dominance of OCDD over the more chlorinated furans. In DE and CE the proﬁle was characterized by a gradient of increasing concentrations of PCDD/F with increasing degree of chlorination, but with a dominance of OCDF over OCDD and HpCDF. Cluster analysis conﬁrmed this categorization of samples into three groups (Figure 3). No correlation was observed between PCDD congeners in the sediment and in the polychaete tissues (p > 0.05); in the case of furans, signiﬁcant correlations (Spearman’s R > 0.9; p < 0.05) were found for the PeCDFs, HpCDFs and two congeners of HxCDFs (namely, 1,2,3,4,7,8-HxCDF and 1,2,3,6,7,8-HxCDF). Spearman’s correlation was not calculated for 2,3,7,8-TCDD and 1,2,3,7,8,9 HxCDD due to the large number of data below detection limits. Sediment Accumulation Factors value ranged between 0.003 (OCDF in site SA) and 11.13 (TCDF in site BR). Hepta- and octachlorinated congeners showed a BSAF about one order of magnitude lower than the less chlorinated congeners (Table 1). Linear regression revealed a signiﬁcant relationship with a decreasing trend between logBSAF and logKOw (F1,83 = 43.878; r2 = 0.289; r = −0.539; p < 0.0001), indicating a tendency to accumulate preferentially the congeners with lower logKOW (Figure 5). Sediment Chemistry The samples with less accumulation, DE and CE, showed a more heterogeneous pattern, with an accumulation of OCDD and less chlorinated PCDF (DE) or OCDD and more chlorinated PCDF (CE) (Figure 4). In any case, diﬀerences in homolog concentrations in these samples are very low. The equivalent toxicity 6TE (WHO-TEQ) in the sediment samples ranged from 2 pg g−1 dw (CE) to 60 pg g−1 dw (SA) and showed the same gradient as the total concentrations: CE < DE < BR < < OS < SA. Tissue Analysis Polychaete tissues were characterized by a variable lipid concentration, ranging from 0.10 to 0.14 g g−1 of tissue, whilst organic carbon content averaged 0.53 g g−1 of tissue (range: 0.52–0.54 g g−1). Welch’s ANOVA highlighted signiﬁcant diﬀerences among the BSAFs calculated for the congeners (F16,25 = 6.108, p < 0.001); Games-Howell test detected signiﬁcant diﬀerences (p < 0.05) between 2,3,7,8-TCDF and several congeners (Figure 6). Real total concentration (6PCDD/F) in polychaete tissues ranged from 33 pg g−1 dw (DE) up to 300 pg g−1 dw (SA) (Table 1). The gradient of real total concentration was as follows: DE < CE < OS < BR < SA. The concentrations of dioxins were quite homogeneous among the samples, ranging from 13 pg g−1 dw (DE) to 30 pg g−1 dw (BR). In samples CE and DE only 1,2,3,7,8-PCDD, 1,2,3,4,6,7,8-HpCDD and OCDD occurred at concentrations above the detection limits (0.25 pg g−1 dw); this latter was also the congener exhibiting the highest concentration in all the samples. As observed in the sediments, 2,3,7,8-TCDD was detectable only in sample SA. Site DE also showed the lowest concentration of furans (21 pg g−1 dw), whilst the highest were detected in the tissues of the polychaetes sampled in the industrial canals BR (111 pg g−1 dw of furans) and SA (281 pg g−1 dw of furans). The congeners occurring at higher concentrations were 2,3,7,8-TCDF and 1,2,3,4,6,7,8,9-OCDF. Concentrations normalized to lipid content ranged from 268 pg g−1 lipids (DE) to 2111 pg g−1 lipids (SA). DISCUSSION The lack of replication did not allow to verify whether among-site diﬀerences are signiﬁcant (especially for the less contaminated CE and DE samples) and may raise issues concerning the analytical variability of the data. However, speciﬁc contamination patterns emerged both in sediments and in polychaete tissues that are consistent with the underlying contamination gradient and other studies (Jimenez et al., 1998; MAG. ACQUE-Thetis, 2006, 2007; Picone, 2006), suggesting that analytical variability is of secondary relevance as compared with environmental variability of PCDD/F contamination in the Venice Lagoon. PCDD/F in the Sediments The equivalent toxicity in the tissues ranged from 3 pg g−1 dw (DE) to 26 pg g−1 dw (SA); the gradient (DE < CE < BR < OS < <SA) was slightly diﬀerent from the one for total concentrations, since OS showed higher equivalent toxicity with respect to BR (10 pg g−1 dw vs. 5 pg g−1 dw), despite lower total concentration (104 pg g−1 dw vs. 141 pg g−1 dw). Sediment total concentrations and toxicity equivalents corroborated expectations for the underlying contamination gradient. PCDD/F in the Sediments The lowest concentration of PCDD/F were measured at the reference site CE and at the estuarine site DE located far from the industrial district, whilst the highest were observed within July 2020 \| Volume 8 \| Article 235 Frontiers in Ecology and Evolution \| www.frontiersin.org 5 7 conge zed to o OS 11 51 51 84 11 ,058 2,227 193 400 197 681 323 ,463 334 ,317 373 ,240 4,493 5,520 0,013 30 1 3 63 4 17 3 40 493 520 013 0 mposite polychaetes samples and Biota-to-Sediment Accumulation Factors (BSAFs) calculated for the 17 Tissue real concentration (pg g−1 dw) Sediment concentration normalize carbon (pg g−1 C) BR CE DE OS SA BR CE DE OS <0.25 <0.25 <0.25 <0.25 1 <19 <27 <16 11 1 2 1 2 2 <19 <27 <16 51 1 1 1 1 1 <19 <27 <16 51 1 2 1 1 2 <19 <27 39 84 1 1 1 1 1 <19 <27 136 11 5 3 2 2 4 1,364 133 686 2,0 22 10 9 8 9 7,519 1,059 2,816 12,2 14 2 5 21 69 <19 <27 68 19 3 2 3 13 28 225 96 107 40 4 2 2 9 22 101 <27 36 19 4 3 2 12 50 473 96 159 68 2 2 1 5 17 151 59 78 32 1 2 1 1 3 1,163 <27 <16 2,4 2 2 1 4 10 54 <27 68 33 10 5 2 12 36 2,345 676 1,780 6,3 2 2 1 3 11 357 133 175 37 68 15 3 9 34 5,543 1,399 4,563 4,24 31 19 13 14 19 8,981 1,324 3,725 14,4 111 37 21 90 281 10,430 2,564 7,049 15,5 142 56 33 104 300 19,411 3,888 10,773 30,0 5 4 3 10 26 27 28 29 30 pg g−1 lipid) BSAF Spe SA BR CE DE OS SA R 8 >0.10 >0.09 >0.12 >0.20 0.27 n.c 14 >0.73 >1.41 >0.89 0.25 0.15 0.69 8 > 0.65 > 1.04 > 0.50 0.08 0.03 0.00 11 > 0.41 > 1.12 0.10 0.05 0.07 0.15 7 > 0.65 > 0.89 0.03 > 0.41 > 0.64 n.c 25 0.03 0.22 0.02 0.01 0.01 0.30 62 0.02 0.09 0.02 0.01 0.01 −0.5 487 > 11.13 > 1.64 0.63 0.94 1.35 0.82 197 0.11 0.24 0.19 0.29 0.13 0.99 155 0.29 > 0.67 0.43 0.40 0.46 0.99 354 0.07 0.27 0.10 0.16 0.10 0.90 117 0.10 0.32 0.10 0.14 0.08 0.90 19 0.01 > 1.41 > 0.50 0.00 0.09 0.00 72 0.32 > 1.79 0.15 0.11 0.08 0.60 256 0.03 0.07 0.01 0.02 0.01 0.90 80 0.05 0.16 0.05 0.07 0.02 0.90 239 0.10 0.11 0.01 0.02 0.00 0.30 134 1,977 2,111 36 ncentrations. nt (pg g− concen ed. Data t (pg g concen ed. Data PCDD/F in the Sediments Before analysis, data were transformed to per mil 2,3,7,8 chlorosubstituted homologs referred to the sum of all the isomers with concentration above detection limits. of a factory for the production of liquid gases (Frignani et al., 2001a); and (type 3) proﬁle containing mainly furans, with a prevailing component of OCDF, probably due to the production of metals, coke and related chemicals in the industrial district. in industrial channels and reported concentrations up to 36,590 pg g−1 dw for dioxins in the layer 35–38 cm depth and up to 19,241 pg g−1 dw for furans in the layer 8–10 cm depth (Frignani et al., 2001a). Later studies conducted by the Venice Water Authority investigated the top layer to a depth of 25 cm (MAG. ACQUE- Thetis, 2007) in the shallows of the inner lagoon facing the industrial district and reported concentrations in the range 13– 8,312 pg g−1 dw of 6PCDD/F and equivalent toxicity (WHO- TEQ) up to 112 pg g−1 dw. These data are in agreement with the concentrations observed in the present study, both for the industrial district (SA, BR) as well as the estuarine sample OS. At this latter sample, the measured concentrations (1,402 pg g−1 dw as 6PCDD/F) were also higher than those reported in the literature (depth 0–20 cm) for the inner lagoon shallows opposite to the estuary and in the proximity of the illegal landﬁll of Campalto (120 pg g−1 dw as 6PCDD/F) (Picone, 2006). These data suggest that the pollution of the area could be due mainly to the urban and industrial discharges upstream, rather than to leakages from the landﬁll. Nevertheless, in the present study only the sample collected from Canale Industriale Sud (SA), showed a proﬁle that could be categorized into one of the three types outlined by Bellucci et al. (2000) since the homologs provided a speciﬁc pattern of furans with a dominance of OCDF, attributable to industrial production of coke, metals and related manufactories (type 3), whilst contribution of dioxins in negligible. In the remaining four samples, the proﬁles did not allow for the identiﬁcation of a prevailing source, but indicate the co-occurrence of multiple sources of contamination. Samples CE and DE were characterized by a pattern of PCDD/F, consistent with atmospheric deposition from industrial sources. However, their proﬁles showed also a noteworthy contribution of OCDD, suggesting a not-negligible contribution of combustion processes unrelated to industrial pollution and boat traﬃc. PCDD/F in the Sediments In contrast, in samples OS and BR the dominance of OCDD over OCDF and HpCDF suggested a major contribution of deposition due to combustion combined with untreated domestic sewage over the deposition of furans from industrial sources. Fewer data are available for the sites DE and CE. In surface sediment (0–5 cm) collected in the estuarine site DE, Jimenez et al. (1998) measured a total 6PCDD/F concentration of 79 pg g−1 dw. The proﬁles of the homologs and the cluster analysis evidenced three distinct patterns of PCDD/F at the ﬁve study sites, that may be related to diﬀerent sources of contamination. In the sediment of the industrial area Bellucci et al. (2000) already identiﬁed three distinct type of homolog proﬁles that ﬁt with the main local sources: (type 1) proﬁle dominated almost entirely by OCDD, produced by combustion processes, untreated domestic sewage, urban wastes and boat engine exhausts; (type 2) proﬁle with the dominance of OCDF (90%) and OCDD (10%) that could be ascribed both to the stripping of vinyl chloride monomer (Stringer et al., 1995) and to the accumulation of atmospheric contaminants caused by both the runoﬀfrom the industrial district and the wastes PCDD/F in the Sediments For BSAFs calculation, data below detection limits were assigned a value equal to ha Data highlighted in bold indicate signiﬁcant correlations. n.c. = not calculated due to occurrence of too S n 0 0 0 n 0 − 0 0 0 0 0 0 0 0 0 Accumulation of PCDD/F in Ragworms Picone et al. one et al. Accumulation of PCDD/F in Ragwor FIGURE 2 \| Homolog proﬁles of PCDDs and PCDFs in sediments. Data are reported as per mil 2,3,7,8 chlorosubstituted homologs referred to the sum of all the somers with concentration above detection limits. FIGURE 2 \| Homolog proﬁles of PCDDs and PCDFs in sediments. Data are reported as per mil 2,3,7,8 chlorosubstituted homologs referred to the sum of all the isomers with concentration above detection limits. FIGURE 2 \| Homolog proﬁles of PCDDs and PCDFs in sediments. Data are reported as per mil 2,3,7,8 chlorosubstituted homologs referred to the sum of all the isomers with concentration above detection limits. the industrial area (SA) and at the estuarine site OS located in the proximity of the illegal landﬁll and also receiving urban and industrial discharges. both for the industrial district and the reference and estuarine samples; nevertheless, the comparison with literature data is often complicated by the diﬀerent depth of the sediment core analyzed in the various studies. For the industrial district, many data refer to the ﬁrst 2 cm of sediments, where concentrations in the range 67–13,642 pg g−1 for dioxins and 592–126,561 pg g−1 for furans were reported (Bellucci et al., 2000; Frignani et al., 2001a); other projects investigated the vertical proﬁle of PCDD/F A review of PCDD/F concentration in the sediments of the Venice Lagoon reported a mean value of 14,000 pg g−1 dw of 6PCDD/F for the industrial channels and 1,000 pg g−1 dw of 6PCDD/F for the inner lagoon (Guerzoni et al., 2007). These data are higher than those observed in the present study, July 2020 \| Volume 8 \| Article 235 Frontiers in Ecology and Evolution \| www.frontiersin.org 8 Picone et al. Accumulation of PCDD/F in Ragworms FIGURE 3 \| Cluster analysis on homolog proﬁle in sediments (left) and tissues (right). Before analysis, data were transformed to per mil 2,3,7,8 chlorosubstituted homologs referred to the sum of all the isomers with concentration above detection limits. FIGURE 3 \| Cluster analysis on homolog proﬁle in sediments (left) and tissues (right). Frontiers in Ecology and Evolution \| www.frontiersin.org PCDD/F in Tissues In general, very few data are available in the literature concerning the ﬁeld bioaccumulation of PCDD/F in polychaetes, although ragworms and lugworms are widely used as a bioindicator for biomarker and bioaccumulation studies (Ruus et al., 2005; Durou et al., 2007a,b) and are a key component of the coastal and estuarine food webs. Studies on H. diversicolor were performed by Nunes et al. (2011), that reported a concentration in tissues of 1.38 pg g−1 ww of 6PCDD/F (81.16 pg g−1 lipid) for the Mondego estuary, Portugal. These data were lower than the concentration normalized to lipids observed both in the industrial district and in the estuarine sites of the Venice Lagoon July 2020 \| Volume 8 \| Article 235 Frontiers in Ecology and Evolution \| www.frontiersin.org 9 cone et al. Accumulation of PCDD/F in Ragworm FIGURE 4 \| Homolog proﬁles of PCDDs and PCDFs in tissues of H. diversicolor. Data are reported as per mil 2,3,7,8 chlorosubstituted homologs referred to the sum of all the isomers with concentration above detection limits. Accumulation of PCDD/F in Ragworms Picone et al. FIGURE 4 \| Homolog proﬁles of PCDDs and PCDFs in tissues of H. diversicolor. Data are reported as per mil 2,3,7,8 chlorosubstituted homologs referred to the sum of all the isomers with concentration above detection limits. FIGURE 4 \| Homolog proﬁles of PCDDs and PCDFs in tissues of H. diversicolor. Data are reported as per mil 2,3,7,8 chlorosubstituted homologs referred to the sum of all the isomers with concentration above detection limits. lagoon: Sfriso et al. (2014) measured concentrations in the range of 40–110 pg g−1 dw for the inner lagoon, corresponding to an equivalent toxicity (WHO-TEQ) of 0.49–1.46 pg g−1 ww; Raccanelli et al. (2004, 2008) reported for the industrial area an equivalent toxicity of 2–9 pg g−1 ww; MAG. ACQUE-Thetis (2006) reported concentrations in the range 40–717 pg g−1 lipid and 9–58 pg g−1 lipid for industrial area and inner lagoon, respectively. In this latter study also the levels of PCDD/F in the muscle of the grass goby Zosterisessor ophiocephalus were in the present study (269–2,111 pg g−1 lipid), and may be related to the diﬀerent levels and kind of pollution sources aﬀecting the two estuaries. In the Venice Lagoon the bioaccumulation of PCDD/F from the sediments has been most often assessed by measuring tissue concentration in clams and ﬁshes, and never in polychaetes. Frontiers in Ecology and Evolution \| www.frontiersin.org PCDD/F in Tissues Various studies have focused on the concentrations of PCDD/F in the Manila clam (Ruditapes philippinarum) collected both in the industrial district and in the inner July 2020 \| Volume 8 \| Article 235 Frontiers in Ecology and Evolution \| www.frontiersin.org 10 Accumulation of PCDD/F in Ragworms Picone et al. FIGURE 5 \| Relationship between log KOW and log BSAF for the 17 congeners of 2,3,7,8 chlorosubstituted dioxins and furans. The solid line represents the linear regression log(BSAF) = 4.12–0.69 log(KOW) (r2 = 0.289), whilst broken lines indicate the 95% conﬁdence interval. FIGURE 5 \| Relationship between log KOW and log BSAF for the 17 congeners of 2,3,7,8 chlorosubstituted dioxins and furans. The solid line represents the linear regression log(BSAF) = 4.12–0.69 log(KOW) (r2 = 0.289), whilst broken lines indicate the 95% conﬁdence interval. H. diversicolor than in the sediments at the same stations. The diﬀerences were up to one order of magnitude for the hepta- and octa-chlorinated congeners, especially at sites DE, OS and SA, whilst the trend was less evident for the hexa-chlorinated congeners. measured, ranging between 14 and 435 pg g−1 lipid for the inner lagoon and 78−640 pg g−1 lipid for the industrial area (MAG. ACQUE-Thetis, 2006). Tissue concentrations in gobies are in agreement with the observed concentrations measured in polychaete tissues in the present study. Other authors also reported data for the thicklip gray mullet Chelon labrosus (1.6−2.6 pg g−1 ww of 6 PCDD/F) and the crab Carcinus aestuarii, both in the industrial district (36 pg g−1 ww of 6 PCDD/F) and in the inner lagoon (1.5–5 pg g−1 ww of 6 PCDD/F) (Jimenez et al., 1998; MAG. ACQUE, 1999). Homolog proﬁles and Spearman’s correlation showed a generally low association between congener concentrations in sediment and polychaete tissues, resulting in large variability among the corresponding BSAF values. On the other hand, the signiﬁcant linear relation observed between logBSAF and logKOW, despite having a low coeﬃcient of determination (r2 = 0.289), outlined polychaete’s tendency to take up preferentially the less chlorinated and less lipophilic congeners, characterized by lower logKOW, as also observed by Kono et al. (2010) in a laboratory study aiming to model the dioxin transfer from sediments to Perinereis nuntia. These authors also evidenced that bioaccumulation of PCDD/F in polychaetes follow similar characteristics to those observed in ﬁshes (Sijm et al., 1993). PCDD/F in Tissues Homolog proﬁles indicated that the polychaetes accumulated OCDD preferentially as concern dioxins, whilst two diﬀerent patterns are evident for furans: at sites CE and BR the dominant congener was the more chlorinated OCDF, whilst in samples SA, OS and also DE there was a relevant contribution of the less chlorinated congeners. In particular, there was a notable contribution provided by 2,3,7,8-TCDF, a typical marker of pollution due to chloralkali plants (Jimenez et al., 1998). The congeners 2,3,7,8-TCDD and 2,3,7,8-TCDF are characterized by high bioaccumulation and low elimination; thus their BSAF tends to be higher than those of the more chlorinated congeners (Sijm et al., 1993; Kono et al., 2010). In the case of H. diversicolor this is evident for 2,3,7,8-TCDF, whose accumulation factors were the highest in all the analyzed sample (except CE) despite the low sediment concentrations; Comparison Between Sediments and Tissues Sediment concentrations were higher than tissue concentrations for all the analyzed PCDD/F, apart from the less chlorinated furan 2,3,7,8-TCDF and 2,3,4,7,8- PeCDF, whose concentrations were higher in the tissues of July 2020 \| Volume 8 \| Article 235 Frontiers in Ecology and Evolution \| www.frontiersin.org 11 Accumulation of PCDD/F in Ragworms Picone et al. FIGURE 6 \| Biota-to-Sediment Accumulation Factors (BSAF) calculated for the 17 congeners of 2,3,7,8 chlorinated dioxins and furans. Boxes indicate mean ± standard error, whiskers mean ± standard deviation. Letter “a” identiﬁes congeners with mean BSAF statistically different from 2,3,7,8-TCDF (after Kruskal-Wallis ANOVA and multiple comparison test). FIGURE 6 \| Biota-to-Sediment Accumulation Factors (BSAF) calculated for the 17 congeners of 2,3,7,8 chlorinated dioxins and furans. Boxes indicate mean ± standard error, whiskers mean ± standard deviation. Letter “a” identiﬁes congeners with mean BSAF statistically different from 2,3,7,8-TCDF (after Kruskal-Wallis ANOVA and multiple comparison test). depuration phase was attributed to the elimination of the gut content, facilitated by a low assimilation eﬃciency for highly chlorinated compounds, whilst the slower phase of the depuration was attributed to elimination from other body compartments. The elimination process in polychaetes has not yet been elucidated, but a signiﬁcant correlation between the elimination constant (k2) and log KOW has been observed by Kono et al. (2010), supporting the hypothesis that more chlorinated and hydrophobic congeners do not bioaccumulate since they are quickly eliminated. In ﬁsh and oligochaetes the more rapid elimination of super-hydrophobic organics as OCDD compared to lower chlorinated PCBs, TCDD and TCDF – also observed in human blood (Flesch-Janys, 1996) – also suggests that OCDD in invertebrates is mainly associated with fast-clearing compartments as cell membranes or blood serum (Kono et al., 2010). for 2,3,7,8-TCDD this trend is not tangible, probably due to the sediment concentrations below detection limits that impeded any further analysis. Preferential accumulation of tetrachlorinated congeners has also been observed by Pruell et al. (2000) for the Nereididae Alitta virens following 70-d laboratory exposure to contaminated sediments: in this case, polychaetes accumulated only 2,3,7,8-TCDD and 2,3,7,8-TCDF, although the sediments were characterized by a range of PCDD/F and also high concentrations of highly chlorinated congeners. On the other hand, 1,2,3,4,6,7,8-HpCDD, HpCDFs, 1,2,3,4,6,7,8,9-OCDD and 1,2,3,4,6,7,8,9-OCDF are congeners characterized by low bioaccumulation, as applies to all the 1,4-substituted and/or 6,9-substituted PCDD/F (Sijm et al., 1993; Kono et al., 2010). Comparison Between Sediments and Tissues Also in this case, similarities in BSAFs may be due to comparable eﬃciency of the digestive ﬂuids in the solubilization of the contaminants, as shown by Mayer et al. (2001) for benzo[a]pyrene. area of Mestre and the illegal landﬁll of San Giuliano. Distinctly diﬀerent homolog proﬁles discriminated between sites inﬂuenced by heterogeneous pollution sources (BR, OS, DE and CE) and the industrial site (SA), where a speciﬁc pattern of furans with a dominance of OCDF attributable to industrial production of coke, metals and related manufactories was observed. In the ragworm tissues, the concentration pattern of the congeners is diﬀerent from those observed in the sediments, especially with regard to furans: in sample CE and BR the polychaetes accumulated mostly 1,2,3,4,6,7,8,9- OCDF whilst in samples SA, OS and DE the major contribution was due to the less substituted congeners and principally 2,3,7,8-TCDF, recognized as a marker of possible contamination deriving from chloralkali industrial plants. In general, H. diversicolor was a good indicator for assessing the transfer of PCDD/F from sediment to biota, but accumulated more eﬃciently the less chlorinated congeners with lower logKOW resulting in a negative correlation between BSAF and KOw, after logarithmic transformation. py Sediment Accumulation Factor data suggest that H. diversicolor may accumulate PCDD/F signiﬁcantly from the sediments and may serve as an indicator for the bioavailability of dioxins and furans from sediments to the polychaete community, especially as concerns the less chlorinated 2,3,7,8-TCDF, 2,3,4,7,8-PeCDF and 2,3,4,7,8-PeCDD. Nevertheless, the BSAFs for PCDD/F in polychaetes are generally low (BSAF < 1 or less) when compared to the BSAFs measured for other organochlorine contaminants (i.e., PCBs and organochlorine pesticides) in a number of species including A. virens, N. incisa, Glycera sp., Perinereis nuntia and also H. diversicolor (Lake et al., 1990; Brannon et al., 1993; Volpi Ghirardini et al., 2004; Kono et al., 2010; Nesto et al., 2010); this trend was also observed in ﬁshes where the apparent lower bioaccumulation of PCDD/F was attributed to lower solubility in the lipids, reduced membrane transport and also biotransformation mediated by cytochrome P450 monooxygenase isozymes (Opperhuizen and Sijm, 1990; Sijm et al., 1993; van der Oost et al., 2003). Biotransformation of PCDD/F in polychaetes has been rarely studied, and no data are available concerning the possible mechanisms involved. Comparison Between Sediments and Tissues Nevertheless, since cytochrome P450 isozymes are active also in polychaetes (Zheng et al., 2013), it cannot be excluded that the lower bioaccumulation of PCDD/F in ragworms could be due to the same causes already identiﬁed for ﬁshes. Further studies are needed, however, to elucidate these possible mechanisms of biotransformation and elimination. g Despite H. diversicolor’s ability to accumulate 2,3,7,8-TCDD, 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF, the BSAFs calculated for the PCDD/F are low as compared with those reported in the literature for other organochlorine compounds (i.e., PCBs and organochlorine pesticides) in a number of species including A. virens, N. incisa, Glycera sp., Perinereis nuntia and also H. diversicolor. Lower BSAFs for PCDD/F may be due to diﬀerent uptake kinetics, or the presence in polychaetes of a more eﬃcient elimination pathway for PCDD/F than for PCBs or pesticides, possibly involving also cyt-P450-mediated biotransformations; further research is needed to identify both biotransformation and elimination processes driving accumulation in polychaetes. Due to the paucity of information on this species, this research not only represents a baseline but also an invitation for further ecotoxicological studies on H. diversicolor. DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the article/supplementary material. AUTHOR CONTRIBUTIONS MP developed the manuscript concept, wrote the manuscript, and took care of the statistical analyses. ED contributed to the development of the study design, supported sampling, took care of data analysis, and commented on the manuscript. DT participated in the development of study design, supported sampling and handling, and commented on the manuscript. IG supported the analysis and commented on the manuscript. AV developed the study design, participated in the manuscript concept development, supported writing and analysis. All authors contributed to the article and approved the submitted version. Comparison Between Sediments and Tissues Both low bioavailability and elimination may contribute to the low accumulation (and also BSAF) of highly chlorinated PCDD/F. Lipophilicity is a major contributor for determining the bioavailability of sediment-bound PCDD/F, as conﬁrmed by the negative relationship of BSAF versus KOW in H. diversicolor (this study) and P. nuntia. However, contaminant molecular size and conformation (speciﬁcally planarity/nonplanarity), sediment characteristics and feeding habits may also play a signiﬁcant role, as observed in oligochaetes (Lyytikäinen et al., 2003). Elimination of highly chlorinated PCDD/F in caddisﬂy larvae and oligochaetes ﬁts a biphasic model (Loonen et al., 1997; Pastershank et al., 1999). A ﬁrst, rapid The BSAFs for PCDD/F observed in the present study with H. diversicolor (0.02–1.41 for dioxins; 0.01 – 11.13 for furans) are at least one order of magnitude higher than those measured in P. nuntia (0.00036–0.22 for dioxins and 0.0002–0.36 for furans) (Kono et al., 2010); moreover, the data are in agreement with the BSAFs reported for other polychaetes such as Nephtys sp. (McFarland et al., 1994) and A. virens (Pruell et al., 1993, 2000; Schrock et al., 1997). In all cases, the BSAF obtained with H. diversicolor for 2,3,7,8- TCDF is higher than those reported for other polychaete species, suggesting a speciﬁc ability of H. diversicolor to July 2020 \| Volume 8 \| Article 235 Frontiers in Ecology and Evolution \| www.frontiersin.org 12 Accumulation of PCDD/F in Ragworms Picone et al. accumulate this congener. Since bioavailability is a crucial factor driving bioaccumulation, and digestive ﬂuids are the primary factors driving solubilization of organic contaminants in polychaetes (Voparil and Mayer, 2000; Ahrens et al., 2001), diﬀerences in BSAFs for 2,3,7,8-TCDF among H. diversicolor and other polychaete species may be due to diﬀerences in gut digestive chemistry. Concentrations and properties of digestive ﬂuids vary broadly among species and it may aﬀect the assimilation eﬃciency for some substances (Ahrens et al., 2001; Mayer et al., 2001). 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CONCLUSION Total PCDD/F concentrations in sediments in Venice Lagoon showed a clear pollution gradient along the investigated sites, with higher levels of contamination in the samples collected in the industrial district (SA) and in the estuarine site receiving urban and industrial discharges from the Total PCDD/F concentrations in sediments in Venice Lagoon showed a clear pollution gradient along the investigated sites, with higher levels of contamination in the samples collected in the industrial district (SA) and in the estuarine site receiving urban and industrial discharges from the July 2020 \| Volume 8 \| Article 235 Frontiers in Ecology and Evolution \| www.frontiersin.org 13 Accumulation of PCDD/F in Ragworms Picone et al. FUNDING The authors are very grateful to Michele Cornello and Alessandra Arizzi Novelli for helping with sampling and handling of sediment and polychaetes. 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Pharmacol. 13, 57–149. doi: 10.1016/S1382-6689(02)00126-6 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. Volpi Ghirardini, A., Arizzi Novelli, A., and Tagliapietra, D. (2005). Sediment toxicity assessment in the Lagoon of Venice (Italy) using Paracentrotus lividus (Echinodermata: Echinoidea) fertilization and embryo bioassays. Environ. Int. 31, 1065–1077. doi: 10.1016/j.envint.2005.05.017 Copyright © 2020 Picone, Delaney, Tagliapietra, Guarneri and Volpi Ghirardini. Frontiers in Ecology and Evolution \| www.frontiersin.org REFERENCES 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. Copyright © 2020 Picone, Delaney, Tagliapietra, Guarneri and Volpi Ghirardini. 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. Volpi Ghirardini, A., Cavallini, L., Delaney, E., Tagliapietra, D., Ghetti, P. F., Bettiol, C., et al. (1999). H. diversicolor, N. succinea and P. cultrifera (Polychaeta: Nereididae) as bioaccumulators of cadmium and zinc from sediments: preliminary results in the Venetian lagoon (Italy). Toxicol. Environ. Chem. 71, 457–474. doi: 10.1080/02772249909358815 July 2020 \| Volume 8 \| Article 235 Frontiers in Ecology and Evolution \| www.frontiersin.org 16
https://openalex.org/W2956048706	https://res.mdpi.com/polymers/polymers-11-01133/article_deploy/polymers-11-01133.pdf?filename=&attachment=1	English	null	Poly (1-butene-ran-ethylene) Monomodal Copolymers from Metallocene Catalysts: Structural and Morphological Differences with Increasing Ethylene Content	Polymers	2,019	cc-by	11,549	Article Poly (1-butene-ran-ethylene) Monomodal Copolymers from Metallocene Catalysts: Structural and Morphological Diﬀerences with Increasing Ethylene Content Carla Marega 1,, Federica Malizia 2 and Stefano Spataro 2 1 Department of Chemical Sciences, via Marzolo 1, University of Padova, 35131 Padova, Italy 2 Basell Polioleﬁne Italia Srl, P.le G. Donegani 12, 44100 Ferrara, Italy Correspondence: carla.marega@unipd.it; Tel.: +39-049-8275233 Carla Marega 1,, Federica Malizia 2 and Stefano Spataro 2 1 Department of Chemical Sciences, via Marzolo 1, University of Padova, 35131 Padova, Italy 2 Basell Polioleﬁne Italia Srl, P.le G. Donegani 12, 44100 Ferrara, Italy Correspondence: carla.marega@unipd.it; Tel.: +39-049-8275233 Received: 4 June 2019; Accepted: 29 June 2019; Published: 3 July 2019 Received: 4 June 2019; Accepted: 29 June 2019; Published: 3 July 2019 Abstract: Samples of random poly(butene-ran-ethylene) copolymers produced with metallocene catalysts were studied in order to elucidate the diﬀerent behaviors of this particular class of materials as a function of increasing ethylene (C2) content. The samples cooled down from the melt are semi-crystalline or amorphous and crystallize in diﬀerent crystal modiﬁcations, depending on the amount of C2. Thermal analysis, X-ray diﬀraction, and microscopic techniques were used to follow the changes of the materials with aging time and to understand the structural and morphological behavior with the aim of highlighting possible peculiar properties, which may be of great interest in the application of such materials in the ﬁeld of Hot Melt adhesives. Keywords: 1-butene/ethylene copolymers; polymorphism; thermal analysis; X-ray diﬀraction; microscopy; hot melt adhesive Keywords: 1-butene/ethylene copolymers; polymorphism; thermal analysis; X-ray diﬀraction; microscopy; hot melt adhesive polymers polymers Polymers 2019, 11, 1133; doi:10.3390/polym11071133 www.mdpi.com/journal/polymers 1. Introduction It is generally considered a metastable form which, and in the presence of low heating rates, tends to crystallize in form II and in form I’ [14]. An increase of temperature promotes the transition III→II [13]. One of the ways to speed up the solid-to-solid transformation II→I is to copolymerize 1-butene with short-chain α-oleﬁns [18–20]. In particular, the presence of ethylene accelerates such transition until the direct crystallization from the melt of the stable form I for the most modiﬁed copolymers. It was observed that, as a function of the amount of comonomer and the experimental conditions (temperature, pressure, cooling rate) in which the molten polymer is cooled, the rate of transformation is not aﬀected by diﬀerent molecular weight, but the presence of ethylene comonomer enhances the rate of the transformation II→I. In particular, the most modiﬁed and defective the copolymer molecules are, the lower the time needed for the interconversion at Troom [20,21]. Using metallocene catalysts that allow to control the content and the type of chain defects (stereo and regio defects), it has been possible to clarify the relationship between the microstructure of the chain and the polymorphic behavior of both PB and 1-butene/ethylene copolymers [22–24]. On one hand, the presence of ethylene promotes the melt-crystallization of form I’, on the other hand, it accelerates the transformation of form II into form I. For low amounts of comonomer (around 1%), the molten polymer crystallizes in form II, which over time turns into Form I at room temperature. Copolymers with an intermediate ethylene content (around 2% wt) crystallize in both forms I’ and II and transform afterwards to form I. On the other hand, more modiﬁed copolymers (with C2 > 3% wt) do not crystallize from the melt, but they crystallize from amorphous state upon aging directly in form I and I’. In particular, for the most modiﬁed copolymers, a peculiar characteristic was found, that is the ability to be undergo "self-sealing" after being cut. This eﬀect has been attributed to the high mobility of the polymer chains due to the low melting temperature of form I’ and to the low degree of crystallinity of the copolymer [25]. The presence of ethylene, in addition to acting on the polymorphic behavior of the PB, involves some variations in the properties of the polymer such as the decrease of the crystallization rate, the degree of crystallinity and the melting temperature. 1. Introduction The ﬁrst synthesis of isotactic Poly(1-butene) (PB) was reported in 1954 by Natta and collaborators [1], starting from heterogeneous solid catalysts (Ziegler–Natta catalysts) already used for the production of isotactic polypropylene, and they obtained a linear semi-crystalline polymer with high stereoregularity from the polymerization of 1-butene. The polymerization of 1-butene can be also carried out using metallocene catalytic systems [2], and the use of such a catalyst ensures a better random and uniform distribution of ethylene with respect to that achieved with Ziegler–Natta catalysts. Therefore, the choice of the catalytic approach, in the synthesis of PB, is an important step in the design of the polymer. Poly(1-butene) exhibits a singular and very complex polymorphic behavior: according to the conditions under which the crystallization process is conducted, five crystalline forms can be obtained [3,4] which differ in terms of unit cell, helical conformation [5,6] (Table 1), thermodynamic stability and the consequent chemical, physical, and mechanical properties. Table 1. Crystallographic data [6] and references therein. Crystal Lattice Helix a (nm) b (nm) c (nm) I/I’ Hexagonal 3/1 1.77 1.77 0.65 II/II’ Tetragonal 11/3 1.46 1.46 2.12 III Orthorhombic 4/1 1.25 0.89 0.76 Table 1. Crystallographic data [6] and references therein. Polymers 2019, 11, 1133; doi:10.3390/polym11071133 Polymers 2019, 11, 1133 2 of 15 Form I is the most thermodynamically stable species at room temperature. Although form II is the most favored species from the kinetic point of view, it is a metastable form which is obtained by cooling the melted polymer and tends to transform spontaneously into form I. Form I’ can be obtained by crystallization from the melt under high pressure conditions [7], in the presence of suitable solvents [4] or by low temperature polymerization [8]. This form is not distinguishable from form I by wide angle X-ray diﬀraction, as they share the same crystalline cell, but they diﬀer by the melting temperature (Tm) (form I’ presents a lower Tm) [9] and they exhibit diﬀerent characteristic peak intensities in IR spectra [4]. Form II’ is obtained by crystallization from the melt at a pressure above 2000 atm [10]. Also, in this case, the diﬀraction pattern is the same as in form II, while Tm is lower. Form I’ and II’ are called ’imperfect’ forms, respectively, of form I and form II. Form III, orthorhombic, can be obtained by precipitation at room temperature, from a solution of the polymer in organic solvents [11–17]. 2. Materials and Methods The samples taken into consideration were obtained from pilot plants in LyondellBasell G. Natta Research Centre in Ferrara (Italy) by using Zr-based metallocene catalysis [2] and they consisted of 1-butene modiﬁed with diﬀerent ethylene (C2) content, as described in Table 2, which gathers the main characteristics of the obtained materials. The polymerization was carried out in liquid monomer of 1-butene at 70 ◦C in a single continuous reactor to produce random copolymers with a weight percentage of ethylene going between 0 and 20% (Scheme S1 in Supporting Information). The use of PB is growing in applications that require softness and tenacity. With the development of new catalysts, like the Zr-based metallocene, we can obtain polymers with lower molecular weight with respect to the standard Ziegler–Natta products and better copolymerize the comonomer content (ethylene in this case), so that real plastomers having no crystallinity could be successfully obtained. The materials have an increasing C2 content (0, 1, 2.4, 2.9, 3.8, 6.8% by weight, see Figure S1) and almost constant viscosity (MFI). This allows us to study the eﬀect of the diﬀerent compositions while ﬁxing the viscosity parameter. As expected, density and Tg are directly linked to C2 content randomized in the chain, while average molecular weight and MFI are ﬁxed. The low polydispersity index is a direct consequence of the catalyst used to polymerize these samples. Table 2. Samples characterization. PB0 PB1 PB2.4 PB2.9 PB3.8 PB6.8 C2 %wt 0 0.8 2.4 2.9 3.8 6.8 MFI g/10’ 40–60 40–60 40–60 40–60 40–60 40–60 Mw * 126151 126724 143923 131228 135570 141250 Mn * 61755 62100 66768 61506 59634 65949 Mw/Mn * 2.0 2.0 2.2 2.1 2.3 2.1 Density Kg/dm3 0.9066 0.9028 0.8978 0.8930 0.8903 0.8817 Tg ** ◦C −11 −12 −21 −22 −22 −27 * Data from Gel Permeation Chromatography (GPC) (Figures S2–S5); ** Data from Dynamic Mechanical Thermal Analysis (DMTA) Table 2. Samples characterization. 1. Introduction Its inﬂuence on the mechanical properties of the copolymer is also noteworthy: the increase in the comonomer content follows the decrease in the glass transition temperature, the hardness and the breaking load while increasing ﬂexibility and elasticity [23,25]. The focus of this paper is to characterize poly (1-butene-ran-ethylene) copolymers, with diﬀerent ethylene contents, which are the building blocks of a more complex system that is generated by using a setup of two-reactor in series. This material has applications as a Hot Melt Adhesive (HMA). Within this application, there is a complex system of end uses that are mainly driven by viscosity and crystallinity, where the polymer is the major part of a blend formed also by a tackiﬁer and a polymeric wax. Today the application is covered by amorphous poly(alpha-oleﬁn)s (APAOs), poly (ethylene-vinyl acetate) (EVA) and propylene and ethylene (C2)-based plastomers [26,27]. The role of a polyoleﬁn in a HMA formula is the core that 3 of 15 Polymers 2019, 11, 1133 gives strength and tackiness. It should have adhesive and cohesive properties, so a combination of two polymers is the ideal system. The analysis has involved several techniques that allowed a structural and morphological characterization of the diﬀerent samples, the ﬁrst was achieved by wide angle X-ray diﬀraction (WAXD) related to thermal analysis (DTA), while the second one was, instead, performed by small angle X-ray diﬀraction (SAXS) and transmission electron microscopy (TEM). 2.1. Thermal Analysis The calorimetric measurements were carried out by using roughly 6 mg of polymers enclosed into aluminum crucibles by means of TA Instrument Q2000 instrument operating under nitrogen atmosphere. The temperature scale and enthalpy were calibrated by using high purity indium as a standard. The main thermal protocol involved a ﬁrst heating of the samples at 10 ◦C/min from room temperature to 180 ◦C and an isothermal step at this temperature for 5 minutes (in order to erase completely their physical and mechanical history) followed by a cooling step at 10 ◦C/min down to −20 ◦C. The DSC crucible was then kept at room temperature and atmospheric pressure for diﬀerent times ready to be melted up to 180 ◦C at 10 ◦C/min after diﬀerent aging times. 2.2. Wide Angle X-ray Diﬀraction 2.2. Wide Angle X-ray Diﬀraction 2.3. Small Angle X-ray Scattering The SAXS measurements were performed in a MBraun system by using CuKα radiation from a Philips PW1830 X-ray generator (The Netherlands). The patterns were recorded by a position sensitive detector in the scattering angular range 0.1–5.0 ◦2θ and corrected for the blank scattering. A constant, continuous background scattering [29] was subtracted and the obtained intensity values eI (s) were smoothed, in the tail region, with the aid of the seI (s) versus 1/s2 plot [30]. Then Vonk’s desmearing procedure [31] was applied and the one-dimensional scattering function was obtained using the Lorentz correction: I1(s) = 4πs2I(s), where I1(s) is the one-dimensional scattering function and I(s) is the desmeared intensity function. The sum of the average thicknesses of the crystalline and amorphous layers was determined as the Bragg identity period L of the function I1(s). 2.2. Wide Angle X-ray Diﬀraction WAXD patterns were recorded in the diffraction angular range 5–35 ◦2θ by using a Bruker D8 Advance Powder Diffractometer (Germany), working in the reflection geometry, and CuKα radiation was used. Polymers 2019, 11, 1133 4 of 15 WAXD analysis was carried out on the ﬁlms of 1 mm thickness, prepared in DTA and conditioned at room temperature and atmospheric pressure for diﬀerent aging time. The application of the least-squares ﬁt procedure elaborated by Hindeleh and Johnson [28] gave the degree of crystallinity by weight (CWAXD), calculated for the samples at the end of the solid–solid transformation. 2.4. Transmission Electron Microscopy TEM images were obtained using a FEI Tecnai 10 (USA), operating at an acceleration voltage of 100 kV and with a resolution of 0.34 nm. Using a cryogenic microtome (Leica EM UC7, Germany), a thin layer (100 nm) was sectioned from the aged sample at −70 ◦C. This instrument operates at a temperature lower than the glass transition temperature of the sample, thus allowing the morphology of the sample to be maintained and improving the cutting conditions. SAXS Data Analysis The evaluation of the SAXS patterns according to some theoretical distribution models [32] was carried out referring to the Hosemann model [33], which assumes the presence of lamellar stacks having an inﬁnite side dimension. This assumption takes into account a monodimensional electron density change along the normal direction to the lamellae. The ﬁtting procedure [32] of the calculated one dimensional scattering function with the experimental one allows one to optimize the values of the thicknesses and distributions of the crystalline and amorphous layers, the long period and the crystallinity, along with their distribution, associated with lamellar stacks. 3.1. Structural Analysis: DTA and WAXD Measurements (a) Endo → 160 140 120 100 80 60 T (°C) t=264h t=0h t=5.5h t=14h t=20h t=42.5h (a) i A h d A Endo → 160 140 120 100 80 60 T (°C) t=264h t=0h t=5.5h t=14h t=20h t=42.5h (a) Figure 1. DTA thermograms (a) and WA Endo → 160 140 120 100 80 60 T (°C) t=264h t=0h t=5.5h t=14h t=20h t=42.5h (a) (b) Figure 1. DTA thermograms (a) and WAXD patterns (b) of PB0 at different aging times. Endo → 160 140 120 100 80 60 T (°C) t=264h t=0h t=5.5h t=14h t=20h t=42.5h Figure 1. DTA thermograms (a) and WAXD patterns (b) of PB0 at diﬀerent aging times. (a) (b) Figure 1. DTA thermograms (a) and WAXD patterns (b) of PB0 at different aging times. Endo → 160 140 120 100 80 60 T (°C) t=264h t=0h t=5.5h t=14h t=20h t=42.5h (a) (b) Figure 1. DTA thermograms (a) and WAXD patterns (b) of PB0 at different aging times. Endo → 160 140 120 100 80 60 T (°C) t=264h t=0h t=5.5h t=14h t=20h t=42.5h (b) (b) rent ag (a) (a) therm Figure 1. DTA thermograms (a) and WAXD patterns (b) of PB0 at different aging times. Figure 1. DTA thermograms (a) and WAXD patterns (b) of PB0 at diﬀerent aging times. Figure 1. DTA thermograms (a) and WAXD patterns (b) of PB0 at different aging times. g g ( ) p ( ) g g (b) (b) (b) (b) f PB1 diff i i (a) (b) Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at different aging times. (a) (b) Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at different aging times. Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at diﬀerent aging times. (a) (b) Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at different aging times. (a) (a) (a) Fi 2 DTA h ( ) d WA (a) (a) (a) (b) (b) (b) Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at different aging times. Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at different aging times. Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at diﬀerent aging times. Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at different aging times. 3.1. Structural Analysis: DTA and WAXD Measurements In this study, the results obtained from wide angle X-ray diﬀraction were compared to those from thermal analysis, as the crystallization procedure applied to the samples were the same for specimens subjected to WAXD as well as DTA analysis. Figures 1–6 show, for every considered material, the collection of DTA heating scans (a), and WAXD patterns (b) recorded after having aged the specimens for diﬀerent times at room temperature and atmospheric pressure. Looking at DTA thermograms, except for samples PB0 and PB1, where just two endothermic peaks are visible, the other samples exhibit quite complex behavior, showing multiple endothermic peaks, which appear and change in intensity as a function of the aging time of the samples. 5 of 15 Polymers 2019, 11, 1133 The ascription of such thermal peaks to the various polymorphic forms of PB was carried out by merging the information obtained from the analysis of the evolution of WAXD patterns of the corresponding sample, as well as from the literature data [23]. Polymers 2019, 11, x FOR PEER REVIEW 5 of 16 Polymers 2019, 11, x FOR PEER REVIEW 5 of 16 Polymers 2019, 11, x FOR PEER REVIEW 5 of 16 (a) (b) Figure 1. DTA thermograms (a) and WAXD patterns (b) of PB0 at different aging times. (a) (b) Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at different aging times. Endo → 160 140 120 100 80 60 T (°C) t=264h t=0h t=5.5h t=14h t=20h t=42.5h Figure 1. DTA thermograms (a) and WAXD patterns (b) of PB0 at diﬀerent aging times. (a) (b) Figure 1. DTA thermograms (a) and WAXD patterns (b) of PB0 at different aging times. (a) (b) Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at different aging times. Endo → 160 140 120 100 80 60 T (°C) t=264h t=0h t=5.5h t=14h t=20h t=42.5h Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at diﬀerent aging times. (a) (b) Figure 1. DTA thermograms (a) and WAXD patterns (b) of PB0 at different aging times. (a) (b) Figure 2. DTA thermograms (a) and WAXD patterns (b) of PB1 at different aging times. Endo → 160 140 120 100 80 60 T (°C) t=264h t=0h t=5.5h t=14h t=20h t=42.5h (b) (b) (b) f PB0 t diff t i ti (b) erns (b) of PB0 at different aging times. 3.1. Structural Analysis: DTA and WAXD Measurements DTA thermograms (a) and WAXD patterns (b) of PB3.8 at different aging times. Figure 5. DTA thermograms (a) and WAXD patterns (b) of PB3.8 at diﬀerent aging times. (a) (b) Figure 5. DTA thermograms (a) and WAXD patterns (b) of PB3.8 at different aging times. (a) (a) (a) (b) (b) (b) Figure 5. DTA thermograms (a) and WAXD patterns (b) of PB3.8 at different aging times. Figure 5. DTA thermograms (a) and WAXD patterns (b) of PB3.8 at different aging times. Figure 5. DTA thermograms (a) and WAXD patterns (b) of PB3.8 at diﬀerent aging times. Figure 5. DTA thermograms (a) and WAXD patterns (b) of PB3.8 at different aging times. s (b) o 3.8 at di e e t agi g ti es. (b) (b) (b) g g (a) (a) (a) (a) (b) Figure 6 DTA thermograms (a) and WAXD patterns (b) of PB6 8 at different aging times (a) (b) Figure 6. DTA thermograms (a) and WAXD patterns (b) of PB6.8 at different aging times. (a) (b) Figure 6. DTA thermograms (a) and WAXD patterns (b) of PB6.8 at different aging times. Figure 6. DTA thermograms (a) and WAXD patterns (b) of PB6.8 at diﬀerent aging times. ( ) (a) (a) (b) (b) (b) (a) (b) Fi 6 DTA th ( ) d WAXD tt (b) f PB6 8 t diff t i ti Figure 6. DTA thermograms (a) and WAXD patterns (b) of PB6.8 at different aging times. Figure 6. DTA thermograms (a) and WAXD patterns (b) of PB6.8 at different aging times. Figure 6. DTA thermograms (a) and WAXD patterns (b) of PB6.8 at diﬀerent aging times. Figure 6. DTA thermograms (a) and WAXD patterns (b) of PB6.8 at different aging times. The homopolymer sample and the one with the lowest amount of C2 (i.e., PB0 and PB1, Figures 1 and 2, respectively) show very similar evolution of crystalline phases during annealing, after their crystallization from the molten state. Both such samples crystallize from the melt mainly into form II hi h th t i f I th i ti i The homopolymer sample and the one with the lowest amount of C2 (i.e., PB0 and PB1, Figures 1 and 2, respectively) show very similar evolution of crystalline phases during annealing, after their crystallization from the molten state. 3.1. Structural Analysis: DTA and WAXD Measurements DTA thermograms (a) and WAXD patterns (b) of PB3.8 at diﬀerent aging times. (a) (b) Figure 4. DTA thermograms (a) and WAXD patterns (b) of PB2.9 at different aging times. (a) (b) Figure 5. DTA thermograms (a) and WAXD patterns (b) of PB3.8 at different aging times. (a) (b) Figure 6. DTA thermograms (a) and WAXD patterns (b) of PB6.8 at different aging times. he homopolymer sample and the one with the lowest amount of C2 (i.e., PB0 and PB1, Figu 2, respectively) show very similar evolution of crystalline phases during annealing, after t llization from the molten state. Both such samples crystallize from the melt mainly into fo i h th t i f I th i ti i Figure 6. DTA thermograms (a) and WAXD patterns (b) of PB6.8 at diﬀerent aging times. The homopolymer sample and the one with the lowest amount of C2 (i.e., PB0 and PB1, Figures 1 a ctively) show very similar evolution of crystalline phases during annealing, after their crystalliza he molten state. Both such samples crystallize from the melt mainly into form II, which then turn I th i ti i (a) (b) Figure 4. DTA thermograms (a) and WAXD patterns (b) of PB2.9 at different aging times. Figure 4. DTA thermograms (a) and WAXD patterns (b) of PB2.9 at diﬀerent aging times. (a) (b) Figure 4. DTA thermograms (a) and WAXD patterns (b) of PB2.9 at different aging times. (a) (b) Figure 4. DTA thermograms (a) and WAXD patterns (b) of PB2.9 at different aging times. (b) (b) (b) f PB2 9 t diff t i ti (b) s (b) of PB2.9 at different aging times. (a) (a) Fi 4 DTA th ( ) d WAX (a) Figure 4. DTA thermograms (a) and WAX (b) (b) fferent (a) (a) A ther Figure 4. DTA thermograms (a) and WAXD patterns (b) of PB2.9 at different aging times. Figure 4. DTA thermograms (a) and WAXD patterns (b) of PB2.9 at diﬀerent aging times. Figure 4. DTA thermograms (a) and WAXD patterns (b) of PB2.9 at different aging times. g g ( ) p ( ) g g (b) (b) (b) (b) f PB3 8 diff i i (a) (a) (a) Fi 5 DTA h ( ) d WA (a) (b) Figure 5. DTA thermograms (a) and WAXD patterns (b) of PB3.8 at different aging times. (a) (b) Figure 5. 3.1. Structural Analysis: DTA and WAXD Measurements ( ) g g (b) (b) (b) (a) (a) (a) (a) (b) Figure 3. DTA thermograms (a) and WAXD patterns (b) of PB2.4 at different aging times. (a) (b) Figure 3. DTA thermograms (a) and WAXD patterns (b) of PB2.4 at different aging times. (a) (b) Figure 3. DTA thermograms (a) and WAXD patterns (b) of PB2.4 at different aging times. Figure 3. DTA thermograms (a) and WAXD patterns (b) of PB2.4 at diﬀerent aging times. (b) (b) (b) (b) (b) (b) ( ) (a) (a) (b) (b) (b) (a) (b) Figure 3 DTA thermograms (a) and WAXD patterns (b) of PB2 4 at different aging times Figure 3. DTA thermograms (a) and WAXD patterns (b) of PB2.4 at different aging times. Figure 3. DTA thermograms (a) and WAXD patterns (b) of PB2.4 at different aging times. Figure 3. DTA thermograms (a) and WAXD patterns (b) of PB2.4 at diﬀerent aging times. 6 of 15 6 of 16 6 of 16 6 of 16 Polymers 2019, 11, 1133 Polymers 2019 11 FOR Polymers 2019, 11, x FOR Polymers 2019, 11, x FOR (a) (b) Figure 4. DTA thermograms (a) and WAXD patterns (b) of PB2.9 at different aging times. (a) (b) Figure 5. DTA thermograms (a) and WAXD patterns (b) of PB3.8 at different aging times. (a) (b) Figure 6. DTA thermograms (a) and WAXD patterns (b) of PB6.8 at different aging times. he homopolymer sample and the one with the lowest amount of C2 (i.e., PB0 and PB1, Figu 2, respectively) show very similar evolution of crystalline phases during annealing, after t lli ti f th lt t t B th h l t lli f th lt i l i t f Figure 4. DTA thermograms (a) and WAXD patterns (b) of PB2.9 at diﬀerent aging times. (a) (b) Figure 4. DTA thermograms (a) and WAXD patterns (b) of PB2.9 at different aging times. (a) (b) Figure 5. DTA thermograms (a) and WAXD patterns (b) of PB3.8 at different aging times. (a) (b) Figure 6. DTA thermograms (a) and WAXD patterns (b) of PB6.8 at different aging times. he homopolymer sample and the one with the lowest amount of C2 (i.e., PB0 and PB1, Figu 2, respectively) show very similar evolution of crystalline phases during annealing, after t llization from the molten state. Both such samples crystallize from the melt mainly into fo Figure 5. 3.1. Structural Analysis: DTA and WAXD Measurements Both such samples crystallize from the melt mainly into form II, which then turns in form I as the ageing time increases. The homopolymer sample and the one with the lowest amount of C2 (i.e., PB0 and PB1, Figures 1 and 2, respectively) show very similar evolution of crystalline phases during annealing, after their crystallization from the molten state. Both such samples crystallize from the melt mainly into form II, which then turns in form I as the ageing time increases. The homopolymer sample and the one with the lowest amount of C2 (i.e., PB0 and PB1, Figures 1 and 2, respectively) show very similar evolution of crystalline phases during annealing, after their crystallization from the molten state. Both such samples crystallize from the melt mainly into form II, which then turns in form I as the ageing time increases. Polymers 2019, 11, 1133 Polymers 2019, 11, 1133 7 of 15 On the other hand, all the other samples were completely amorphous after the controlled solidiﬁcation from the melt and their successive structural evolution depended on the amount of C2. In particular, sample PB2.4 shows the typical development of form I that starts after the decreasing of form II, while the samples with the higher amount of C2 (i.e., PB2.9, PB3.8 and PB6.8) undergo to a spontaneous solid-phase transformation directly in form I. However, it must be noted that, while the evolution of diﬀraction patterns is quite simple to understand, the thermal proﬁles exhibit multiple endothermic peaks whose nature is not straightforward and immediately associated with the various polymorphic phases of PB. InthecaseofsamplePB2.4, lookingatthecollectionofDTAandWAXDdata(Figure3), theendotherms present at temperatures below 60 ◦C can be attributed to form II without any reasonable doubt due to the fact that they disappear as the annealing time increases. The other endotherms present at higher temperatures would, instead, be associated with form I, as diffraction data clearly show. However, in order to understand if the presence of multiple endotherms is due to the phenomenon of melting and crystallization of metastable crystals, followed by the crystallization of a more perfect one, or the melting of population of crystals with different lamellar thickness, DTA melting runs, collected at different heating rates on completely aged samples, have been executed (Figure S1). 3.1. Structural Analysis: DTA and WAXD Measurements Since the heating rate has no influence on the relative temperature position of endothermic peaks as well as on the relative melting enthalpies, such endotherms cannot be attributed to the relaxation process of metastable crystals, but to the presence of crystals of form I with different lamellar thicknesses [34]. On the other hand, the small endothermic peak around 40 ◦C which appears after some annealing time is ascribed to form I’. In fact, this form is not distinguishable from form I in the diffraction pattern because these two forms present the same crystalline cell, but it is known [3,4] that they differ in the melting temperature. In particular, form I’ melts at a lower temperature with respect to form I, because it is characterized by a less perfect three-dimensional order [23]. Diﬀraction patterns of samples PB2.9, PB3.8 and PB6.8 show just the presence of diﬀraction peaks which are typical of crystallographic planes of form I. However, also for such samples, the thermal proﬁles are much more complicated, exhibiting several endothermic peaks. The assignation of these peaks has been carried out studying the thermal proﬁles of the completely aged samples obtained changing the heating rate. The endothermic peaks of samples PB2.9 and PB3.8 (Figure S2), tend to get closer as the heating rate increases, meaning that the phenomenon of molecular reorganization occurs during the thermal scanning. In contrast, the endothermic peaks of sample PB6.8 (Figure S3) do not change both their relative temperature positions and enthalpies. This implies that the two endotherms at about 40 ◦C and 50 ◦C are related to the presence of form I’ and form I, respectively. Table 3 summarizes the melting peak temperatures, assigned to the diﬀerent polymorphic forms, together with the associated enthalpies and the degree of crystallinity calculated both by DTA and WAXD analysis, for all the considered materials. Table 3. Melting temperatures (TmI’, TmI, TmII), melting enthalpy (∆HI), degree of crystallinity (CDSC, CWAXD) and half time of the II→I phase transformation (t 1/2) of the examined samples. Table 3. Melting temperatures (TmI’, TmI, TmII), melting enthalpy (∆HI), degree of crystallinity (CDSC, CWAXD) and half time of the II→I phase transformation (t 1/2) of the examined samples. Table 3. Melting temperatures (TmI’, TmI, TmII), melting enthalpy (∆HI), degree of crystallinity (CDSC, CWAXD) and half time of the II→I phase transformation (t 1/2) of the examined samples. 3.1. Structural Analysis: DTA and WAXD Measurements C2 in the copolymer speeds up the phase transition: the sample PB2.4 transforms into form I faster than the others [35]. It is also possible to observe that the ∆HI and the TmI are inﬂuenced by the percentage of C2 present in the sample, in particular, as the C2 content increases, both parameters decrease. The same thing also occurs for the melting temperature related to form II, in the samples in which it is detectable. As for the Tm of form I’, it is not aﬀected by the amount of C2, it remains in fact stable even at rather high C2 content. y g y y g amount of C2 in the copolymer speeds up the phase transition: the sample PB2.4 transforms into form I faster than the others [35]. It is also possible to observe that the ΔHI and the TmI are influenced by the percentage of C2 present in the sample, in particular, as the C2 content increases, both parameters decrease. The same thing also occurs for the melting temperature related to form II, in the samples in which it is detectable. As for the Tm of form I’, it is not affected by the amount of C2, it remains in fact stable even at rather high C2 content. The presence of ethylene produces an important modiﬁcation in the structure of the sample. In particular, the increase of ethylene amount causes a signiﬁcant decrease of the melting temperatures of form I and form II as well as of the degree of crystallinity; at the same time, it favors the kinetic of the spontaneous transformation of form II in form I. On the other hand, samples with C2 content equal or greater than 2.9%wt do not crystallize by cooling the melt to room temperature, but the amorphous samples crystallize directly in stable form I and I’ by aging at room temperature. The presence of ethylene produces an important modification in the structure of the sample. In particular, the increase of ethylene amount causes a significant decrease of the melting temperatures of form I and form II as well as of the degree of crystallinity; at the same time, it favors the kinetic of the spontaneous transformation of form II in form I. 3.1. Structural Analysis: DTA and WAXD Measurements Figure 7. WAXD patterns of sample PB2.4 (a) and PB2.9 (b) subjected to pressure. (Red arrows indicate the (101)III peak after completion of phase transformation). The presence of the crystalline form III is quite difficult to identify because its most intense peak, at about 2θ=12.2 °, corresponding to the family of planes (101) III [36], overlaps with that of form II (2θ = 11.9 °). Nevertheless, what has allowed its identification in the diffraction profiles was the strange behavior of this peak with the increasing of aging time; in fact, an increase in intensity was noticed as the aging time increased, until a constant intensity was reached. This behavior could not be attributed to form II, which exhibits a decrease in intensity with increasing aging time. Further confirmation of the presence of the form III is given by the existence of a peak at about 2θ = 14.2 ° [36], which is typical of this crystalline form. The presence of the crystalline form III is quite diﬃcult to identify because its most intense peak, at about 2θ = 12.2 ◦, corresponding to the family of planes (101) III [36], overlaps with that of form II (2θ = 11.9 ◦). Nevertheless, what has allowed its identiﬁcation in the diﬀraction proﬁles was the strange behavior of this peak with the increasing of aging time; in fact, an increase in intensity was noticed as the aging time increased, until a constant intensity was reached. This behavior could not be attributed to form II, which exhibits a decrease in intensity with increasing aging time. Further conﬁrmation of the presence of the form III is given by the existence of a peak at about 2θ = 14.2 ◦[36], which is typical of this crystalline form. yp y As can be seen from Figure 7a in this case, unlike what can be seen in the same sample not subjected to pressure (Figure 3b), the peak at 11.9 ° is already visible at t = 0, its intensity increases, at first, with increasing aging time and after about six hours it decreases and finally remains constant. This behavior is due to the overlap of the peaks of form II and III: at the beginning form II is the predominant one, as aging proceeds, it is converted into form I and, therefore, only form III contributes to the intensity of the peak. 3.1. Structural Analysis: DTA and WAXD Measurements On the other hand, samples with C2 content equal or greater than 2.9%wt do not crystallize by cooling the melt to room temperature, but the amorphous samples crystallize directly in stable form I and I’ by aging at room temperature. An unexpected behavior was noted for the samples PB2.4 and PB2.9, which under speciﬁc conditions showed the presence of form III. Although not strictly related to the purpose of the study reported in this paper, we found interesting the experimental observations concerning the presence of a form that has always been detected only starting from PB solution [11,12]. This occurred by applying a pressure of about 0.006 kg/cm2 on the samples immediately after the thermal treatment and prior to the WAXD analysis. Figure 7a,b show the diﬀraction proﬁles collected. An unexpected behavior was noted for the samples PB2.4 and PB2.9, which under specific conditions showed the presence of form III. Although not strictly related to the purpose of the study reported in this paper, we found interesting the experimental observations concerning the presence of a form that has always been detected only starting from PB solution [11,12]. This occurred by applying a pressure of about 0.006 kg/cm2 on the samples immediately after the thermal treatment and prior to the WAXD analysis. Figures 7a,b show the diffraction profiles collected. (b) Intensity (a.u.) 35 30 25 20 15 10 5 Diffraction angle (°2θ) PB2.9 t=168h PB2.9 t=1h PB2.9 t=6h PB2.9 t=8h PB2.9 t=13h PB2.9 t=24h PB2.9 t=72h (a) (b) Figure 7. WAXD patterns of sample PB2.4 (a) and PB2.9 (b) subjected to pressure. (Red arrows indicate the (101)III peak after completion of phase transformation). Intensity (a.u.) 35 30 25 20 15 10 5 Diffraction angle (°2θ) PB2.4 t=48h PB2.4 t=0h PB2.4 t=3h PB2.4 t=6h PB2.4 t=9h PB2.4 t=20h Intensity (a.u.) 35 30 25 20 15 10 5 Diffraction angle (°2θ) PB2.9 t=168h PB2.9 t=1h PB2.9 t=6h PB2.9 t=8h PB2.9 t=13h PB2.9 t=24h PB2.9 t=72h Figure 7. WAXD patterns of sample PB2.4 (a) and PB2.9 (b) subjected to pressure. (Red arrows indicate the (101)III peak after completion of phase transformation). (a) Intensity (a.u.) 35 30 25 20 15 10 5 Diffraction angle (°2θ) PB2.4 t=48h PB2.4 t=0h PB2.4 t=3h PB2.4 t=6h PB2.4 t=9h PB2.4 t=20h (b) (a) Figure 7. WAXD patterns of sample PB2.4 (a) and PB2.9 (b) subjected to pressure. (Red arrows indicate the (101)III peak after completion of phase transformation). 3.1. Structural Analysis: DTA and WAXD Measurements PB0 PB1 PB2.4 PB2.9 PB3.8 PB6.8 TmII ◦C 101.3 87.3 53.5 - - - TmI ◦C 117.3 104.6 87.1 73.9 64.1 48.9 TmI’ ◦C - - 37.2 36.1 38.8 38.3 ∆HI J/g 72.3 63.3 47.7 46.7 40.0 25.5 CDSC % 58 51 37 36 31 20 CWAXD % 52 45 33 31 23 17 t 1/2 h 27.4 11.3 8.0 - - - PB0 PB1 PB2.4 PB2.9 PB3.8 PB6.8 TmII ◦C 101.3 87.3 53.5 - - - TmI ◦C 117.3 104.6 87.1 73.9 64.1 48.9 TmI’ ◦C - - 37.2 36.1 38.8 38.3 ∆HI J/g 72.3 63.3 47.7 46.7 40.0 25.5 CDSC % 58 51 37 36 31 20 CWAXD % 52 45 33 31 23 17 t 1/2 h 27.4 11.3 8.0 - - - Both from the results obtained by WAXD and thermal analysis, it can be observed that the increase in the ethylene content causes a decrease in the degree of crystallinity. In addition, a higher amount of 8 of 15 at the Polymers 2019, 11, 1133 Both from the C2 in the copolymer speeds up the phase transition: the sample PB2.4 transforms into form I faster than the others [35]. It is also possible to observe that the ∆HI and the TmI are inﬂuenced by the percentage of C2 present in the sample, in particular, as the C2 content increases, both parameters decrease. The same thing also occurs for the melting temperature related to form II, in the samples in which it is detectable. As for the Tm of form I’, it is not aﬀected by the amount of C2, it remains in fact stable even at rather high C2 content. y g y y g amount of C2 in the copolymer speeds up the phase transition: the sample PB2.4 transforms into form I faster than the others [35]. It is also possible to observe that the ΔHI and the TmI are influenced by the percentage of C2 present in the sample, in particular, as the C2 content increases, both parameters decrease. The same thing also occurs for the melting temperature related to form II, in the samples in which it is detectable. As for the Tm of form I’, it is not affected by the amount of C2, it remains in fact stable even at rather high C2 content. 3.2. Morphological Analysis: SAXS and TEM 3.2. Morphological Analysis: SAXS and TEM The morphological investigation was carried out using two diﬀerent techniques: small angle X-ray scattering and transmission electron microscopy. Both techniques, by exploiting diﬀerent phenomena, provide information related to the morphology, and thus, it is possible to compare the data obtained via these methods. Using SAXS, the information related to the lamellar stacks is determined by elaborating the diﬀraction data using theoretical models. In contrast, the analysis of the TEM images can provide information regarding the morphology of a sample. The morphological investigation was carried out using two different techniques: small angle X-ray scattering and transmission electron microscopy. Both techniques, by exploiting different phenomena, provide information related to the morphology, and thus, it is possible to compare the data obtained via these methods. Using SAXS, the information related to the lamellar stacks is determined by elaborating the diffraction data using theoretical models. In contrast, the analysis of the TEM images can provide information regarding the morphology of a sample. SAXS patterns of all samples were collected at the end of the aging process. Figure 8a,b shows, for the samples PB1 and PB2.4, the experimental and calculated diﬀraction patterns, the latter of which were obtained by reproducing the experimental data by means of theoretical models. The choice of the model leads to the reconstruction of the experimental proﬁle through the optimization of the morphological parameters. g p g g p gy p SAXS patterns of all samples were collected at the end of the aging process. Figure 8 (a, b) shows, for the samples PB1 and PB2.4, the experimental and calculated diffraction patterns, the latter of which were obtained by reproducing the experimental data by means of theoretical models. The choice of the model leads to the reconstruction of the experimental profile through the optimization of the morphological parameters. (a) (a) (b) Figure 8. SAXS pattern (dotted line) and trace calculated by fitting procedure (solid line) of PB1 (a) and PB2.4 (b). Figure 8. SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB1 (a) and PB2.4 (b). (b) (b) (a) Figure 8. SAXS pattern (dotted line) and trace calculated by fitting procedure (solid line) of PB1 (a) and PB2.4 (b). Figure 8. SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB1 (a) and PB2.4 (b). Polymers 2019, 11, 1133 Polymers 2019 11 x FOR Polymers 2019, 11, 1133 Polymers 2019 11 x FOR 9 of 15 9 of 16 Therefore, from the diﬀraction patterns it is possible to see that, at the end of the solid-phase transformation, there is a coexistence of form I and form III. Therefore, from the diffraction patterns it is possible to see that, at the end of the solid-phase transformation, there is a coexistence of form I and form III. Sample PB2.9 (Figure 7b), due to the higher amount of C2, does not show the peak of form II. In this case, form I and form III are formed immediately from the amorphous and remain stable over time. Sample PB2.9 (Figures 7b), due to the higher amount of C2, does not show the peak of form II. In this case, form I and form III are formed immediately from the amorphous and remain stable over time. What has been found is that form III is not only obtainable from a PB solution in organic solvents [11,12], but also from the melt by pressure of the newly solidiﬁed sample. However, this occurs only for copolymer, as the examined ones, having intermediate amounts of C2, a not very high molecular weight, and a narrow molecular weight distribution (Table 2). These latter aspects could cause a greater mobility of the chains which, in combination with a favorable ethylene content, would lead to the formation of phase III, remaining stable over time at room temperature. What has been found is that form III is not only obtainable from a PB solution in organic solvents [11,12], but also from the melt by pressure of the newly solidified sample. However, this occurs only for copolymer, as the examined ones, having intermediate amounts of C2, a not very high molecular weight, and a narrow molecular weight distribution (Table 2). These latter aspects could cause a greater mobility of the chains which, in combination with a favorable ethylene content, would lead to the formation of phase III, remaining stable over time at room temperature. 3.1. Structural Analysis: DTA and WAXD Measurements As can be seen from Figure 7a in this case, unlike what can be seen in the same sample not subjected to pressure (Figure 3b), the peak at 11.9 ◦is already visible at t = 0, its intensity increases, at ﬁrst, with increasing aging time and after about six hours it decreases and ﬁnally remains constant. This behavior is due to the overlap of the peaks of form II and III: at the beginning form II is the predominant one, as aging proceeds, it is converted into form I and, therefore, only form III contributes to the intensity of the peak. 3.2. Morphological Analysis: SAXS and TEM 3.2. Morphological Analysis: SAXS and TEM The profiles calculated for PB0 and PB1 samples (Figure 8a) were obtained by applying a model considering stacks of infinite dimension, and a Gaussian function was used for the distribution of the amorphous and crystalline areas. In both cases the best results are obtained using only one lamellar population. The proﬁles calculated for PB0 and PB1 samples (Figure 8a) were obtained by applying a model considering stacks of inﬁnite dimension, and a Gaussian function was used for the distribution of the amorphous and crystalline areas. In both cases the best results are obtained using only one lamellar population. Regarding the calculated profiles of PB2.4 (Figure 8b), PB2.9, PB3.8, and PB6.8 samples, the same model has been applied, but it was necessary to use two different lamellar populations to obtain a good fitting. Regarding the calculated proﬁles of PB2.4 (Figure 8b), PB2.9, PB3.8, and PB6.8 samples, the same model has been applied, but it was necessary to use two diﬀerent lamellar populations to obtain a good ﬁtting. All the morphological data obtained by SAXS analysis are reported in Table 4. All the morphological data obtained by SAXS analysis are reported in Table 4. 10 of 15 Polymers 2019, 11, 1133 Table 4. Morphological parameters of the lamellar stacks obtained by SAXS analysis of the samples: long period (L), number of lamellae (N), thickness of the crystalline (Y), and amorphous layer (Z), along with their relative distributions, (σY/Y = σZ/Z, σL/L) and the degree of crystallinity by volume (ΦSAXS). Sample N Y1 Z1 L1 Y2 Z2 L2 ΦSAXS (nm) (nm) (nm) (nm) (nm) (nm) (%) PB0 ∞ 7.6 6.0 13.6 - - - 56 PB1 ∞ 5.3 7.3 12.6 - - - 42 PB2.4 ∞ 3.2 6.5 9.7 2.6 5.5 8.1 33 PB2.9 ∞ 2.8 7.1 9.9 2.2 5.5 7.7 28 PB3.8 ∞ 2.9 7.6 10.5 2.5 6.4 8.9 28 PB6.8 ∞ 3.3 10.7 14.0 2.7 8.7 11.4 24 σY1/Y1 σZ1/Z1 σL1/L1 σY2/Y2 σZ2/Z2 σL1/L2 0.50 0.50 0.35 - - - 0.45 0.45 0.33 - - - 0.35 0.35 0.26 0.36 0.36 0.27 0.34 0.34 0.27 0.40 0.40 0.30 0.34 0.34 0.27 0.33 0.33 0.26 0.40 0.40 0.32 0.41 0.41 0.41 TEM images were also acquired for the samples at the end of the aging process. Polymers 2019, 11, 1133 Polymers 2019, 11, 1133 The measurement of the long period was obtained from the Fourier transform (FFT) of the signal: after selecting an area within the acquired image, with a processing program associated with the instrument, the FFT provided information regarding the variation of the signal as a function of the frequency (see sample PB1, Figure 10). If there is some periodicity in the morphology of the material, two spikes were observed in the FFT of the signal (white dots in Figure 10), which denote the existence of areas in which the signal repeats with a higher frequency. The distance between the center and the spike provides the average long period measurement. Polymers 2019, 11, x FOR PEER REVIEW 12 of 16 Figure 10. TEM image of PB1 (a), FFT signal (b), lamella image (c). Figure 10. TEM image of PB1 (a), FFT signal (b), lamella image (c). Polymers 2019, 11, x FOR PEER REVIEW 12 of 16 Figure 10 TEM image of PB1 (a) FFT signal (b) lamella image (c) Polymers 2019, 11, x FOR PEER REVIEW Figure 10. TEM image of PB1 (a), FFT signal (b), lamella image (c). Figure 10. TEM image of PB1 (a), FFT signal (b), lamella image (c). Fi TEM f PB1 ( ) FFT l (b) l ll ( ) Together with TEM analysis, the optical microscopy observation in polarized light was carried out on PB1, which is the copolymer presenting the higher degree of order. This further study allows us to explain more clearly the correlation between spherulitic and lamellar morphology. Figure 11a shows the images obtained through POM (Polarized Optical Microscopy), at two different magnifications, where the spherulitic morphology of the PB1 sample is clearly visible. Observing Figure 11b, collected by TEM in the region of the spherulite indicated in the red box, it is possible to highlight the lamellar morphology inside the spherulite. Together with TEM analysis, the optical microscopy observation in polarized light was carried out on PB1, which is the copolymer presenting the higher degree of order. This further study allows us to explain more clearly the correlation between spherulitic and lamellar morphology. Figure 11a shows the images obtained through POM (Polarized Optical Microscopy), at two different magnifications, where the spherulitic morphology of the PB1 sample is clearly visible. 3.2. Morphological Analysis: SAXS and TEM 3.2. Morphological Analysis: SAXS and TEM The measurement of the lamellar thicknesses, in order to obtain an average value more representative of the whole material, was carried out in diﬀerent areas of the image, and on diﬀerent images for each sample. Figure 9 shows the TEM image obtained for the PB1 sample, at two diﬀerent magniﬁcations, in which the lamellar morphology is clearly visible. Polymers 2019, 11, x FOR PEER REVIEW 11 of 16 Figure 9. TEM image of PB1 at different magnification Figure 9. TEM image of PB1 at diﬀerent magniﬁcation. Fi 9 TEM i f PB1 diff ifi i Figure 9. TEM image of PB1 at diﬀerent magniﬁcation. 11 of 15 Polymers 2019, 11, 1133 Observing Figure 11b, collected by TEM in the region of the spherulite indicated in the red box, it is possible to highlight the lamellar morphology inside the spherulite. g g ( ) g ( ) g ( ) Together with TEM analysis, the optical microscopy observation in polarized light was carried out on PB1, which is the copolymer presenting the higher degree of order. This further study allows us to explain more clearly the correlation between spherulitic and lamellar morphology. Figure 11a shows the images obtained through POM (Polarized Optical Microscopy), at two different magnifications, where the spherulitic morphology of the PB1 sample is clearly visible. Observing Figure 11b, collected by TEM in the region of the spherulite indicated in the red box, it is possible to highlight the lamellar morphology inside the spherulite. (a) (b) Figure 11. POM image of PB1 (a) at different magnification, and TEM image of the selected area (b). Analyzing the data shown in Table 5, which collects the results obtained by SAXS and TEM, it is (a) (b) Figure 11. POM image of PB1 (a) at different magnification, and TEM image of the selected area (b). Analyzing the data shown in Table 5 which collects the results obtained by SAXS and TEM it is Figure 11. POM image of PB1 (a) at diﬀerent magniﬁcation, and TEM image of the selected area (b). (a) Figure 11 POM image of PB1 (a) at diff (a) (b) of the se (b) (a) age of P (a) g g ( ) g g ( ) Analyzing the data shown in Table 5 which collects the results obtained by SAXS and TEM it is Figure 11. POM image of PB1 (a) at different magnification, and TEM image of the selected area (b). Figure 11. POM image of PB1 (a) at diﬀerent magniﬁcation, and TEM image of the selected area (b). Polymers 2019, 11, 1133 Polymers 2019, 11, 1133 Polymers 2019, 11, 1133 12 of 15 Analyzing the data shown in Table 5, which collects the results obtained by SAXS and TEM, it is possible to draw interesting considerations. Analyzing the data shown in Table 5, which collects the results obtained by SAXS and TEM, it is possible to draw interesting considerations. Table 5. Comparison of morphological parameters obtained by SAXS and TEM analysis Table 5. Polymers 2019, 11, 1133 Comparison of morphological parameters obtained by SAXS and TEM analysis Sample * SAXS TEM Y L Y Ymin Ymax L (nm) (nm) (nm) (nm) (nm) (nm) PB0 7.6 13.6 9.1 7.4 12.0 13.2 PB1 5.3 12.6 5.6 4.7 6.6 12.4 PB2.9 2.8 2.2 9.9 7.7 4.3 3.4 5.7 10.1 PB3.8 2.9 2.5 10.5 8.9 3.5 2.5 3.9 9.0 PB6.8 3.3 2.7 14.0 11.4 4.3 3.1 5.3 13.5 * Sample PB2.4, which, after aging, has a behavior overlapping that of sample PB2.9, has not been analyzed by TEM. The parameters determined by SAXS, show a good agreement with those obtained by the microscopic analysis. Regarding PB0 and PB1 samples, the long period values obtained by the two techniques are very similar, also the lamellar thickness values obtained by SAXS are included in the interval determined by TEM analysis. y y More difficult is the comparison of the other samples, which are characterized by the presence of two lamellar populations in the SAXS patterns. This morphology, in fact, was not revealed by TEM images. Using microscopy, it is possible to visualize the lamellar stacks, but not to carry out a statistical analysis on large areas of the sample that could lead to the identification of two distinct families of lamellar stacks containing lamellae and amorphous layers with average thicknesses such as to determine different long periods. Instead, the determination of the dimensions of the crystalline lamellae as well as of the long period has to be done by calculating an average value on all the stacks present without the possibility of discriminating those belonging to one or the other family. This then leads to a value of long period that is the average on all the stacks present in the images. Meanwhile, by applying SAXS, which allows analysis of a macroscopic area of the material, it is possible to discriminate the two lamellar populations and the two different values of long period characteristic of each one. In Table 5, it can be seen that, for samples PB2.9, PB3.8, and PB6.8, the long period calculated by TEM, is similar to that determined by SAXS for one of the two populations and that the lamellar thickness is generally overestimated. Polymers 2019, 11, 1133 This is probably due to the fact that the elaboration of the signal of TEM images is limited to very small areas of the sample and the contrast between dark and light areas is not enough to allow highly accurate measurements of the lamellar thicknesses. Starting from the knowledge of the crystalline forms present in the samples, the two identiﬁed lamellar populations were attributed to forms I and I’; the samples presenting the double population are indeed those in which both phase I and phase I’ were observed. These crystalline forms, although presenting the same cell have, possess a diﬀerent three-dimensional organization: form I’ has a low degree of order [23]. This characteristic probably has a great inﬂuence on the order at a morphological level as well, and so, form I and I’ are distinguishable by SAXS but not by WAXD. The population presenting the values of crystalline thickness and of higher periodicity (and therefore more similar to the homopolymer) was attributed to the form I, the other as a consequence to the form I’. From the integration of the areas of the calculated patterns, corresponding to the two populations, it was also possible to determine which of the two was more present in the samples. In all three samples, the prevailing population is the one with the highest lamellar thickness and periodicity, and therefore it can be concluded that, in all these samples, form I prevails. 4. Conclusions The goal of this paper was the structural and morphological study of poly (1-butene-ran-ethylene) copolymers with diﬀerent amount of ethylene, a characteristic which, as it is well known, inﬂuences the crystallization behavior and, consequently, the ﬁnal properties of materials. Polymers 2019, 11, 1133 13 of 15 Our interest for such samples lies in the fact that they are the building blocks of biphasic materials currently produced by using two reactors in series for hot melt adhesive application. In fact, the two diﬀerent phases should deliver their individual properties to the whole ﬁnal material. Therefore, the knowledge of their characteristics and behaviors should help in designing materials with the desired combination of adhesive and cohesive strength. The structural study (WAXD and DTA) of the samples provided insight regarding the characteristic behaviors as a function of the ethylene content: for the homopolymer (PB0) and the sample with low C2 content (PB1) it was possible to observe the presence of the crystalline form II already in the polymer solidiﬁed from the melt and at short aging times and, with increasing time, the transformation into form I. The PB2.4 sample solidiﬁes in an amorphous form and, with time, turns into form II and I, the latter increases over time while form II decreases. It has been conﬁrmed that the increase of C2 accelerates the transformation II→I but decreases the crystallization rate. The samples with higher C2 content (PB2.9, PB3.8, and PB6.8) crystallize, starting from the amorphous solid, directly in form I. All samples, starting from PB2.4, present form I’, which can be distinguished from form I, due to the lower melting temperature. Thus, the C2 content clearly modiﬁes the crystallization process, a propensity to solidify in the amorphous phase is noted and the crystallization takes place after a certain time of aging. Moreover, if the C2 amount is suﬃciently high the material crystallizes directly in form I and I’. It was conﬁrmed that the increase of C2 accelerates the transformation, but decreases the crystallization rate. A peculiar behavior was detected for the samples PB2.4 and PB2.9: by applying a modest pressure on the newly solidiﬁed material, form III was produced. It is possible to hypothesize that for random copolymers having such a molecular weight and distribution, an amount of ethylene between 2.3 and 3.0% by weight can lead to the generation of the form III starting from the melt. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4360/11/7/1133/s1, Scheme S1: General polymerization process, Figure S1: FTIR spectra of samples PB1, PB2.4, PB2.9, PB3.8, PB6.8, Figure S2: GPC curve of sample PB0, Figure S3: GPC curve of sample PB1, Figure S4: GPC curves of samples PB2.4, PB2.9, PB3.8, Figure S5: GPC curve of sample PB6.8, Figure S6: DTA thermograms of PB2.4 at diﬀerent heating rate, Figure S7: DTA thermograms of PB2.9 at diﬀerent heating rate, Figure S8: DTA thermograms of PB3.8 at diﬀerent heating rate, Figure S9: DTA thermograms of PB6.8 at diﬀerent heating rate, Figure S10: SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB0 Figure, Figure S11: SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB2.4, Figure S12: SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB2.9, Figure S13: SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB3.8, Figure S14: SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB6.8, S15: TEM image of PB0 (a), TEM image of the selected area and FFT signal (b), Figure S16: TEM image of P2.90 (a), TEM image of the selected area and FFT signal (b), Figure S17: TEM image of P3.8 (a), TEM image of the selected area and FFT signal (b), Figure S18: TEM image of P6.8 (a), TEM image of the selected area and FFT signal (b). 4. Conclusions By morphological analysis (SAXS and TEM), it was possible to determine the parameters relating to the lamellar stacks in the different samples. This investigation showed that the increase in the C2 content causes a noticeable decrease in the size of the crystalline lamella starting from the sample containing 2.4% of C2, which also results in a decrease in the long period, but which increases in the most modified sample in ethylene (PB6.8) and therefore shows a periodicity very similar to that of the homopolymer. This is a consequence of the increase in the thickness of the amorphous areas, as is evident considering the degree of crystallinity ΦSAXS, which decreases as the percentage of comonomer increases. Of particular interest is the identiﬁcation of a lamellar population characteristic of the form I’ and distinguishable from that of the form I, which allows us to establish that, although the two forms share the same crystalline structure, they can be distinguished by the diﬀerent lamellar morphology as well as for the melting temperature. In particular, the presence of form I’ can be considered essential for industrial products, like HMA, whose applications beneﬁt from the presence of a phase with a low melting temperature. 4. Conclusions Acknowledgments: The authors would like to thank Roberta Marchini and Marco Casinelli (Basell Polioleﬁne Italia, Ferrara) for their assistance in samples preparation and microscopy measurements. 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. 4. Conclusions Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4360/11/7/1133/s1, Scheme S1: General polymerization process, Figure S1: FTIR spectra of samples PB1, PB2.4, PB2.9, PB3.8, PB6.8, Figure S2: GPC curve of sample PB0, Figure S3: GPC curve of sample PB1, Figure S4: GPC curves of samples PB2.4, PB2.9, PB3.8, Figure S5: GPC curve of sample PB6.8, Figure S6: DTA thermograms of PB2.4 at diﬀerent heating rate, Figure S7: DTA thermograms of PB2.9 at diﬀerent heating rate, Figure S8: DTA thermograms of PB3.8 at diﬀerent heating rate, Figure S9: DTA thermograms of PB6.8 at diﬀerent heating rate, Figure S10: SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB0 Figure, Figure S11: SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB2.4, Figure S12: SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB2.9, Figure S13: SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB3.8, Figure S14: SAXS pattern (dotted line) and trace calculated by ﬁtting procedure (solid line) of PB6.8, S15: TEM image of PB0 (a), TEM image of the selected area and FFT signal (b), Figure S16: TEM image of P2.90 (a), TEM image of the selected area and FFT signal (b), Figure S17: TEM image of P3.8 (a), TEM image of the selected area and FFT signal (b), Figure S18: TEM image of P6.8 (a), TEM image of the selected area and FFT signal (b). 14 of 15 Polymers 2019, 11, 1133 Author Contributions: Author Contributions: Sample preparation, S.S.; DTA, WAXD measurements, F.M.; SAXS measurements, C.M.; TEM and POM measurements, S.S.; data elaboration and conceptualization, C.M., F.M., S.S.; writing-original draft preparation, C.M.; writing-review and editing, C.M., F.M., S.S. 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https://openalex.org/W2278415057	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0142397&type=printable	English	null	Effects of Persistent Atrial Fibrillation-Induced Electrical Remodeling on Atrial Electro-Mechanics – Insights from a 3D Model of the Human Atria	PloS one	2,015	cc-by	12,684	Editor: Alexander V Panfilov, Gent University, BELGIUM Received: June 20, 2015 Accepted: October 21, 2015 Published: November 25, 2015 Copyright: © 2015 Adeniran 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. RESEARCH ARTICLE Ismail Adeniran1, David H. MacIver1,2, Clifford J. Garratt3, Jianqiao Ye4, Jules C. Hancox1,5, Henggui Zhang1* Ismail Adeniran1, David H. MacIver1,2, Clifford J. Garratt3, Jianqiao Ye4, Jules C. Hancox1,5, Henggui Zhang1* 1 Biological Physics Group, School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom, 2 Taunton & Somerset Hospital, Somerset, United Kingdom, 3 Manchester Heart Centre, Manchester Royal Infirmary, Manchester, United Kingdom, 4 Department of Engineering, Lancaster University, Lancaster, United Kingdom, 5 School of Physiology and Pharmacology, and Cardiovascular Research Laboratories, University of Bristol, Bristol, United Kingdom * henggui.zhang@manchester.ac.uk OPEN ACCESS Citation: Adeniran I, MacIver DH, Garratt CJ, Ye J, Hancox JC, Zhang H (2015) Effects of Persistent Atrial Fibrillation-Induced Electrical Remodeling on Atrial Electro-Mechanics – Insights from a 3D Model of the Human Atria. PLoS ONE 10(11): e0142397. doi:10.1371/journal.pone.0142397 * henggui.zhang@manchester.ac.uk Aims Atrial stunning, a loss of atrial mechanical contraction, can occur following a successful car- dioversion. It is hypothesized that persistent atrial fibrillation-induced electrical remodeling (AFER) on atrial electrophysiology may be responsible for such impaired atrial mechanics. This simulation study aimed to investigate the effects of AFER on atrial electro-mechanics. Editor: Alexander V Panfilov, Gent University, BELGIUM Introduction Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia [1,2] and has an increase in incidence and prevalence with each decade of adult life [3,4]. AF may be precipi- tated by a variety of cardiac or non-cardiac diseases which cause abnormalities in cardiac electrophysiology and in turn act as a substrate for the development of the arrhythmia [5]. Cur- rent treatments for atrial fibrillation to restore sinus rhythm include external and internal Direct Current (DC) cardioversion, chemical cardioversion (pharmacological intervention), and radiofrequency ablation [1–4]. Atrial stunning is the loss of mechanical atrial contraction following a successful cardiover- sion, which is maximal immediately after cardioversion and can take up to 6 weeks for normal atrial contraction to re-establish [6]. A long period of atrial stunning may cause an increased risk of thromboembolism. Atrial stunning occurs rarely following spontaneous cardioversion in paroxysmal arrhyth- mias. Previous studies have also shown that factors delaying return of normal atrial mechanical function include duration of atrial fibrillation, presence of structural heart disease, atrial pres- sures and atrial size [6–8]. However, the exact mechanisms causing impaired atrial mechanics, as occurs in atrial stunning are unknown. Some postulated mechanisms include tachycardia induced atrial cardiomyopathy, accumulation of cytosolic calcium and atrial hibernation [6–8]. It is unknown why atrial stunning occurs frequently for chronic AF but rarely for paroxys- mal AF patients [6–8]. The intrinsic electrophysiological properties of the atria are altered dur- ing chronic AF due to the atrial fibrillation induced electrical remodelling (AFER) [9–13]. Multiple clinical electrophysiological and experimental studies have shown that electrical remodelling is characterised by an abbreviated atrial action potential (AP) morphology, which is associated with underlying changes to the density and kinetics of some membrane ionic cur- rents and to cellular Ca2+ handling processes [10–12,14–16]. Chronic atrial fibrillation can also cause atrial structural remodelling, which is characterised by down-regulation and heteroge- neous expression of connexin proteins that form intercellular gap junctions (responsible for the AP conduction), as well as the presence of severe fibrosis, accumulation of fatty deposits and fibre disorganisation [9,10,13,17–19]. All of these factors may contribute to decreases in the AP conduction velocity and increases in conduction anisotropy and heterogeneity. We hypothesised that the impaired atrial mechanics as seen in atrial stunning after a suc- cessful cardioversion to chronic atrial fibrillation might be due to AFER during chronic fibrilla- tion, which impairs the mechanical contraction of the human atria. Methods and Results A 3D electromechanical model of the human atria was developed to investigate the effects of AFER on atrial electro-mechanics. Simulations were carried out in 3 conditions for 4 states: (i) the control condition, representing the normal tissue (state 1) and the tissue 2–3 months after cardioversion (state 2) when the atrial tissue recovers its electrophysiological properties after completion of reverse electrophysiological remodelling; (ii) AFER-SR condi- tion for AF-remodeled tissue with normal sinus rhythm (SR) (state 3); and (iii) AFER-AF condition for AF-remodeled tissue with re-entrant excitation waves (state 4). Our results indicate that at the cellular level, AFER (states 3 & 4) abbreviated action potentials and reduced the Ca2+ content in the sarcoplasmic reticulum, resulting in a reduced amplitude of the intracellular Ca2+ transient leading to decreased cell active force and cell shortening as compared to the control condition (states 1 & 2). Consequently at the whole organ level, atrial contraction in AFER-SR condition (state 3) was dramatically reduced. In the AFER-AF condition (state 4) atrial contraction was almost abolished. Copyright: © 2015 Adeniran et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the project grant from Engineering and Physical Science Research Council UK (EP/J00958X/1; EP/I029826/1 and EP/J009482/1). Competing Interests: The authors have also declared that no competing interests exist. 1 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics Conclusions This study provides novel insights into understanding atrial electro-mechanics illustrating that AFER impairs atrial contraction due to reduced intracellular Ca2+ transients. PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Regional Single Cell Models The atria are composed of several electrically distinct regions (Fig 1A). This regional electrical heterogeneity is thought to play a large role in the genesis and maintenance of atrial arrhyth- mias [24–26]. Data for human atrial electrophysiology are scarce, particularly for action poten- tial variations in the different regions within the atria. Most studies involving the investigation of the ionic mechanisms underlying regional action potential variations in the atria have been carried out on other mammals, mainly dog [27–30]. In this study, we modified the well established Courtemanche-Ramirez-Nattel (CRN) model [31] of the human atrial cell to couple electrophysiology to mechanics. As the CRN model provides detailed descriptions of cellular electrical APs in the right atrium (RA), the model was first modified to incorporate experimental data from the literature [27–29] on the heterogeneous ionic currents across the atria as implemented in previous modelling studies [32,33]. Relative changes in maximum ion current conductances for the different regions of the atria are listed in Table 1. Specifically, the conductances of the following ion channel currents were rescaled based on experimental data [27,29,34,35] to reproduce AP morphologies in various parts of the atria: L- type Ca2+ current (ICaL), transient outward K+ current (Ito), rapidly-activated potassium cur- rent (IKr), ultra rapid delayed rectifier current (IKur) and the fast Na+ current (INa). The scale factors of the conductances in the RA and left atrium (LA), Bachmann’s bundle (BB), crista ter- minalis (CT), pectinate muscles (PM), right and left atrial appendages (RAA and LAA respec- tively), tricuspid and mitral valve rings (TVR and MVR respectively) and atrial septum are summarised in Table 1. Then the CRN model was modified to incorporate the electrical-mechanical coupling. Due to the lack of detailed experimental data on which to base the development of biophysically detailed equations for the electrical-mechanical coupling of the human atrial myocytes, this was done by coupling the CRN electrophysiological model to the Rice et al. myocyte contrac- tion model [36], which describes the mechanics of a cardiac myocyte. This model was chosen as it is based on the cross-bridge cycling model of cardiac muscle contraction and is able to rep- licate a wide range of experimental data including steady-state force-sarcomere length (F-SL), force-calcium and sarcomere length-calcium relations [36]. The Rice et al. myocyte contraction model [36] was developed for guinea-pig myocytes. Electrical Remodeling Effects on Atrial Electro-Mechanics Computational models provide a powerful tool to study cardiac function [20–23]. Being constructed from and validated against experimental data, they provide a means for quantita- tively predicting the functional roles of altered molecular dynamics and ionic channels on car- diac functions in a systematic fashion that is difficult to achieve in an experimental setting. Therefore, in this study, we developed a novel 3D anatomical model of the human atria with coupled electrical and mechanical dynamics at cellular and tissue levels. Using the multi-scale models we investigated the functional impact of AFER on atrial electrical and mechanical activ- ities in order to elucidate the mechanism underlying atrial stunning. The principal contribu- tions of this study are: (i) the development of a new family of biophysically detailed, electrical- mechanically coupled models of the human atrium at cellular and 3D anatomical levels; (ii) investigation of the functional impacts of AFER on the electrical and mechanical dynamics of the atria; and (iii) elucidation of possible mechanisms underlying the impaired mechanical contraction as seen in atrial stunning in patients with persistent atrial fibrillation. PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Introduction However, due to the complexity of the atrial system, the functional impact of AFER on atrial electro-mechanical dynamics has not yet been investigated in such a way that the ionic mechanisms underlying atrial stunning can be elucidated. Though previous electrophysiological studies have identified some cellular and sub-cellular changes associated with chronic AF, such as abbreviated action potential duration (APD) and reduced amplitude of the intracellular Ca2+ transient [10–12,14– 16], changes at the whole organ level emerge from both cellular (i.e, single cell behaviours) and intercellular (i.e., cell-to-cell interactions) dynamic processes. 2 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Regional Single Cell Models In order to validate its use for human atrial myocytes, we followed our previous studies [20,37–39] to simulate the force-calcium relation- ship in the modified CRN model with incorporation of the Rice et al. electrical-mechanical 3 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Fig 1. 3D anatomical atrial geometry. (A) Segmented atria from two different views (left: top view and right: view into the atrial cavities). All regions are labelled. (B) Fibre orientations of the atria from two different views (left: top view and right: view into the atrial cavities). SAN: Sinoatrial Node, PV: Pulmonary Vein, CT: Crista Terminalis, IAS: Inter-atrial Septum, TVR: Tricuspid Valve Ring, MVR: Mitral Valve Ring, BB: Bachmann’s Bundle, PM: Pectinate Muscle, RS (Right Superior), RI (Right Inferior), LS (Left Superior), LI (Left Inferior). doi:10.1371/journal.pone.0142397.g001 Electrical Remodeling Effects on Atrial Electro-Mechanics Electrical Remodeling Effects on Atrial Electro-Mechanics Fig 1. 3D anatomical atrial geometry. (A) Segmented atria from two different views (left: top view and right: view into the atrial cavities). All regions are labelled. (B) Fibre orientations of the atria from two different views (left: top view and right: view into the atrial cavities). SAN: Sinoatrial Node, PV: Pulmonary Vein, CT: Crista Terminalis, IAS: Inter-atrial Septum, TVR: Tricuspid Valve Ring, MVR: Mitral Valve Ring, BB: Bachmann’s Bundle, PM: Pectinate Muscle, RS (Right Superior), RI (Right Inferior), LS (Left Superior), LI (Left Inferior). Fig 1. 3D anatomical atrial geometry. (A) Segmented atria from two different views (left: top view and right: view into the atrial cavities). All regions are labelled. (B) Fibre orientations of the atria from two different views (left: top view and right: view into the atrial cavities). SAN: Sinoatrial Node, PV: Pulmonary Vein, CT: Crista Terminalis, IAS: Inter-atrial Septum, TVR: Tricuspid Valve Ring, MVR: Mitral Valve Ring, BB: Bachmann’s Bundle, PM: Pectinate Muscle, RS (Right Superior), RI (Right Inferior), LS (Left Superior), LI (Left Inferior). Fig 1. 3D anatomical atrial geometry. (A) Segmented atria from two different views (left: top view and right: view into the atrial cavities). All regions are labelled. (B) Fibre orientations of the atria from two different views (left: top view and right: view into the atrial cavities). SAN: Sinoatrial Node, PV: Pulmonary Vein, CT: Crista Terminalis, IAS: Inter-atrial Septum, TVR: Tricuspid Valve Ring, MVR: Mitral Valve Ring, BB: Bachmann’s Bundle, PM: Pectinate Muscle, RS (Right Superior), RI (Right Inferior), LS (Left Superior), LI (Left Inferior). Fig 1. PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics Table 1. Scale factors ion channel conductivities for the different regions of the atria relative to the base model of [31]. Modified from [32]. Heterogeneity Source GNa Gto GCaL GKr GKur RA/PM Base model 1.00 1.00 1.00 1.00 1.00 CT upper endo [29] 1.00 1.00 1.67 1.00 1.00 CT upper epi [27] 1.00 0.50 1.67 1.00 1.00 CT lower endo [27] 1.00 0.68 1.67 1.00 1.00 CT lower epi [27] 1.00 0.34 1.67 1.00 1.00 BB (RA part) [27,29] 1.00 1.00 1.67 1.00 1.00 TVR [29] 1.00 1.00 0.67 1.53 1.00 MVR [29,34] 1.00 1.00 0.67 2.44 1.00 RAA [29] 1.00 0.68 1.06 1.00 1.00 LAA [29,34] 1.00 0.68 1.06 1.60 1.00 LA [34] 1.00 1.00 1.00 1.60 1.00 SEP [35] 1.50 1.00 0.25 1.00 0.67 RA: Right Atrium PM: Pectinate Muscle, CT: Crista Terminalis, endo: endocardium, epi: epicardium, BB: Bachmann’s Bundle, TVR:Tricuspid Valve Ring doi:10.1371/journal.pone.0142397.t001 nel conductivities for the different regions of the atria relative to the base model of [31]. Modified from [32]. tors ion channel conductivities for the different regions of the atria relative to the base model of [31]. Modifi Table 1. Scale factors ion channel conductivities for the different regions of the atria relative to the base mo Crista Terminalis, endo: endocardium, epi: epicardium, BB: Bachmann’s Bundle, TVR:Tricuspid Valve Ring coupling equations. The simulated data replicated the experimental data on the force-calcium relationship from human atrial myocytes [40]. As in our previous studies [20,37–39], the intracellular calcium concentration [Ca2+]i from the single cell electrophysiology models (EP) was used as the coupling link to the myofilament mechanics model (MM). [Ca2+]i produced as dynamic output from the EP models during the AP served as input to the MM model from which the amount of calcium bound to troponin is calculated. Regional Single Cell Models 3D anatomical atrial geometry. (A) Segmented atria from two different views (left: top view and right: view into the atrial cavities). All regions are labelled. (B) Fibre orientations of the atria from two different views (left: top view and right: view into the atrial cavities). SAN: Sinoatrial Node, PV: Pulmonary Vein, CT: Crista Terminalis, IAS: Inter-atrial Septum, TVR: Tricuspid Valve Ring, MVR: Mitral Valve Ring, BB: Bachmann’s Bundle, PM: Pectinate Muscle, RS (Right Superior), RI (Right Inferior), LS (Left Superior), LI (Left Inferior). doi:10.1371/journal.pone.0142397.g001 4 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 The resulting formulation of the myoplasmic Ca2+ concentration in the electrome- chanically coupled model is: dCai dt ¼ Cm ðICaL þ IbCa þ IpCa 2INaCaÞ 2VmyoF þ Vnsr ðIleak IupÞ þ 0:5IrelVjsr Vmyo dTroptotCa dt 1 1000 1 þ ½CMDNmaxKm;CMDN ð½Ca2þi þ Km;CMDNÞ 2 ð1Þ dCai dt ¼ Cm ðICaL þ IbCa þ IpCa 2INaCaÞ 2VmyoF þ Vnsr ðIleak IupÞ þ 0:5IrelVjsr Vmyo dTroptotCa dt 1 1000 1 þ ½CMDNmaxKm;CMDN ð½Ca2þi þ Km;CMDNÞ 2 ð1Þ ð1Þ where Cm is the membrane cell capacitance per unit surface area, Vnsr is the network sarcoplas- mic reticulum (SR) volume, Vjsr is the junctional SR volume, Vmyo is the cytoplasmic volume, Ileak is the SR Ca2+ leak current, Iup is the Ca2+ uptake current into the NSR, IbCa is the back- ground Ca2+ current, IpCa is the sarcoplasmic Ca2+ pump current, INaCa is the Na+/Ca2+ exchanger current, ICaL is the L-type inward Ca2+ current, Irel is the Ca2+ release current from the JSR, F is the Faraday constant, [CMDN]max is the total calmodulin concentration in the myoplasm, Km,CMDN is the Ca2+ half-saturation constant for calmodulin and dTroptoCa dt is the rate of Ca2+ binding to troponin. To model the effect of electrophysiological remodelling associated with AF, we followed the work of Colman et al. [41], which was based on an extensive review of the available experimen- tal data regarding AF remodelling of the main ion channels and sarcoplasmic reticulum pro- cesses. Table 2 summarises the modifications made to the control/healthy electromechanical single cell models to generate a family of AF-remodelled single cell models. 5 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics Table 2. Implementation of AF remodelling. Process Model ICaL -70% IKur -50% Ito -65% IK1 +100% IKs +100% IKAch No change INaCa +55% IKr No change SERCA +50% RyR +300% SR Ca2+ leak +25% Maximum channel conductances of the control model were changed based on an extensive review of available experimental data following the work of [41]. SERCA = Sarco/endoplasmic Ca2+-ATPase. doi:10.1371/journal.pone.0142397.t002 Table 2. Implementation of AF remodelling. Model Maximum channel conductances of the control model were changed based on an extensive review of available experimental data following the work of [41]. SERCA = Sarco/endoplasmic Ca2+-ATPase. doi:10.1371/journal.pone.0142397.t002 3D tissue model of electro-mechanical coupling 3D anatomical model: The 3D anatomical model was reconstructed and segmented based on anatomical features from the Visible Female dataset [42] (Fig 1A). As the intrinsic heterogene- ity of electrophysiological properties of atrial tissue plays an important role in ensuring the right timing sequences of depolarization and repolarization pattern in the atria, the 3D model considers different electrical properties for different regions of the atria: the sinoatrial node (SAN), left atrium (LA), right atrium (RA), crista terminalis (CT), pectinate muscles (PM), lim- bus fossa ovalis, Bachmann’s bundle (BB), right inferior isthmus, pulmonary veins (PV), right atrial appendage (RAA), left atrial appendage (LAA), inter-atrial septum (SEP), tricuspid valve ring (TVR) and mitral valve ring (MVR). Fibre orientation was incorporated using a novel semi-automatic rule-based approach and was validated against patient-specific volumetric models derived from CT, MRI and photographic data [43,44] (Fig 1B). 3D electrophysiological model: Electrical excitation wave propagation in the 3D human atria was modeled by a monodomain representation [20,45–47] of cardiac tissue with a modifi- cation (i.e., the incorporation of the Right Cauchy Green deformation tensor, C) to take into account the effect of the deforming atrial tissue, similar to previous studies [20,37,48–50]. Cm dV dt ¼ ðIion þ IstimÞ þ r ðDC1rVÞ ð2Þ ð2Þ where Cm is the cell capacitance per unit surface area, V is the membrane potential, Iion is the sum of all transmembrane ionic currents from the electromechanics single cell model, Istim is an externally applied stimulus and D is the diffusion tensor. The tissue conductivities that make up the diffusion tensor, D, were chosen so that the activation time and conduction veloci- ties of the atria matched those of well established atrial models [26,32,51]. The conductivity ratio (parallel to ﬁbre direction:transverse direction) was set to 3:1. The longitudinal (along the ﬁbres) and transverse conductivities were 1.26 mm2 ms-1 and 0.42 mm2 ms-1 respectively reproducing the heterogeneous conduction velocities of well established atrial models [32,51] and human experimental data [52]. 3D electro-mechanical coupling model: To develop a 3D electromechanical model of the human atria, the family of electromechanically coupled single cell atrial models developed in this study was incorporated into a 3D atrial geometry. 3D electro-mechanical coupling model: To develop a 3D electromechanical model of the human atria, the family of electromechanically coupled single cell atrial models developed in this study was incorporated into a 3D atrial geometry. 3D tissue model of electro-mechanical coupling 6 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics We modelled cardiac tissue mechanics within the theoretical framework of nonlinear elas- ticity [53,54] as an inhomogeneous, anisotropic, nearly incompressible material similar to pre- vious studies [49,55–60]. The coupling between cardiac mechanics and electrical excitation propagation is addressed in a framework in which the electrical action potential dictates the active strain of the muscle [58–60]. We adopted an active strain approach in this study as opposed to the active stress approach [48,49,61]. Unlike the active stress approach, the active strain approach requires no tuning to provide the observed deformation when fibre contrac- tion is included in the equations. In addition, frame invariance and rank-one ellipticity are inherited from the corresponding properties of the standard strain energy of the material [58,60,62] whereas rank-one ellipticity cannot be ensured when large deformations occur for a specific active stress form [60,63]. We used a two-field variational principle with the deformation u and the hydrostatic pres- sure p as the two fields [54,64,65]. p is utilized as the Lagrange multiplier to enforce the near incompressibility constraint. Thus, the total potential energy function P for the mechanics problem is formulated as: Pðu; pÞ ¼ Pintðu; pÞ þ PextðuÞ ð3Þ ð3Þ where Pint(u,p) is the internal potential energy or total strain energy of the body and Pint(u) is the external potential energy or potential energy of the external loading of the body. The deformation gradient F is a tensor that maps elements from the undeformed configura- tion to the deformed configuration [53,54]. Following [58,66], we multiplicatively decompose F into a microscopic (active) component and a macroscopic elastic (passive) component: F ¼ FeFo ð4Þ ð4Þ The active component Fo measures the length change of the tissue due to muscle contraction while the passive component Fe accounts for the passive mechanical response of the tissue and possible tension due to external loads. PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 where: ð11Þ Q ¼ C2E2 11 þ C3ðE2 22 þ E2 33 þ E2 23Þ þ 2C4ðE12E21 þ E13E31Þ ð11Þ following previous modelling studies [20,68]. C1 = 0.831 kPa, C2 = 14.31, C3 = 4.49 and C4 = 10. Eij are the components of the Green-Lagrange strain tensor. following previous modelling studies [20,68]. C1 = 0.831 kPa, C2 = 14.31, C3 = 4.49 and C4 = 10 E th t f th G L t i t We applied a constant pressure boundary condition of 1.07 kPa on the endocardial surface of the atria (Γendo) and to prevent rigid body rotations, we set the displacement u = 0 on the subset of the epicardial surface (Γepi) in the left atria, which is normally in close proximity to the thoracic spine and between the RIPV and the RSPV (Fig 1). Solving the 3D electromechanics model: The electromechanics problem consists of two sub- problems: the electrophysiology problem (Eq 2) and the mechanics problem (Eq 3). The electrophysiology problem (Eq 2) was solved with a Strang splitting method [69] ensuring that the solution is second-order accurate. It was discretized in time using the Crank-Nicholson method [70], which is also second-order accurate and discretized in space with Finite Elements [70–73]. Iion in (Eq 2) represents the single cell electromechanics model from which the active strain (Eq 6) input to the 3D mechanics model for contraction was obtained. This was done via an L2 projection from the finite element space of the electrophysiology mesh to the finite ele- ment space of the mechanics mesh. The system of ordinary differential equations (ODE) com- posing Iion was solved with a combination of the Rush-Larsen scheme [74] and the CVODE solver [75,76]. The mechanics problem (Eq 3) was also solved using the Finite element Method using the automated scientific computing library, FEniCS [77]. The resulting non-linear system of equa- tions was solved iteratively using the Newton-Raphson method to determine the equilibrium configuration of the system. FEniCS [77], which is a problem-solving environment for auto- mated solution of finite element computations allows a close relation between the mathemati- cal notation and the source code, and automated differentiation of both the constitutive law and the residual equation. It provides functionality that can be used to linearize the nonlinear residual equation (linear form) automatically for use with the Newton-Raphson method. 3D tissue model of electro-mechanical coupling With the vector field f denoting the unique direction of the fibres in the undeformed state of the atria, the microscopic active component of the deformation tensor F takes the form: Fo ¼ I þ gðSLÞf f ð5Þ ð5Þ where I is the identity tensor, SL is the sarcomere length of the electromechanical single cell, γ is a scalar ﬁeld that represents the intensity of the contraction, i.e., the active strain: where I is the identity tensor, SL is the sarcomere length of the electromechanical single cell, γ is a scalar ﬁeld that represents the intensity of the contraction, i.e., the active strain: g ¼ SL SL0 SL0 ð6Þ ð6Þ where SL0 is the resting sarcomere length. Thus, γ>0 denotes elongation, and γ<0 denotes contraction. The elastic component Fe is formulated as: The elastic component Fe is formulated as: Fe ¼ FF1 o ð7Þ ð7Þ and the corresponding Right Cauchy-Green strain tensor is: Ce ¼ FT e Fe ð8Þ ð8Þ \| DOI:10.1371/journal.pone.0142397 November 25, 2015 7 / 24 7 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics The associated Green-Lagrange strain tensor is: Ee ¼ 1 2 ðCe IÞ ð9Þ ð9Þ To characterise the constitutive behaviour of cardiac tissue, for the strain energy function W, we used the Guccione constitutive law [67] given by: W ¼ WðFeÞ ¼ C1eQ ð10Þ ð10Þ where: Fea- tures of the Unified Form Language (UFL) [77,78] used in the FEniCS [77] project permit problems to be posed as energy minimization problems, making it unnecessary to compute an expression for a stress tensor, or its linearization, explicitly. The value of the Right Cauchy Green Tensor C was then used to update the diffusion coefficient tensor in Eq 1. Over a typical finite element domain, P2 elements [71–73] were used to discretize the displacement variable u, while the pressure variable p was discretized with P1 elements [71–73]. This P2–P1 mixed finite element has been proven to ensure stability [77,79,80] and an optimal convergence rate [73,79,81]. The algorithm for solving the full electro-mechanics problem is as follows: 1. Determine the initial deformation and obtain the value of the Right Cauchy Green Tensor C. 2. While time < end time of simulation: 8 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics a. Solve the electrophysiology problem for Δtmechanics = 1 ms with C as input and active strain γ as output (Δtelectrophysiology = 0.01 ms). a. Solve the electrophysiology problem for Δtmechanics = 1 ms with C as input and active strain γ as output (Δtelectrophysiology = 0.01 ms). a. Solve the electrophysiology problem for Δtmechanics = 1 ms with C as input and active strain γ as output (Δtelectrophysiology = 0.01 ms). b. Project γ from the electrophysiology mesh onto the mechanics mesh. c. Solve the mechanics problem with γ as input and C as output. Meshes and Computation: The atrial electrophysiology mesh consisted of 2736128 tetrahe- dra and 566234 vertices while the atrial mechanics mesh consisted of 42752 tetrahedra and 14349 vertices. The two meshes were also checked to ensure good mesh quality metrics. The single cell ODEs were parallelised using OpenMP while the Conjugate Gradient method with the Symmetric successive overrelaxation preconditioner (Electrophysiology) and the parallel sparse direct solver MUMPS (Mechanics) were used for Linear Algebra via PETSc [82,83]. The simulations were carried out on an Intel Xeon CPU E5-2687W @ 3.10 GHz with 32 hyper- threaded cores and 64 GB of memory. It took about 48 hours to simulate a period of 1000 ms electro-mechanical activities of the model. where: Methods of stimulus, and measurement of atrial volume: For the single cell simulations, APs were elicited with an S1-S2 protocol consisting of 1000 S1 stimuli followed by a single S2 stimulus at a frequency of 1 Hz. For the 3D simulations, in order to guard against any drift in the steady state values of the ion concentrations in the model, the electromechanical single cell models described in section 2.1 were pre-paced for a 1000 beats before being incorporated into the tissue model. Atrial volume was computed as a surface integral using the Ostrogradsky formula [84]: VINNERðφÞ ¼ ð φðOINNERÞ dv ¼ 1 3 ð @φðGENDOÞ x nds ð12Þ ð12Þ where φ is the deformation, OINNER is the volume enclosed by the atrial endocardium ΓENDO and n is the outward unit normal vector. Modelling different states of the atria In order to investigate the functional impact of AFER on atrial electromechanical dynamics, we implemented 3 simulation conditions mimicking 4 states of atrial tissue. These include: (i) the control condition, which mimics the healthy and normal atrial tissue (state 1), as well as the tissue about 6 weeks after successful cardioversion (state 2). This assumption is reasonable as the atria normally restore their mechanical contraction about 6 weeks after successful car- dioversion with atrial stunning [6–8,85]. This time period is sufficient for atrial tissue to go through reverse electrical remodeling after cardiac arrhythmias as shown in some experimental animal studies [86]; (ii) AFER-SR condition that simulates the AF-remodeled atrial tissue with normal sinus rhythm (SR) (state 3). This condition mimics the state of atrial tissue just after a successful cardioversion such that the tissue electrophysiology is remodelled; and (iii) AFER-AF condition simulating the AF-remodeled tissue with multiple reentrant excitation waves (state 4). This condition mimics the atrial tissue during chronic atrial fibrillation, in which both tissue electrophysiological remodeling and re-entrant wavelets are present. Single Cell Electromechanical Simulations Fig 2 shows the simulated electromechanical activities of the right atrial baseline model under control (states 1 & 2) and AFER conditions (states 3 & 4) for AP (Fig 2A), ½Ca 2þ i (Fig 2B), SL (Fig 2C) and normalised active force (Fig 2D). In simulations, AFER abbreviated the atrial 9 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics Fig 2. Electromechanical properties of the baseline, isolated RA single cell under control (dark) and AFER (dashed) conditions. (A) Action potential. (B) Cytosolic Ca2+ concentration. (Inset) Comparison of our simulation results (grey bars) with experimental data (symbols) on APD90 [10–12,14,87,88] and cytosolic Ca2+ reduction [88,90,91]. (C) Sarcomere length shortening and (D) Active force normalised to maximum of Control. doi:10 1371/journal pone 0142397 g002 Fig 2. Electromechanical properties of the baseline, isolated RA single cell under control (dark) and AFER (dashed) conditions. (A) Action potential. (B) Cytosolic Ca2+ concentration. (Inset) Comparison of our simulation results (grey bars) with experimental data (symbols) on APD90 [10–12,14,87,88] and cytosolic Ca2+ reduction [88,90,91]. (C) Sarcomere length shortening and (D) Active force normalised to maximum of Control. doi:10.1371/journal.pone.0142397.g002 action potential duration. The measured APD90 (action potential duration at 90% repolarisa- tion) values were 274 ms and 188 ms under control and AFER conditions respectively. This represented about 31% APD abbreviation by AFER, which was within the range of observed APD abbreviation in atrial cells of AF patients as compared to those of patients in sinus rhythm action potential duration. The measured APD90 (action potential duration at 90% repolarisa- tion) values were 274 ms and 188 ms under control and AFER conditions respectively. This represented about 31% APD abbreviation by AFER, which was within the range of observed APD abbreviation in atrial cells of AF patients as compared to those of patients in sinus rhythm 10 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics [10–12,14,24,87,88]. In simulations, AFER reduced the systolic ½Ca 2þ i level by 25% and the dia- stolic ½Ca 2þ i level by ~50% (Fig 2B), which was also within the range of observed reduction in systolic ½Ca 2þ i transient amplitude in atrial cells of AF patients as compared to those of patients with normal sinus rhythm [14,88–91]. Single Cell Electromechanical Simulations Fig 2B inset shows a comparison of the simulation APD90 and systolic ½Ca 2þ i level reduction (grey bars) with relevant experimental data (sym- bols) from literature [10–12,14,87,88]. The reduced ½Ca 2þ i transient amplitude led to a decreased sarcomere length shortening (Fig 2C) and consequently, to an 82% decrease in active force (Fig 2D). This reduction in active force is close to clinical data showing that in AF patients the average force of atrial contraction was reduced by about 75% [92]. The match between simulation data and clinical data at the cellular level for the reduced active force also provides validation for the use of the single cell model for the electro-mechanical coupling. The mechanisms for the reduced amplitude and diastolic level of ½Ca 2þ i are attributable to the effects of AFER on the Ca2+ signalling processes as shown in Fig 3. The ﬁgure plotted the time courses under control (states 1 & 2) and AFER conditions (states 3 & 4) for AP (Fig 3A), ½Ca 2þ i (Fig 3B), sarcoplasmic reticulum Ca2+ content (CaSR; Fig 3C), the L-type Ca2+ current density, (ICaL; Fig 3D), the Na+/Ca2+ exchanger current density (INaCa; Fig 3E), the ﬂux of Ca2+ uptake into the sarcoplasmic reticulum (Jup; Fig 3F) and the ﬂux of Ca2+ release from the sarco- plasmic reticulum (Jrel; Fig 3G). It was shown that AFER reduced the CaSR content (by ~50%) (Fig 3C), which was accompanied by a reduced ICaL (Fig 3D) that led to a reduced trigger for Ca2+ release from the sarcoplasmic reticulum, an increased Na+-Ca2+ exchange current, and a functionally reduced sarcoplasmic reticulum Ca2+ uptake and release though both maximal activities were increased by the AFER (see Table 1). All of these changes were collectively responsible for the AFER-induced reduction in the systolic and diastolic levels of ½Ca 2þ i . AFER also altered the electromechanical activities of other types of atrial cells as shown in Fig 4, which were created from the right atrial baseline model (Fig 2). In the figure, the electro- mechanical activities of different atrial cell types in the control condition (Fig 4A and 4D) are compared with those in the AFER (Fig 4E and 4H) conditions. Single Cell Electromechanical Simulations The APs from the single cell family under both the control (Fig 4A) and the AFER conditions (Fig 4A) exhibited heteroge- neous morphologies with significant differences in the diastolic and systolic ½Ca 2þ i levels (Fig 4B and 4F), SL shortening (Fig 4C and 4G), active force (Fig 4D and 4H) and APD90, the latter ranging from 181 ms in the PV to 325 ms in the BBRA (Table 3). For all of the cell types, AFER abbreviated APD, reduced the diastolic and systolic level of [Ca2+]i, leading to reduced cell length shortening and the production of the active force. In simulations, AFER induced marked and heterogeneous reduction in the active force among variant cell types, ranging from a 57% reduction in the BBRA to 97% in the PV. There was negligible contraction in the SEP. Table 3 summarises the characteristics of the electromechanical activities for different cell models and the changes between the control and AFER conditions. Note that the range of sim- ulated reduction in active force (57–97%) among variant types of atrial cells is also close to the clinical data of reduced force of atrial contraction observed in AF patients [92]. 3D Electro-mechanical simulations in the 3D model Atrial contraction is an integral action of a large population of myocytes that are electrically and mechanically coupled. Due to the complex geometry, heterogeneity and anisotropic prop- erties of atrial tissue, it cannot be assumed that behaviour at the single cell level necessarily translates into similar activity at the organ level. In particular, reduction in cell shortening as observed in Fig 4 cannot automatically be interpreted as leading to reduction of the contraction volume of the atria, which is normally measured at the clinical setting: cells with differing 11 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics Fig 3. AFER effects on the Ca2+ signalling processes of the baseline, isolated RA single c control (dark) and AFER (dashed) conditions. (A) Action Potential. (B) Cytosolic Ca2+ concen SR Ca2+ content. (D) L-type Ca2+ current (ICaL) density. (E) Na+/Ca2+ exchanger current density Flux of Ca2+ uptake into the SR (Jup) and (G) Flux of Ca2+ release from the SR (Jrel) Inset: Magn Jrel in the first 20 ms. doi:10.1371/journal.pone.0142397.g003 Fig 3. AFER effects on the Ca2+ signalling processes of the baseline, isolated RA single cell under control (dark) and AFER (dashed) conditions. (A) Action Potential. (B) Cytosolic Ca2+ concentration. (C) SR Ca2+ content. (D) L-type Ca2+ current (ICaL) density. (E) Na+/Ca2+ exchanger current density (INaCa). (F) Flux of Ca2+ uptake into the SR (Jup) and (G) Flux of Ca2+ release from the SR (Jrel) Inset: Magnified view of Jrel in the first 20 ms. doi:10 1371/journal pone 0142397 g003 Fig 3. AFER effects on the Ca2+ signalling processes of the baseline, isolated RA single cell under control (dark) and AFER (dashed) conditions. (A) Action Potential. (B) Cytosolic Ca2+ concentration. (C) SR Ca2+ content. (D) L-type Ca2+ current (ICaL) density. (E) Na+/Ca2+ exchanger current density (INaCa). (F) Flux of Ca2+ uptake into the SR (Jup) and (G) Flux of Ca2+ release from the SR (Jrel) Inset: Magnified view of Jrel in the first 20 ms. doi:10.1371/journal.pone.0142397.g003 doi:10.1371/journal.pone.0142397.g003 12 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics electrophysiological and electromechanical properties are coupled, which will therefore influ- ence timing of electrical propagation and therefore mechanical activity. In addition, passive stress arising from tissue contraction may also influence atrial contraction at the whole organ Fig 4. Electrical Remodeling Effects on Atrial Electro-Mechanics Table 3. Electromechanical properties of the family of single cell atrial models. Cell Type APD90 (Control) (ms) APD90 (AF) (ms) ½Ca 2þ i % Change (Control to AF) Active Force % Change (Control to AF) RA/PM 274 188 25%# 82%# LA 234 167 26%# 86%# CT/BBLA 322 213 34%# 57%# BBRA 325 215 33%# 56%# TVR 250 149 29%# 93%# MVR 218 130 28%# 92%# RAA 272 189 26%# 78%# LAA 229 167 26%# 82%# PV 181 145 43%# 97%# SEP 189 125 26%# 0%# RA: Right Atrium, LA: Left Atrium, PM: Pectinate Muscle, CT: Crista Terminalis, BB: Bachmann’s Bundle, TVR: Tricuspid Valve Ring, MVR: Mitral Valve Ring, RAA: Right Atrial Appendage, LAA: Left Atrial Appendage, PV: Pulmonary Vein, SEP: Septum d i 10 1371/j l 0142397 t003 Table 3. Electromechanical properties of the family of single cell atrial models. Active Force % Change (Control to AF) RA: Right Atrium, LA: Left Atrium, PM: Pectinate Muscle, CT: Crista Terminalis, BB: Bachmann’s Bundle, TVR: Tricuspid Valve Ring, MVR: Mitral Valve Ring, RAA: Right Atrial Appendage, LAA: Left Atrial Appendage, PV: Pulmonary Vein, SEP: Septum RA: Right Atrium, LA: Left Atrium, PM: Pectinate Muscle, CT: Crista Terminalis, BB: Bachmann’s Bundle, TV Ring, RAA: Right Atrial Appendage, LAA: Left Atrial Appendage, PV: Pulmonary Vein, SEP: Septum organ level. Therefore, we performed further simulations using realistic human 3D anatomical atrial geometry with consideration of the intrinsic electrical heterogeneity in the atria. At the whole organ level, AFER impaired atrial mechanical contraction as shown in Fig 5, in which the sequence of electrical wave initiation and propagation, and the resultant contraction of the atria are shown for control (states 1 & 2; S1 Video), AFER tissue in the normal sinus rhythm (i.e, 1Hz) (AFER-SR) (middle panels for state 3; S2 Video) and AFER tissue with reen- trant excitation wave condition (AFER-AF) (right panels for state 4; S3 Video). In all the cases, the deforming mesh was superimposed on an undeformed mesh (grey) in order to show the atrial contraction. At 0 ms, excitation was initiated from the SAN for both the control and AFER-SR conditions. At 100 ms, under the control condition, the whole atria had almost completely activated while in AFER, due to the slower electrical wave conduction, large parts of the right and left atria (blue-coloured) were yet to be activated. AFER caused a delayed atrial activation. 3D Electro-mechanical simulations in the 3D model Electromechanical properties of the isolated regional atrial single cell types under control (A-D) and AFER (E-H) conditions. (A,E) Action potential. (B,F) Cytosolic Ca2+ concentration. (C,G) Sarcomere length shortening and (D,H) Active force normalised to maximum of Control (Note the different scales between control and AFER). doi:10.1371/journal.pone.0142397.g004 Fig 4. Electromechanical properties of the isolated regional atrial single cell types under control (A-D) and AFER (E-H) conditions. (A,E) Action potential. (B,F) Cytosolic Ca2+ concentration. (C,G) Sarcomere length shortening and (D,H) Active force normalised to maximum of Control (Note the different scales between control and AFER). doi:10.1371/journal.pone.0142397.g004 doi:10.1371/journal.pone.0142397.g004 electrophysiological and electromechanical properties are coupled, which will therefore influ- ence timing of electrical propagation and therefore mechanical activity. In addition, passive stress arising from tissue contraction may also influence atrial contraction at the whole organ level. Thus, it is necessary to investigate the functional consequences of AFER at the 3D atrial electrophysiological and electromechanical properties are coupled, which will therefore influ- ence timing of electrical propagation and therefore mechanical activity. In addition, passive stress arising from tissue contraction may also influence atrial contraction at the whole organ level. Thus, it is necessary to investigate the functional consequences of AFER at the 3D atrial PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 13 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 At 200 ms, there was significant atrial contraction under the control condition but compara- tively negligible contraction under AFER-SR. There was a delayed onset of atrial contraction in the AFER condition. At 300 ms, relaxation was underway under the control condition, but in the AFER-SR con- dition, relaxation was almost complete, suggesting a shortened contraction time course under AFER condition due to an abbreviated APD and the significant reduction in SR loading (Fig 3C) leading to an abbreviated time course of the intracellular Ca2+ transient. Fig 5 also shows the time course of the atrial volume change during atrial excitation and contraction under both control and AFER-SR conditions. Volume change (i.e., atrial contrac- tion) was reduced by 76.3% in the AFER-SR condition as compared to the control condition. Such a reduction in the atrial contraction volume was attributable to the reduction in active force at the cellular level (~80%) in all of the atrial cell types in the AFER condition (Table 3 and Fig 4). Note that at the 3D whole atria level, the simulation result of 76.3% reduction in atrial contraction corresponds with clinical data of 75% reduction of atrial contraction in AF patients [92], which provides further validation of the cellular and 3D organ models used for the electro-mechanical coupling of the human atria. Further simulations were carried out to investigate the functional impact of AFER on atrial contraction while there were rapid reentrant excitation waves in the atria (AFER-AF; Fig 5, 14 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics Fig 5. Snapshots of atrial electromechanical contraction superimposed on an atrial mesh (grey) under control an cardioversion (Left; states 1 & 2), AFER-SR (Middle; AF-remodelled tissue under sinus rhythm; state 3) and AFE with multiple re-entrant excitation waves; state 4) conditions. 0 ms: Before electrical activation. 100 ms: Almost com condition compared to slower activation in AFER-SR (middle) and AFER-AF (right) conditions. 200 ms: Control (left) con AFER-SR (middle) and AFER-AF (right) conditions with negligible contraction. 300 ms: Control (left) and AFER-SR (mid repolarisation and re-entrant wavelets in the AFER-AF (right) condition. 700 ms: Complete repolarisation and tissue rela Fig 5. Discussion In this study, we have developed a new biophysically detailed, 3D anatomical model of the elec- tromechanical activity of the human atria. This incorporates accurate anatomical structures (Fig 1A) including fibre orientations (Fig 1B), which were validated against patient-specific vol- umetric models derived from CT, MRI and photographic data [43,44]. The 3D geometry itself is based on the Visible Female dataset [42]. The 3D human atrial electromechanical model also incorporates the intrinsic electrophysiological heterogeneity of the human atria through the integration of a family of heterogeneous, cellular models for the electrical APs of human atrial myocytes (Fig 3A and 3D). These cellular models were also modified to reflect ionic channel changes during chronic AF-remodelling (Fig 3E and 3H). Electrical Remodeling Effects on Atrial Electro-Mechanics (middle) conditions but multiple sustaining re-entrant wavelets in the AFER-AF (right) condition. Bottom: Time course of computed atrial volume during electrical excitation for control (blue), AFER-SR (green) and AFER-AF (red) conditions. (middle) conditions but multiple sustaining re-entrant wavelets in the AFER-AF (right) condition. Bottom: Time course of computed atrial volume during electrical excitation for control (blue), AFER-SR (green) and AFER-AF (red) conditions. (middle) conditions but multiple sustaining re-entrant wavelets in the AFER-AF (right) condition. Bottom: Time course of computed atrial volume during electrical excitation for control (blue), AFER-SR (green) and AFER-AF (red) conditions. doi:10.1371/journal.pone.0142397.g005 doi:10.1371/journal.pone.0142397.g005 right panels). In this case, reentrant excitation waves were initiated by a premature stimulus applied to a localized region (IAS) at a 250 ms delay from the initiation of sinoatrial node exci- tation. Before the premature stimulus, the activation pattern and the mechanical contraction time course were the same as those in the AFER-SR condition. After the premature stimulus, sustained reentrant excitation wavelets developed, which were uncoordinated across the atria, leading to diminished mechanical contraction of the atria as illustrated by an almost flattened time course of the atrial volume. Fig 6 shows the time course of AP (Fig 6A), [Ca2+]i (Fig 6B), SL (Fig 6C) and active force (Fig 6D) recorded from a localized site of the atria under control, AFER-SR and AFER-AF con- ditions. It was shown that as compared to the control condition, AFER impaired atrial mechan- ical activity by reducing the diastolic and systolic levels of [Ca2+]i, leading to reduced cell length shortening and less active force production. For the AFER tissue with rapid reentrant excitation activity, though the diastolic [Ca2+]i level was elevated because of Ca2+ accumulation as compared to the sinoatrial node rhythm, the systolic [Ca2+]i level was markedly reduced, which is responsible for the reduced cell length shortening and the product of the active force. Snapshots of atrial electromechanical contraction superimposed on an atrial mesh (grey) under control and the tissue 2–3 months after cardioversion (Left; states 1 & 2), AFER-SR (Middle; AF-remodelled tissue under sinus rhythm; state 3) and AFER-AF (Right; AF-remodelled tissue with multiple re-entrant excitation waves; state 4) conditions. 0 ms: Before electrical activation. 100 ms: Almost complete activation under control (left) condition compared to slower activation in AFER-SR (middle) and AFER-AF (right) conditions. 200 ms: Control (left) condition undergoing contraction, AFER-SR (middle) and AFER-AF (right) conditions with negligible contraction. 300 ms: Control (left) and AFER-SR (middle) conditions undergoing repolarisation and re-entrant wavelets in the AFER-AF (right) condition. 700 ms: Complete repolarisation and tissue relaxation in control (left) and AFER-SR Fig 5. Snapshots of atrial electromechanical contraction superimposed on an atrial mesh (grey) under control and the tissue 2–3 months after cardioversion (Left; states 1 & 2), AFER-SR (Middle; AF-remodelled tissue under sinus rhythm; state 3) and AFER-AF (Right; AF-remodelled tissue with multiple re-entrant excitation waves; state 4) conditions. 0 ms: Before electrical activation. 100 ms: Almost complete activation under control (left) condition compared to slower activation in AFER-SR (middle) and AFER-AF (right) conditions. 200 ms: Control (left) condition undergoing contraction, AFER-SR (middle) and AFER-AF (right) conditions with negligible contraction. 300 ms: Control (left) and AFER-SR (middle) conditions undergoing repolarisation and re-entrant wavelets in the AFER-AF (right) condition. 700 ms: Complete repolarisation and tissue relaxation in control (left) and AFER-SR PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 15 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Effects of AFER on atrial electro-mechanics To investigate the effects of AFER on atrial electro-mechanics, simulations have been per- formed for 3 conditions mimicking 4 states. At the cellular level, AFER (state 3 & 4) abbrevi- ated atrial action potential duration and reduced Ca2+ loading in the sarcolemma reticulum, which resulted in reduced diastolic and systolic [Ca2+]i levels, leading to reduced cell shorten- ing and active force production. Impaired electro-mechanical activity at the cellular level was reflected by impaired mechani- cal contraction of the atria at the whole organ level for the AFER-SR condition (state 3). In simulations, AFER-SR resulted in a reduction in the emptying fraction of the atria as demon- strated by results shown in Fig 5. In the AFER-SR, the conduction of atrial excitation was slo- wed down, leading to delayed onset but earlier completion of atrial contraction as compared to the control tissue. The overall contraction volume was markedly reduced (Fig 5). Such impaired mechanical contraction is attributable in our model to the reduced level of diastolic and systolic [Ca2+]i levels, resulting in reduced cell length shortening and production of the active force (Fig 6). In our simulations of the AFER-AF tissue (state 4), multiple rapid reentrant excitation waves were initiated and sustained. Due to uncoordinated regional excitation waves, the PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 16 / 24 Electrical Remodeling Effects on Atrial Electro-Mechanics Fig 6. Electromechanical properties of a RA cell in the 3D atria under control (blue), AFER-SR (sinus rhythm, green) and AFER-AF (red) conditions. (A) Action potential. (B) Cytosolic Ca2+ concentration. (C) Sarcomere length shortening and (D) Active force normalised to maximum of Control. doi:10.1371/journal.pone.0142397.g006 Fig 6. Electromechanical properties of a RA cell in the 3D atria under control (blue), AFER-SR (sinus rhythm, green) and AFER-AF (red) conditions. (A) Action potential. (B) Cytosolic Ca2+ concentration. (C) Sarcomere length shortening and (D) Active force normalised to maximum of Control. doi:10.1371/journal.pone.0142397.g006 doi:10.1371/journal.pone.0142397.g006 17 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Electrical Remodeling Effects on Atrial Electro-Mechanics mechanical contraction of the atria is significantly diminished even though the diastolic [Ca2+]i level was elevated arising from Ca2+ accumulation (Figs 5 and 6). Relevance to Previous Studies This study is the first to develop a 3D electromechanical model of the human atria with hetero- geneous, electromechanically coupled, regional single cell models. It is also the first to investi- gate the cellular mechanisms responsible for weak atrial contraction in AFER tissue. Fritz et al. [93] developed a 3D electromechanical model of the human atria, but that study used a geo- metric model and appears to not consider atrial cellular heterogeneity, as it used just the right atrial CRN single cell model. Also it did not investigate AF [93]. Chapelle et al. [94] and Collin et al. [95] developed a surface-based electrophysiology model of the atria, treating the atria as a shell structure. Their model relied on detailed asymptotic analysis, reduced the computational cost of simulating a 3D atria and they presented strong convergence results of the numerical solution. However, they did not consider atrial deformation. Coudiere et al. [96] et al. also developed a two-layered model of atrial electrophysiology with two coupled, superimposed lay- ers with the introduction of 3D heterogeneity. They used the model to explore numerical con- vergence for vanishing thicknesses. This model also reduced computational cost relative to a 3D model. It is important to note that while multiscale simulations of atrial electromechanics are rare, there are several studies involving the ventricles and the whole heart such as the Multi-scale, Multi-physics Heart Simulator, “UT Heart” [97], the parallel multiphysics code for Computa- tional Biomechanics, ALYA Red [98,99], the interactive framework for rehearsal of and train- ing in cardiac catheter ablation from INRIA [100] and several others [57,68,101–103]. Clinical Implications Altered electro-mechanical function has important clinical implications. The failure of a return of atrial contraction following successful cardioversion may result in reduced exercise capacity and to an increased risk of thrombo-embolism and stroke. In addition, atrial stunning is associ- ated with a greater risk of recurrence of atrial fibrillation [104], perhaps related to lower atrial pressures and less atrial stretch. PLOS ONE \| DOI:10.1371/journal.pone.0142397 November 25, 2015 Conclusion We have developed an anatomically accurate, 3D biophysically detailed, electro-mechanical model of the human atria, which incorporates a suite of electro-mechanical single cell models comprising the different regions of the human atria. The model also incorporates fibre orienta- tions validated against patient-specific data. Using the model, we have investigated the func- tional consequences of AFER for atrial contraction. We have shown that chronic AF-induced remodeling of ionic channel and intracellular Ca handling may be responsible for weak atrial contraction, a phenomenon as seen in atrial stunning after successful cardioversion. Electrical Remodeling Effects on Atrial Electro-Mechanics the LA and RA but realistically, it varies throughout the cardiac cycle and may increase follow- ing the onset of atrial fibrillation. The use of a computational fluid dynamics model enabling the consideration of blood flow would allow the use of a more realistic pressure profile. The model does not take into consideration contact between the atria and the ventricles or the effect of ventricular motion on atrial mechanics, which all likely play a significant role in determining atria function. We did not investigate the effect of stunning on the LA and RA individually. It is possible that it affects both differently [8]. Therefore, this should be a subject of future study. Note that in simulations we have assumed that atrial tissue would take about 6 weeks (a time required for restoring atrial mechanical contraction after cardioversion) to complete reverse electrical remodeling, and simulated the tissue at that time using the same condition as healthy tissue. Though in animal models it has been demonstrated that reverse cardiac electrical remodeling process may take approximately the same time period [109], in human, due to multiple factors (structural, pathological and physiological) associated with atrial fibrillation, such an assumption may be too idealised. Whilst it is important that these potential limitations are stated, they do not fundamentally alter the principal conclusions of this study. Author Contributions Conceived and designed the experiments: HZ IA. Performed the experiments: IA HZ. Ana- lyzed the data: HZ IA JCH DM CJG JY. Contributed reagents/materials/analysis tools: HZ IA JCH DM CJG JY. Wrote the paper: IA HZ JCH DM CJG JY. S2 Video. Atrial electrical wave propagation and mechanical contraction under the atrial fibrillation-induced electrical remodelling condition under sinus rhythm (AFER-SR). (AVI) S3 Video. Atrial electrical wave propagation and mechanical contraction under the atrial fibrillation-induced electrical remodelling condition with atrial fibrillation re-entrant exci- tation waves (AFER-AF). (AVI) Supporting Information S1 Video. Atrial electrical wave propagation and mechanical contraction in the normal tis- sue (state 1) and the tissue 2–3 months after cardioversion (state 2). (AVI) S1 Video. Atrial electrical wave propagation and mechanical contraction in the normal tis- sue (state 1) and the tissue 2–3 months after cardioversion (state 2). (AVI) S2 Video. Atrial electrical wave propagation and mechanical contraction under the atrial fibrillation-induced electrical remodelling condition under sinus rhythm (AFER-SR). (AVI) S3 Video. Atrial electrical wave propagation and mechanical contraction under the atrial fibrillation-induced electrical remodelling condition with atrial fibrillation re-entrant exci- tation waves (AFER-AF). (AVI) Limitations In addition to acknowledged limitations of both the CRN electrophysiology model [31] on which the atrial single cells were based and of the Rice et al. [36] mechanics model, all the other regional cell models used here are inherited from Colman et al. [41] atrial models, the limita- tions of which have been discussed in our previous study.[41] The CRN electrophysiology model [31] is based on human experimental data whilst the Rice et al. myofilament model [36] is based on data from rat and rabbit. Ideally, all model parameters should be derived from human data, but as is often the case, there are a very limited amount of human experimental data to use to constrain the myofilament model and indeed the electromechanical model. This is a limitation but a necessary model compromise. This approach is consistent with that adopted in prior studies from our own group and others [41,105–107]. 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https://openalex.org/W2584343984	https://digitalcommons.usf.edu/context/kip_articles/article/6691/viewcontent/K26_05548_Bernard_et_al_2017_Ecology_and_Evolution.pdf	English	null	Winter behavior of bats and the progression of white‐nose syndrome in the southeastern United States	Ecology and evolution	2,017	cc-by	9,299	University of South Florida University of South Florida Digital Commons @ University of Digital Commons @ University of South Florida South Florida KIP Articles KIP Research Publications January 2017 Winter behavior of bats and the progression of white-nose Winter behavior of bats and the progression of white-nose syndrome in the southeastern United States syndrome in the southeastern United States Riley Bernard Gary F. McCracken Follow this and additional works at: https://digitalcommons.usf.edu/kip_articles K E Y W O R D Sii K E Y W O R D S acoustic activity, bats, echolocation, white-nose syndrome, winter captures Recommended Citation Recommended Citation Bernard, Riley and McCracken, Gary F., "Winter behavior of bats and the progression of white-nose syndrome in the southeastern United States" (2017). KIP Articles. 5692. https://digitalcommons.usf.edu/kip_articles/5692 This Article is brought to you for free and open access by the KIP Research Publications at Digital Commons @ University of South Florida. It has been accepted for inclusion in KIP Articles by an authorized administrator of Digital Commons @ University of South Florida. For more information, please contact digitalcommons@usf.edu. Received: 10 November 2016 \| Revised: 21 November 2016 \| Accepted: 22 December 2016 Received: 10 November 2016 \| Revised: 21 November 2016 \| Accepted: 22 December 2016 DOI: 10.1002/ece3.2772 Received: 10 November 2016 DOI: 10.1002/ece3.2772 Winter behavior of bats and the progression of white-nose syndrome in the southeastern United States Riley F. Bernard1 \| Gary F. McCracken2 1Department of Ecosystem Science and Management, Pennsylvania State University, University Park, PA, USA Abstract Understanding the winter behavior of bats in temperate North America can provide insight into how bats react to perturbations caused by natural disturbances such as weather, human-induced disturbances, or the introduction of disease. This study measured the activity patterns of bats outside of their hibernaculum and asked how this winter activity varied by time, temperature, bat species, body condition, and WNS status. Over the course of three winters (2011–2013), we collected acoustic data and captured bats outside of five hibernacula in Tennessee, United States. During this time, Pseudogymnoascus destructans, the causative agent of white-nose syndrome, became established in hibernacula throughout the region, allowing us to track disease- related changes in the winter behavior of ten bat species. We determined that bats in the southeastern United States were active during winter regardless of disease. We recorded activity outside of hibernacula at temperatures as low as −13°C. Although bat activity was best determined by a combination of variables, the strongest factor was mean daily temperature (R2 = .2879, F1,1450 = 586.2, p < .0001). Bats that left the hibernacula earlier in evening had lower body condition than those that left 2–4 hr after sunset (F7,932 = 7.225, p < .0001, Tukey HSD, p < .05). The number of daytime emergences from hibernacula, as determined via acoustic detection, increased the longer a site was P. destructans positive (F3,17 808 = 124.48, p < .0001, Tukey HSD, p < .05). Through the use of passive acoustic monitoring and monthly captures, we determined that winter activity was driven by both ambient temperature and the pres- ence of P. destructans. Abstract 2Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN, USA Correspondence Riley F. Bernard, Department of Ecosystem Science and Management, Pennsylvania State University, University Park, PA, USA. Email: rbernar3@vols.utk.edu Correspondence Riley F. Bernard, Department of Ecosystem Science and Management, Pennsylvania State University, University Park, PA, USA. Email: rbernar3@vols.utk.edu Funding information Basically Bats Wildlife Conservation, Inc. White-Nose Syndrome Research Scholarship; Department of Ecology and Evolutionary Biology at the University of Tennessee; United States Geological Survey. © 2017 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Ecology and Evolution 2017; 7: 1487–1496 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.i 1 \| INTRODUCTION 1 in desert regions of the southwestern United States (US; Boyles et al. 2006). Previous studies of winter activity of bats in Ontario, New England, Indiana, and Missouri found that individuals arousing during winter months mostly flew within the hibernaculum, and rarely left the cave (Griffin 1945; Whitaker and Rissler, 1992; Boyles et al. 2006). These findings reinforce the assumption that bats enter hibernation sites in the fall and are not observed on the landscape until mid-spring. Winter activity is documented for several species of North American bats that use roost sites with variable microclimates such as foliage, bark, or man-made structures (Boyles, Dunbar, and Whitaker, 2006). However, information on winter behavior of North American cave- roosting bats is based largely on studies at latitudes above 40°N, or \| 1487 www.ecolevol.org 1487 Ecology and Evolution 2017; 7: 1487–1496 1488 BERNARD and McCRACKEN confirmed in M. grisescens, they were thought to be vulnerable to the disease due to their propensity for forming high-density clusters during hibernation (U.S. Fish and Wildlife Service, 2009). Myotis gris- escens are known to hibernate in eight caves in Alabama, Arkansas, Kentucky, Missouri, and Tennessee (USFWS, 1982, 1997). In vitro growth curves suggest that P. destructans may reproduce more quickly in cave environments that maintain more moderate temperatures of 10–15°C in winter (Verant et al. 2012), which could result in increased virulence in southern hibernacula. Prior to the white-nose syndrome (WNS) disease epizootic, it was suggested that bats leave hibernacula in winter infrequently, and do so to switch roosts or in search of water or food (Boyles et al. 2006). However, as WNS became established in the northeast, behaviors such as daytime and cold-weather flight during winter became indic- ative of infection. To date, no studies have looked at the winter be- havior of bats in the lower latitudes of the southeastern USA, where warmer temperatures and available insects may allow for sustained activity through winter. The importance of understanding the winter behavior of bats in the USA has become better appreciated due to WNS. First identified in February 2006 in a cave in New York, this disease has spread across eastern North America as far west as Missouri, south into Mississippi, and north into Canada, and was recently found further west in Washington state. To date, WNS has killed over 5 million bats of seven hibernating species (USFWS, 2014). 1 \| INTRODUCTION Mortality as high as 90%–100% has been documented at some northeastern hibernacula, leading to possible regional extinction of several once-common species (Frick et al. 2010; Langwig et al. 2012). The highest rates of morbidity and mortality at sites in the northeast have been documented following the second and third winters after initial visual detection of the fungus (Titchenell 2012; Knudsen, Dixon & Amelon 2013). Our study measured the activity patterns of bats outside of caves during winter and investigated how this activity varied by time, temperature, bat species, body condition, and WNS status. To address these questions, we looked at the following hypotheses: (1) Bats will remain active throughout winter in the southeast due to warmer ambient temperatures than in the northeast and (2) Length of WNS infection at hibernacula will affect the body condition and winter activity of cave-roosting bats. Passive acoustic monitoring and bat captures during winter outside of caves allowed us to ad- dress these questions without adding additional stress to bats within hibernacula. 2.1 \| Study area Bat activity was monitored at three hibernacula in Middle and East Tennessee during January – May 2012 (year 1), and five hibernacula during October – May 2012–2013 (year 2) and 2013–2014 (year 3; Figure 1). Blount cave is the largest known M. sodalis hibernaculum in Tennessee, with an estimated 9,500 individuals in winter 2012– 2013 (Flock 2013). Hawkins and Warren caves are two of the largest M. grisescens hibernacula in the state and also contain small popula- tions of M. sodalis. Recent hibernacula surveys estimated 150,000 and 500,000 endangered M. grisescens at Hawkins and Warren caves, respectively. Campbell and White caves contain Corynorhinus rafines- quii Lesson, Eptesicus fuscus Beauvois, M. leibii Audubon and Bachman, M. lucifugus Le Conte, M. septentrionalis, and M. sodalis (Holliday 2012). White cave had approximately 700 M. grisescens during win- ter 2012–2013. Four bat species hibernated in Blount cave, and ap- proximately six species of bat (n = <10,000 individuals) were counted during hibernacula surveys at Campbell cave (Samoray, 2011; Flock, 2013). Tennessee is among several southeastern states that contain hi- bernacula used by threatened and endangered species, including Myotis sodalis Miller & Allen, M. septentrionalis Trouessart, and M. gris- escens Howell. The fungus was first detected in Tennessee during winter 2009–2010 at the largest M. sodalis hibernacula in the state (Samoray 2011; Carr et al. 2014). By winter 2015–2016, 54% of the counties in Tennessee (n = 52/95) were deemed WNS positive, with several high-priority M. sodalis hibernacula experiencing declines in bat populations (Campbell 2016). Myotis septentrionalis appears to be one of the hardest hit species in Tennessee, having experienced declines varying from 69% to 98.5% at known hibernacula (TWRA, unpublished data). Although declines due to WNS have not been 2 \| MATERIALS AND METHODS White-nose syndrome is caused by the psychrophilic (cold- tolerant) fungus, Pseudogymnoascus destructans Minnis & Lindner (Lorch et al. 2011; Minnis & Lindner 2013), a non-native pathogen from Eurasia (Leopardi, Blake & Puechmaille 2015; Hoyt et al. 2016). Pseudogymnoascus destructans invades the muzzle, wings, and tail membrane of bats during torpor when their immune systems are likely suppressed (Moore et al. 2011; Field et al. 2015). Invasion of the wing tissue by P. destructans can cause dehydration, electrolyte imbalance, and increased arousal frequency leading to critical loss of fat stores needed to survive hibernation (Cryan et al. 2010; Reeder et al. 2012; Warnecke et al. 2012, 2013, Verant et al. 2014, 2016). Changes in win- ter behavior associated with WNS have been documented, with bats roosting in exposed regions of cave entrances or leaving hibernacula during the day, and flying during cold winter nights (Turner, Reeder & Coleman 2011; Foley et al. 2011; Carr, Bernard & Stiver 2014). To date, seven bat species have been confirmed with the disease via his- topathology, with five additional species confirmed with P. destructans DNA on their epidermis (Meteyer et al. 2009; USFWS, 2014; Bernard et al. 2015). 2.2 \| Acoustic activity Ultrasonic bat detectors were deployed near the entrance of each hi- bernaculum. Acoustic data were recorded in zero-crossing mode onto 4 Gb compact flash (CF) cards (year 1) and 32 Gb secure digital (SD) cards (years 2 and 3). Detectors were placed approximately 25–40 m from each cave entrance. During year 1, three Anabat II detectors with CF ZCAIM units (Zero-Crossing Analysis Interface Module; Titley Electronics, Ballina, New South Wales, Australia) were used at Blount, Campbell, and Warren caves. Microphones were housed BERNARD and McCRACKEN 1489 FIGURE 1 Cave locations within county boundaries in Tennessee, United States. Color coding of counties corresponds to the year Pseudogymnoascus destructans was confirmed using either real-time PCR (Muller et al. 2013) or histopathology (Meteyer et al. 2009) FIGURE 1 Cave locations within county boundaries in Tennessee, United States. Color coding of counties corresponds to the year Pseudogymnoascus destructans was confirmed using either real-time PCR (Muller et al. 2013) or histopathology (Meteyer et al. 2009) & Warren caves; n = 361 acoustic days), 4 October 2012 – 7 May 2013 (Blount, Campbell, Hawkins, Warren & White caves; n = 1,004 acoustic days), and at these same caves 2 October 2013 – 13 May 2014 (n = 1,001 acoustic days). During the three winters, acoustic monitoring was conducted on 538 days at Blount cave, 498 days at Campbell cave, 384 days at Hawkins cave, 543 days at Warren cave, and 403 days at White cave. Acoustic monitoring for the last 2 years of the study was from the end of fall swarming through spring emergence. Temperature was recorded at each site at 15-min inter- vals using HOBO U-series data loggers with an accuracy of ± 0.21°C (Onset Computer Corporation, Bourne, MA, USA). Each temperature meter was 2.5 m above the ground and 10–15 m from the acoustic detector. in waterproof “bat hats” with reflector plates (Arnett, Hayes & Huso 2006) and secured on PVC pipes approximately 3 m above ground level. All electronic equipments (Anabat II detector, ZCAIM and two 12-volt batteries in parallel) were enclosed in large utility boxes (Duluth Trading Company, Belleville, WI, USA) located at the base of the PVC pipe. Due to technical difficulties with aging Anabat II and ZCAIM units, SM2Bat+ detectors (Wildlife Acoustics, Maynard, Massachusetts, USA) programed to record data in zero-crossing, were used in years 2 and 3. 2.2 \| Acoustic activity Detectors were redeployed at Blount, Campbell, and Warren caves and added to White and Hawkins caves. SM2Bat+ detectors were secured to a 30.5 × 20 cm piece of plywood and at- tached to a 5-cm-diameter PVC pipe using U-bolts. The SMX-US microphone was attached to a 1-m cord and fastened at a 45° angle to the top of the PVC pipe. The SM2Bat+ detectors were powered by a 6-volt external battery enclosed in utility boxes at the base of the entire unit. the spread of P. destructans among sites. Mist nets were deployed 30 min before civil sunset and remained open for 5 hr, until 30 bats were captured, or temperatures dropped below freezing (0°C). Bats were held individually in paper bags and placed in a large insulated box with four hand warmers (HotHands®, Dalton, Georgia, USA) for 30–60 min prior to processing. Each captured bat was identified to species, and reproductive condition (scrotal, pregnant, lactating, post- lactating, nonreproductive), right forearm length (mm), weight (g), and wing damage index (WDI) score were recorded. Guano was collected when possible. Wing damage was classified from 0 to 3, where WDI score = 0 indicates no obvious scaring or discoloration on the mem- branes, WDI score = 1 denotes light damage covering less than 50% of the membranes, WDI score = 2 indicates moderate damage greater than 50% of the wing membrane covered in scar tissue, and WDI score = 3 signifies heavy damage with deteriorated wing membranes and necrotic tissue (Reichard & Kunz 2009). In year 3, bat wings were examined using ultraviolet (UV) light to check for lesions indicative of WNS. Under UV light, damage caused by P. destructans infiltration will fluoresce yellow orange (UV positive), allowing for confirmation of WNS infection without submitting the bat for histological confir- mation (Turner et al. 2014). Ultraviolet examination was not used in years 1 and 2 of our study because the method had not yet been sub- stantiated. To asses overall health of each bat, body condition indices (BCI) were calculated by dividing weight by forearm length (Speakman & Racey 1986). All species, except Lasiurans and Corynorhinus, were banded with either a 2.4-mm or 2.9-mm numbered forearm band and released on site. R (version 3.2.4; R Core Development Team, 2016). Negative bi- nomial GLMMs were employed to account for overdispersion and repeated sampling. Differences in bat acoustic activity among years were tested using daily and hourly “bat pass” totals. Fixed effects included year, sampling season (winters 2011–2012, 2012–2013, and 2013–2014), month, hibernation period (early—[October 1 – December 15], mid—[December 16 – February 15], late—[February 16 – April 30]), time of day (day or night), hour (0:00 to 23:00 hr), mean daily temperature (24-hr period), temperature at time of emer- gence (dusk), relative humidity, moon illumination, and year since P. destructans confirmation at each cave. Year since P. destructans confirmation was based on the detection of fungal DNA on epider- mal swab samples or histology of voucher specimens (USGS National Wildlife Health Center 2013). Nonparametric Kruskal–Wallis and Mann–Whitney–Wilcoxon tests were used to examine differences between species in frequency of capture and BCI among month, site, across seasons, and year since P. destructans confirmation. Post hoc pairwise comparisons using Tukey and Kramer (Nemenyi) tests were used to determine differences among factors using the package PMCMR (version 4.1). 3.1 \| Acoustic activity Useable acoustic data were collected on 88.4% or 2,091 of 2,366 detector nights. Loss of data resulted from malfunctioning Anabat/ ZCAIM units, SM2Bat+ external AC adaptor failure and a microphone cord failure. The recordings contained a total of 566,000 bat passes with bat activity recorded on 1,695 of the 2,091 recording days. Mean activity at sites varied considerably (Table 1), presumably due to dif- ferences in species compositions and population sizes at each loca- tion. Daily bat activity was best predicted by the model that included the interaction between month and temperature at emergence with the additive effects of year since P. destructans was confirmed and moon illumination (Table 2). Hourly bat activity was best described by the model that included the interaction of time of day (day or night classification) with mean hourly temperature, and year since P. de- structans was confirmed (Table 2). All capture and handling techniques were approved by the University of Tennessee Institute of Animal Care and Use Committee (protocol 2026-0514) and are consistent with the guidelines issued by the American Society of Mammalogists (Sikes, 2016). 2.3 \| Bat captures During years 2 and 3, bats were captured near the entrance of each cave once a month using 6-, 9-, and 12-meter mist nets (Avinet, Dryden, New York, USA; 75/2 mesh size, 2.6 m high, 4 shelves, black polyester for bats). Nets were deployed for a total of 6,899.1 net hr/ m2 over both winters. Each site had designated equipment to prevent Because WNS-affected bats in the northeast are often active during daytime, bat acoustic activity and temperature were recorded 24 hr/day. Data cards and batteries were replaced monthly. Acoustic data were collected from 4 January to 30 May 2012 (Blount, Campbell BERNARD and McCRACKEN 1490 °C Blount Day 3.55 ± 0.34 15.03 ± 0.18 5.34 ± 0.29 8.95 ± 0.18 0.15 ± 0.07b 11.67 ± 0.19 Night 21.74 ± 0.84 9.42 ± 0.17 6.69 ± 0.27 4.41 ± 0.16 8.64 ± 0.96b 6.48 ± 0.16 Campbell Day 19.88 ± 3.16 No temp meter 0.15 ± 0.02 8.49 ± 0.16 1.63 ± 0.11 9.34 ± 0.21 Night 131.56 ± 8.33 No temp meter 11.22 ± 0.75 6.25 ± 0.12 17.67 ± 0.71 5.47 ± 0.15 Hawkins Day 0.50 ± 0.21 11.02 ± 0.21 0.50 ± 0.09 13.44 ± 0.26 Night 7.54 ± 0.80 4.08 ± 0.13 29.95 ± 1.37 4.83 ± 0.19 Warren Day 7.39 ± 0.88 No temp meter 0.63 ± 0.10 10.65 ± 0.24 1.38 ± 0.25 5.57 ± 0.26 Night 87.13 ± 2.73 No temp meter 24.34 ± 0.96 7.33 ± 0.19 18.35 ± 1.15 5.23 ± 0.21 White Day 0.25 ± 0.06 Meter broke 0.10 ± 0.01 5.81 ± 0.17 Night 7.78 ± 0.44 Meter broke 2.48 ± 0.18 5.20 ± 0.14 TABLE 2 Top five models identifying variables that best explain daily and hourly acoustic activities of bats during winter in the southeastern United States. Daily acoustic activity is the average number of bat calls per 24-hr period, whereas hourly acoustic activity is the average number of bat calls per hour. Akaike information criterion (AIC) values identify the best-fit model. Degrees of freedom (df), change in AIC (ΔAIC), and weight of evidence (wi) for the models relating bat activity to abiotic variables are shown for all sites and seasons in all years. Models receiving substantial empirical support (ΔAIC ≤ 2.0) are in bold face type Explanatory variables in model df ΔAIC wi Daily acoustic activity Month * Emerg. Temp. + Yr Pd Confirmed + Moon Illumination 3 0.0 1.0 Month + Emerg. Temp. + Yr Pd Confirmed + Moon Illumination 4 19.1 0.0 Month * Emerg. Temp. + Moon Illumination 2 161.9 0.0 Month + Emerg. Temp. + Moon Illumination 3 165.7 0.0 Hibernation Period + Emerg. Temp. + Yr Pd Confirmed + Moon Illumination 4 181.8 0.0 Hourly acoustic activity Time of Day * Mean Temp. + Yr Pd Confirmed 2 0.0 1.0 Time of Day * Mean Temp. 1 25.9 0.0 Time of Day + Mean Temp. + Yr Pd Confirmed 3 995.3 0.0 Time of Day + Mean Temp. 2 1035.6 0.0 Hour * Mean Temp. TABLE 1 Mean number of bat calls per period ± SE and mean temperature (°C) ± SE per site from January through April 2012 (aindicates truncated season) and October through April 2012–2013 and 2013–2014. Mean number (#) of bat calls and temperature from sunrise to sunset are reported as “Day” and those from sunset to sunrise as “Night”. Hawkins and White caves were added to the sampling regime in winters 2012–2013 and 2013–2014. Mean activity at Blount cave during winter 2013–2014 is high due high call activity in October 2013 (b). The temperature meter at White cave malfunctioned in winter 2012–2013 TABLE 1 Mean number of bat calls per period ± SE and mean temperature (°C) ± SE per site from January through April 2012 (aindicates truncated season) and October through April 2012–2013 and 2013–2014. Mean number (#) of bat calls and temperature from sunrise to sunset are reported as “Day” and those from sunset to sunrise as “Night”. Hawkins and White caves were added to the sampling regime in winters 2012–2013 and 2013–2014. Mean activity at Blount cave during winter 2013–2014 is high due high call activity in October 2013 (b). The temperature meter at White cave malfunctioned in winter 2012–2013 Cave Time of day Winter survey year 2011–2012a 2012–2013 2013–2014 Mean # bat calls Mean temp. °C Mean # Bat Calls Mean temp. °C Mean # bat calls Mean temp. + Yr Pd Confirmed 2 5699.7 0.0 * Indicates interaction between two variables. (+) indicates additive effects. * Indicates interaction between two variables. * Indicates interaction between two variables. (+) indicates additive effects. number of bat calls recorded per day (mean ± SE; 1,252.53 ± 39.14 bat calls) was recorded at caves that were P. destructans negative during the first year of monitoring (Campbell and Warren caves; chi-square = 325.72, df = 4, p < .0001). Four caves experienced sig- nificant declines in mean nightly bat calls each year following P. de- structans confirmation (F2, 39 845 = 1,051.63, p < .0001, Tukey HSD, p < .05). However, nightly bat activity at Hawkins cave increased significantly between years 2 and 3 (Wilcoxon = 4010.5, p < .0001), despite similar mean nightly temperatures for both years (Table 1). The number of daytime emergences from hibernacula increased the longer a site was P. destructans positive (F3,17 808 = 124.48, p < .0001, Tukey HSD, p < .05). 2.4 \| Data analysis The highest Generalized linear mixed models (GLMM) with site as a random effect were calculated using the package lme4 (version 1.1-11) in \| 1491 BERNARD and McCRACKEN TABLE 1 Mean number of bat calls per period ± SE and mean temperature (°C) ± SE per site from January through April 2012 (aindicates truncated season) and October through April 2012–2013 and 2013–2014. Mean number (#) of bat calls and temperature from sunrise to sunset are reported as “Day” and those from sunset to sunrise as “Night”. Hawkins and White caves were added to the sampling regime in winters 2012–2013 and 2013–2014. Mean activity at Blount cave during winter 2013–2014 is high due high call activity in October 2013 (b). The temperature meter at White cave malfunctioned in winter 2012–2013 1491 BERNARD and McCRACKEN 2.4 \| Data analysis Acoustic activity was quantified as the number of files or “bat passes” recorded per 24-hr period. A “bat pass” was defined as a file contain- ing a search-phase echolocation sequence of ≥2 echolocation pulses (Gannon, Sherwin & Haymond 2003). This metric is an index of overall activity and does not correlate to the numbers of individual bats ac- tively flying outside of the cave (Kunz et al. 2007; Schwab & Mabee 2014). All call files were analyzed in AnalookW (version 3.9c, Titley Scientific) using antinoise filters. Filters were used to eliminate files that contained calls with less than two pulses, calls with short dura- tion (<4 ms) or low frequency noise (i.e., wind, insect noise, or rain). Once all noise files were removed, each file was manually vetted to determine whether the remaining call files fit the “bat pass” criteria. Because we were attempting to quantify bat activity at each site re- gardless of species, and due to the difficulties in distinguishing the closely similar calls of several Myotine species (Barclay 1999; Britzke et al. 2011), species identification of each call was not attempted for this study. Bat calls were recorded on nights where temperatures at emer- gence were below 0°C on 23 nights at Blount cave, 27 nights at Campbell cave, eight nights at Hawkins cave, three nights at Warren cave, and three nights at White cave. The lowest temperature at which bats were detected was −13°C (n = 2 calls), and bats were not detected on nights when the temperature at emergence was below −8°C (n = 49 nights). Bat activity was significantly positively correlated with mean daily temperature (R2 = .2879, F1,1450 = 586.2, p < .0001). The highest mean numbers of nightly calls were recorded during year 1, with a reduction in total calls recorded in subsequent years (Table 1). There was an increase in daytime activity at Blount cave in year 2, 2 years post-WNS confirmation, followed by a sharp decline in total acoustic activity by the end of year 3. However, at all sites, night- time acoustic activity always exceeded daytime activity. Species Winter 2012/13 Winter 2013/14 Females Males Total Females Males Total CORA 1 3 4 2 3 5 EPFU 7 13 20 6 13 19 LABO 1 2 3 2 1 3 LANO 0 0 0 1 2 3 MYGR 31 133 164 54 128 182 MYLE 17 35 52a 10 40 51a MYLU 7 5 12 3 16 19 MYSE 39 103 142 24 32 56 MYSO 25 27 52 6 39 45 PESU 33 49 82 8 25 33 Species acronym code: CORA, Corynorhinus rafinesquii; EPFU, Eptesicus fuscus; LABO, Lasiurus borealis; LANO, Lasionycteris noctivagans; MYGR, Myotis grisescens; MYLE, Myotis leibii; MYLU, Myotis lucifugus; MYSE, Myotis septentrionalis; MYSO, Myotis sodalis; PESU, Perimyotis subflavus. aOne MYLE from each year escaped prior to collecting any biometric information, including sex. TABLE 3 Total bat captures by species in 2012–2013 and 2013–2014. Nets were deployed for a total of 6,899.1 net hr/m2 (winter 2012–2013 = 3,182.4 net hr/m2, winter 2013–2014 = 3,716.7 net hr/m2) at a capture rate of approximately 0.137 bats per net hr per m2 TABLE 3 Total bat captures by species in 2012–2013 and 2013–2014. Nets were deployed for a total of 6,899.1 net hr/m2 (winter 2012–2013 = 3,182.4 net hr/m2, winter 2013–2014 = 3,716.7 net hr/m2) at a capture rate of approximately 0.137 bats per net hr per m2 Species acronym code: CORA, Corynorhinus rafinesquii; EPFU, Eptesicus fuscus; LABO, Lasiurus borealis; LANO, Lasionycteris noctivagans; MYGR, Myotis grisescens; MYLE, Myotis leibii; MYLU, Myotis lucifugus; MYSE, Myotis septentrionalis; MYSO, Myotis sodalis; PESU, Perimyotis subflavus. aOne MYLE from each year escaped prior to collecting any biometric information, including sex. g/mm, n = 269), 4 years (0.197 ± 0.005 g/mm, n = 51), and 3 years (0.190 ± 0.004 g/mm, n = 206). Bats captured at caves that had yet to be confirmed as positive for P. destructans exhibited the lowest mean BCI (0.182 ± 0.003 g/mm, n = 309). Individuals identified as UV fluorescence-negative (n = 477) had higher mean BCI than UV fluorescence-positive bats (n = 68; t137.8 = −7.31, p < .0001). captured on a night where the mean nightly temperature was 0°C. Across all seasons, the most bats were captured in October (n = 190) and April (n = 222), the first and last months of the hibernation period. 4 \| DISCUSSION This is the first study to demonstrate that bats are active throughout winter in the southeastern USA. Activity throughout winter was not restricted to bats in WNS-infected caves; however, increases in activ- ity associated with low temperatures and diurnal flight were attrib- uted to the disease. Variation in body condition across all species was found throughout a capture session, across seasons, and in relation to how long a hibernaculum was P. destructans positive. Across all seasons, body condition (BCI) was highest in October, declined during December – February, and began to increase during March and April, the last two months of hibernation (F6, 312 = 42.9653, p < .0001). There were no significant differences in mean BCI be- tween males and females. Mean BCI for all species captured during January through April of year 3 was significantly higher than for the same months in year 2 (t940 = 7.69, p < .0001; Figure 2). Bats captured in the first 2 hr after sunset had the lowest BCI on av- erage, with those captured several hours after emergence having higher mean BCI (F7,932 = 7.225, p < .0001). Individuals that pro- vided fecal samples had a higher mean BCI than those that did not defecate while in captivity (t940 = 3.57, p = .0004). All 290 fecal pel- lets collected contained recently consumed prey (as described by Dunbar et al. 2007; R. F. Bernard, V. A. Brown, E. V. Willcox, & G. F. McCracken, unpublished). There was no significant difference be- tween mean BCI and WDI of bats captured during the study. Body condition varied in relation to how long a site was P. destructans pos- itive (F4, 937 = 12.87, p < .001; Tukey HSD, p < .05). Bats captured at caves positive for 2 years had the highest mean BCI (0.222 ± 0.005 g/mm, n = 107), followed by sites positive for 1 year (0.206 ± 0.003 More bats were captured during winter 2012–2013 (year 2; n = 531) than in winter 2013–2014 (year 3; n = 416; chi-squared = 51.19, p < .0001). There was a significant decline in the number of individu- als captured at a site as the time since confirmation of P. destructans increased (chi-squared = 1387.01, p < .0001). Most bats captured during the study period (n = 871) had a WDI = 0. Sixty-four individu- als (6.75%) scored a WDI = 1, with the remaining individuals (n = 10; 1.15%) scoring WDI = 2–3. Of these 10 bats, four were M. grisescens, four were M. septentrionalis, and two were P. subflavus (Reboredo- Segovia, A. Reboredo Segovia, R. F. Bernard, E. V. Willcox, R. T. Jackson, M. Patton, & G. F. McCracken, unpublished). 3.2 \| Bat captures A total of 947 individuals of 10 species were captured (Table 3). These included 648 males, 297 females, and two M. leibii of unknown sex due to escape. Almost all bats captured (n = 936) were cave hiber- nators; however, eight Lasiurus borealis Müller and three Lasionycteris noctivagans La Conte were captured; both are migratory species that do not typically use caves as hibernacula. Netting attempts were most successful on mild nights where mean temperatures ranged between 11 and 15°C. Lasiurus borealis was captured on nights when average temperatures during the 5 hr following emergence were above 11°C, with all other species captured as low as 8°C and one M. grisescens 1492 BERNARD and McCRACKEN 4.1 \| Winter activity In October of year 3, bats at Blount Cave were recorded at similar numbers to the previous year; however, activity dropped off significantly by January, with no bats recorded at the cave after February. Over 80% of the bats cap- tured at Blount cave in March and April of year 3 had visible signs of WNS, including wing damage or ultraviolet (UV) fluorescence (Turner et al. 2014) and lower than average body condition. At Campbell cave, the total number of bats captured decreased significantly by the third year of sampling, with an increase in the number of P. sub- flavus found roosting outside of the cave. We did not see the same mortality effects of WNS as seen in the northeast in years 2–3 until 4–5 years after the disease was detected in the southeast. Therefore, hibernating bat populations in the southeast may continue to expe- rience delayed declines caused by WNS due to regional differences in climate-related life-history traits such as shorter hibernation peri- ods (Sherwin, Montgomery & Lundy 2013), abbreviated torpor bouts (Brack and Twente, 1985; Twente, Twente, and Brack, 1985; Jonasson & Willis 2012), and foraging opportunities throughout winter (Bernard et al., unpublished). during winter in more mild southern climates, and fecal samples from bats captured outside of caves verify that bats were leaving the hi- bernacula to forage (Bernard et al., unpublished). We captured bats representing every species of cave-dwelling bat known to hibernate in the region, as well as tree and foliage-roosting species, demonstrating that it is not only the latter that remain active during winter (Boyles et al. 2006). Both cave and tree or foliage-roosting species captured were active throughout winter at a range of temperatures, indicating that bats overwintering in the southeast exhibit different behaviors than northern populations (Boyles et al. 2006). This is an important factor for understanding latitudinal differences in WNS susceptibility and mortality (Bernard et al., unpublished). We found evidence for a strong correlation of temperature with winter activity of bats in all 3 years at all study caves. Winter tem- peratures at each site varied significantly during the study period with the warmest temperatures recorded in year 1. While activity levels were impacted by temperature, they were confounded by disease and differed by species. The apparent effects of WNS are seen in the seasonal activity for the species most affected by the disease (i.e., M. lucifugus, M. 4.1 \| Winter activity Hibernating mammals arouse periodically throughout winter to pre- sumably maintain physiological balance and activate suppressed im- mune systems (Prendergast et al. 2002; Field et al. 2015); however, prior to the arrival of WNS, bats in northern latitudes were rarely found leaving the hibernacula (Whitaker and Rissler, 1992; Schwab & Mabee 2014). Czenze et al. (2013) noted that M. lucifugus in Canada (53°N 99°W) entered a hibernaculum in mid-September and remained there until mid-May, showing no signs of emergence. Nevertheless, moder- ate bat activity has been reported throughout winter in some high- latitude regions that experience mild oceanic climates (Park, Jones & Ransome 2000; Hope & Jones 2012; Burles et al. 2014). Continental and Great Lake effects during winter in the northeast where WNS has caused the most dramatic population declines, generally result in severe weather. We recorded and captured bats that were active \| 1493 BERNARD and McCRACKEN FIGURE 2 Mean body condition (body mass (g)/forearm length (mm)) of seven bat species captured at all five hibernacula. Individuals captured in winter 2012–2013 (open diamonds) had significantly lower body condition from mid- to late hibernation (January through April) when compared to individuals captured during the same period in winter 2013–2014 (black squares). Species acronym code: EPFU, Eptesicus fuscus; MYGR, Myotis grisescens; MYLE, Myotis leibii; MYLU, Myotis lucifugus; MYSE, Myotis septentrionalis; MYSO, Myotis sodalis; PESU, Perimyotis subflavus BERNARD and McCRACKEN 1493 FIGURE 2 Mean body condition (body mass (g)/forearm length (mm)) of seven bat species captured at all five hibernacula. Individuals captured in winter 2012–2013 (open diamonds) had significantly lower body condition from mid- to late hibernation (January through April) when compared to individuals captured during the same period in winter 2013–2014 (black squares). Species acronym code: EPFU, Eptesicus fuscus; MYGR, Myotis grisescens; MYLE, Myotis leibii; MYLU, Myotis lucifugus; MYSE, Myotis septentrionalis; MYSO, Myotis sodalis; PESU, Perimyotis subflavus significant declines were not documented until year 3 of this study, which was nearly 5 years after the first confirmed WNS infections. By the second year of monitoring, bats hibernating at caves with large populations of WNS-affected species exhibited expected disease- related behavioral changes. Bats at Blount cave were recorded flying during the day (Carr, Bernard & Stiver 2014), with the number of calls recorded in April of year 2 equivalent to mid-hibernation levels, indi- cating reduced activity during spring emergence. 5 \| CONCLUSIONS In the southeastern USA, bats are active outside of hibernacula and feed on insects throughout winter regardless of WNS disease status. Activity was found to be strongly correlated with temperature; however, activity during daytime and subfreezing temperatures increased following the confirmation of P. destructans at a site, suggesting that vulnerable bat species in the southeast exhibit behaviors similar to those seen at WNS- infected hibernacula in the northeast. The onset of aberrant behavior and mortality occurred 4–5 years after WNS confirmation, which was substantially later than what has been documented in the North. We found regional differences in the bat faunas affected by WNS. Caves dominated by M. septentrionalis, M. sodalis, and P. subflavus displayed increased aberrant behaviors and reduced activity toward the end of year 3, whereas M. grisescens populations showed no such effects. We also identified differences in how bats prepare for winter, with individu- als in the southeast entering hibernation with lower mean BCI than bats studied in the northeast. Mild winter climates and available insects in the southeast can allow for shorter hibernation and continued foraging, likely reducing the demands of fat storage prior to winter. The regional differences in bat behavior we identified in this study provide a better understanding of the adaptive variations of bats to climatic perturba- tions and diseases. These findings are relevant to how bat populations across North America will react to infection by P. destructans and can allow for more targeted intervention and disease management. Winter activity and capture rates documented at Hawkins, Warren, and White caves, which are dominated by M. grisescens, remained con- sistent throughout our study to pre- or early WNS detection years. We credit the decreases in acoustic activity during the second and third years of sampling to colder temperatures rather than to the effects of WNS. Myotis grisescens are the largest bodied Myotine species in east- ern North America and hibernate in clusters of 100,000 to over one million individuals (USFWS, 2009). Living in large colonies is expected to promote density-dependent transmission of the pathogen through increased contact rates (Ryder et al. 2007; Langwig et al. 2012); how- ever, M. grisescens are currently surviving hibernation without any obvious declines due to WNS (Flock 2014; USFWS, 2014). Studies examining load and prevalence of P. destructans on hibernating bat populations have found that less than 20% of M. grisescens sampled are infected with the fungus (Bernard et al., unpublished; Janicki et al. 2015). Myotis grisescens are the only eastern Myotis species to roost in caves year round (Decher & Choate 1995). They preferentially hiber- nate at temperatures as low as 1–9°C (Tuttle 1976; Tuttle & Kennedy 2005) and roost in dense clusters of more than 1,800 bats/m2 (Gore 1992). Although WNS has been confirmed in M. grisescens, we believe their large body size and relaxed energetic constraints for hibernating through shorter winters may prevent P. destructans from reproducing at disease-inducing levels. While these concepts have yet to be inves- tigated, they could provide insight as to why M. grisescens populations are so far persisting during the WNS epizootic. hibernation period can change sex ratios frequently. Although we net- ted at each site once per month throughout winter, a single sex ratio determination for a hibernacula may not accurately reflect the true value for the entire hibernation season. While sample sizes were small, we saw no obvious gender bias in C. rafinesquii which uses abbreviated torpor during hibernation and continue to forage throughout winter (Johnson et al. 2012), or in L. borealis and L. noctivagans, which hiber- nate in more thermally unstable environments (Table 3). These obser- vations suggest that both sexes of these species may be similarly active in winter in foraging or searching for alternative roosts. 4.2 \| Bat captures and body condition Over the course of two winter seasons, we captured twice as many males as females flying outside of each hibernaculum. One explana- tion for sex bias in captures of bats active in winter is the “thrifty female hypothesis” (Jonasson and Willis 2011). This theory suggests that adult females minimize energy loss by relying more on deep tor- por during hibernation, whereas adult males have more energy to ex- pend and rely less on torpor. The capture of more active males than females is consistent with this idea. However, we did not detect any difference in body condition between males and females, nor did we find any seasonal patterns in the rate of decline in BCI for males versus females, as would be expected if females were remaining in caves to preserve energy. The “thrifty female hypothesis” may best explain sex ratio bias in bats hibernating in more northern latitudes and may func- tion best where winters are more severe and intermittent foraging is not feasible like it is in the southeast. 4.1 \| Winter activity septentrionalis, and P. subflavus). However, changes in acoustic activity at caves dominated by M. grisescens, which are as of yet, little affected, were linked to temperature, not disease. 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DATA ACCESSIBILITY and%20White%20Nose%20Syndrome%20Surveillance.pd Our data are archived on figshare at https://figshare.com/s/4b88aacae 7998650f127. Flock, B. (2014). Bat Population Monitoring and White-Nose Syndrome Surveillance. Retrieved from http://www.tnbwg.org/Files/14-07%20 2014%20Bat%20Hibernacula%20Surveys%20and%20WNS%20 Monitoring.pdf.ff Foley, J., Clifford, D., Castle, K., Cryan, P. M., & Ostfeld, R. S. (2011). Investigating and managing the rapid emergence of white-nose syndrome, a novel, fatal, infectious disease of hibernating bats. Conservation Biology, 25, 223–231. GRSM-2013-SCI-1053; GRSM-2014-SCI-1053) and state (TWRA- 3716; TDEC-2011-031) permits to capture and handle bats at winter hibernacula for this study. Field, K. A., Johnson, J. S., Lilley, T. M., Reeder, S. M., Rogers, J., Behr, M. J., & Reeder, D. M. (2015). The white-nose syndrome transcriptome: Activation of anti-fungal host responses in wing tissue of hibernating little brown Myotis. PLoS Pathogens, 11, e1005168. Flock, B. (2013). Tennessee Bat Population Monitoring and White Nose Syndrome Surveillance. Retrieved from http://www.tnbwg.org/ Files/13-22%202013%20Bat%20Population%20Monitoring%20 and%20White%20Nose%20Syndrome%20Surveillance.pdf. ACKNOWLEDGMENTS Funding for this project was provided by Basically Bats Wildlife Conservation, Inc. White-Nose Syndrome Research Scholarship, Department of Ecology and Evolutionary Biology at the University of Tennessee, and United States Geological Survey. We thank Anna Chow, Max Cox, Neil Giffen, Reilly Jackson, Devin Jones, Kitty McCracken, Mariah Patton, Ana Reboredo-Segovia, and Emma Willcox for help in the field, property access, and field help from Great Smoky Mountain National Park, Tennessee Wildlife Resources Agency (TWRA), Tennessee Chapter of The Nature Conservancy, Tennessee Department of Environmental Conservation, and the US Fish and Wildlife Service. We also acknowledge Megan Nelson (AmeriCorps Intern at GRSM) and Dr. John Zobel (UTK FWF) for their statistical guidance. We obtained both federal (USFWS TE-71613A; An alternative explanation may be due to disproportionate sex ratios of bats within hibernacula (Whitaker & Gummer 2000; Parsons et al. 2003). Previous studies suggest sex ratios at winter sites are biased toward males at more northern roosts and become more evenly dis- tributed the farther south a hibernacula is located, with sites in Florida reported to have an equal distribution of males and females (Davis 1959; Tinkle & Milstead 1960; Elder & Gunier 1978). Samanie Loucks and Caire (2007), however, described hibernating colonies of M. velifer in Oklahoma (n = 42 caves) to be female-biased throughout winter, suggesting seasonal movements of males and females throughout the 1495 BERNARD and McCRACKEN CONFLICT INTEREST None declared. 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W2801133876.txt	https://asthmarp.biomedcentral.com/track/pdf/10.1186/s40733-018-0039-4	en		Single nucleotide polymorphisms in asthma candidate genes TBXA2R, ADAM33 FCER1B and ORMDL3 in Pakistani asthmatics a case control study	Asthma research and practice	2,018	cc-by	4,314	Saba et al. Asthma Research and Practice (2018) 4:4 https://doi.org/10.1186/s40733-018-0039-4 RESEARCH Open Access Single nucleotide polymorphisms in asthma candidate genes TBXA2R, ADAM33 FCER1B and ORMDL3 in Pakistani asthmatics a case control study Nusrat Saba1,3, Osman Yusuf2, Sadia Rehman1, Saeeda Munir1, Amna Noor4, Muhammad Saqlain3, Atika Mansoor1 and Ghazala Kaukab Raja3 Abstract Background: Genetic variations in different loci and genes are important in asthma pathogenesis. There is much importance of various immunological pathways in the IgE secretion regulation. Alterations in any main part of these pathways can increase the risk of asthma development. Polymorphisms in these genetic markers can effect certain pathways which predict the asthma susceptibility. In the present study, SNPs directly or indirectly affecting the immunological process pathways are selected. Methods: This study was conducted to determine association of 16 SNPs in 10 candidate genes with asthma in Pakistani population in 333 asthmatic cases and 220 healthy controls. Genotyping was performed using the Sequenom Mass ARRAY iPLEX platform (14 SNPs) and TaqMan assay (2 SNPs). Results: The minor allele at two of the SNPs showed association with protection from asthma, rs1131882 in TBXA2R gene (OR 0.73, 95% CI 0.52–1.01, P = 0.05) and rs2280091 in the ADAM33 gene (OR 0.69, 95% CI 0.50–0.97, P = 0.03). For FCER1B gene, rs2583476 the asthmatic male gender had higher TT genotype counts as compared to controls (OR = 1.86, 95% CI = 1.09–3.17, p = 0.01). In rs11650680 of ORMDL3 gene the CT genotype is more prevalent in female asthma cases in comparison with female controls (OR = 1.99, 95% CI = 1.02–3.89, p = 0.03). Conclusions: This data suggests that variations at TBXA2R and ADAM33 genes are found to be associated with asthma susceptibility in Pakistan. FCER1B gene is associated with male and ORMDL3 in female asthmatics. These genetic markers can be important source of asthma risk in Pakistani population. Keywords: Asthma, Genetic polymorphisms, Pakistan, Genetic markers Background Asthma is a disease of the lower airways that is remarkably heterogeneous between affected individuals [1]. According to defination of GINA (Global Initiative for Asthma) "asthma is a disorder defined by its clinical, physiological and pathological characteristics. Asthma is the chronic (long-lasting) inflammatory disease of the airways in which many cells and cellular pathways are Correspondence: nusratsaba@yahoo.co.in 1 Institute of Biomedical and Genetic Engineering, G-9/1, Islamabad, Pakistan 3 Department of Biochemistry, Pir Mehar Ali Shah Arid Agriculture University Rawalpindi, Rawalpindi, Pakistan Full list of author information is available at the end of the article involved". In those susceptible to asthma, this inflammation causes the airways to spasm and swell periodically so that the airways narrow. The individual then must wheeze or gasp for air. Obstruction to air flow either resolves spontaneously or responds to a wide range of treatments, but continuing inflammation makes the airways hyper-responsive to stimuli such as cold air, exercise, dust mites, pollutants in the air, and even stress and anxiety. According to recent report of GINA "Asthma is the problem worldwide with an estimated 300 million affected individuals. Nonetheless, based on the standardized methods for assessing asthma symptoms, it appears that global prevalence of asthma ranges from 1-16% of © 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. Saba et al. Asthma Research and Practice (2018) 4:4 the population in different countries". Social and economical factors are integral to understanding asthma and its care, from the perspective of both the individual person with asthma and the healthcare provider. The costs of asthma depends on its prevalence, the individual patient’s level of asthma control, the extent to which exacerbations are avoided, and the cost of medical care and medications. According to the latest WHO data published in may 2014 Asthma Deaths in Pakistan reached 10,817 or 0.96% of total deaths. The age adjusted Death Rate is 10.30 per 100,000 of population ranks Pakistan number 41 in the world. Many biological pathways and genes in those pathways have been implicated in asthma pathogenesis. Variants in over 100 genes have been associated with asthma, but all with small individual effect sizes. It is likely that many genes act in concert to determine individual-specific risks for asthma [2]. The genetic factors add approximately 79% alone in asthma pathogenesis [3]. The causes of asthma are not fully understood. Both genetic and environmental factors are involved, but how these factors interact to confer risk is still largely unknown. We selected 16 single nucleotide polymorphisms (SNPs) in 10 genes for genotyping in Pakistani asthma cases and controls. Page 2 of 6 gene and genome-wide studies, we selected 16 single nucleotide polymorphisms (SNPs) in 10 genes for genotyping in Pakistani asthma cases and controls. The selection of genes is purely rely on the immunological and allergic pathways and have been reported in some populations to be strongly associated with asthma. These SNPs from these genes are selected as these are found to have strong effect in either pathogenesis or protection of asthma in some of closely related populations. And we want to check that this is the same result as previously reported or our population has different association. Quality checks and statistical analyses Hardy-Weinberg equilibrium (HWE) was determined in the entire sample and separately in the cases and controls all the SNPs were in Hardy-Weinberg equilibrium. Multiple logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for each SNP. Linkage disequilibrium (LD) between the 16 SNPs in our sample was determined using matSpD program: (http://gump.qimr.edu.au/general/daleN/matS pD/). There was no LD between SNPs. Therefore all tests were considered to be independent. Results Demographic and genotyping characteristics Methods Patient population and study design The ethical review committee of the parent organization approved this project (ERC-08-01). Written informed consent was obtained from all participants. Asthmatic subjects were recruited from Islamabad and Lahore. The final sample included 333 Pakistani adult subjects with an asthma diagnosis provided by a pulmonologist. Two hundred non-asthmatic healthy controls were recruited from the general population to be similar to the cases with respect to ethnicity and proportions of males and females. Blood sample collection, DNA extraction, and genotyping A venous blood sample was obtained from each study participant, and genomic DNA was extracted from whole blood using a standard phenol chloroform extraction protocol [4]. Fourteen SNPs were genotyped using a Sequenom iPLEX assay and two SNPs were genotyped using TaqMan assays and analyzed on an ABI 7900 HT Fast Real Time PCR (Applied Biosystems, USA). All genotyping was performed at the University of Chicago USA. SNP and gene selection We selected SNPs in genes that are involved and implicated in asthma risk. Genes involved in immunological and allergic pathways are important in asthma pathogenesis. Therefore, based on results of previous candidate The asthma cases included 148 (44.5%) males and 185 (55.5%) females; the mean age was 40 ± SE = 0.93 years. The controls included 88 males (44.0%) males and 112 (56.0%)) females with mean age of 30 ± SE = 0.97 years. The genotyping methods, call rates, minor allele frequencies, and Hardy-Weinberg p-value calculated as a group are shown for all 16 SNPs in Table 1. Allelic and genotypic associations Four SNPs showed evidence for an association with asthma at a p < 0.05. Results of all analyses are shown in Additional file 1: Table S1. The minor alleles at SNPs in two genes, TBXA2R and ADAM33, were more frequent in the controls compared to the asthmatics. The odds ratio (OR) for the minor (A) allele at rs1131882 in TBXA2R was 0.73 (95% CI 0.52– 1.01). There were more AA homozygotes in the controls compared to cases. The OR for the minor (G) allele at rs2280091 in ADAM33 G was 0.69 (95% CI 0.5–0.97). There were more GG homozygotes in controls. Gender based association between SNPs and asthma All age and gender adjusted genotyped data was also explored for gender based disease association. Two of the SNPs showed gender based associations with asthma (Table 2). FCER1B gene SNP rs2583476 showed a significant genotype distribution in males. The homozygous TT and CC genotype frequencies in cases were; TT Saba et al. Asthma Research and Practice (2018) 4:4 Page 3 of 6 Table 1 Single Nucleotide Polymorphisms (SNPs) genotyped in asthma patients GENE Chr location rs # Literature cited Genotyping technique Call Rate Allele (minor/major) MAF HWE p-value ADRB2 5 rs1042713 [19] iPLEX 0.962 A/G 0.432 0.566 HLA-G 6 rs1063320 [20] Taqman 0.993 C/G 0.257 0.090 NOS3 7 rs1799983 [21] iPLEX 0.969 T/G 0.185 0.655 NOS3 7 rs1800779 [22] iPLEX 0.964 G/A 0.197 0.233 FCER1B 11 rs2583476 [23] iPLEX 0.966 C/T 0.480 0.191 NOS1 12 rs2682826 [24] iPLEX 0.968 T/C 0.292 0.327 ORMDL3 17 rs11650680 [25, 26] iPLEX 0.969 T/C 0.220 0.602 GSDMA 17 rs3894194 [27] iPLEX 0.955 T/C 0.491 0.767 ORMDL3 17 rs7216389 [26] Taqman 0.901 C/T 0.413 0.199 ORMDL3 17 rs8079416 [27] iPLEX 0.958 T/C 0.486 0.775 TBXA2R 19 rs1131882 [28] iPLEX 0.964 A/G 0.188 0.247 TFGB1 19 rs1800469 [29] iPLEX 0.957 T/C 0.353 0.022 TBXA2R 19 rs4523 [30, 31] iPLEX 0.955 C/T 0.480 0.380 ADAM33 20 rs2280091 [32] iPLEX 0.944 G/A 0.192 0.060 ADAM33 20 rs528557 [11, 33] iPLEX 0.908 G/C 0.393 0.163 ADAM33 20 rs543749 [11] iPLEX 0.940 T/G 0.189 0.334 a MAF minor allele frequency, HWE Hardy-Weinberg equilibrium a SNP not in HWE so excluded from further analysis 37.5% and CC 24.3% as compared with controls; TT 24% and CC 27.7% respectively. The asthmatic males had higher TT genotype counts as compared to controls (OR = 1.99, 95% CI = 1.02–3.89, p = 0.03). In females, a significant association was observed in rs11650680 (ORMDL3). In case-control data, the CT genotype frequency in female cases is 36% and 23% in control female subjects. The CT genotype is more prevalent in female asthmatics as compared to female control group (OR = 1.86, 95% CI = 1.09–3.17, p = 0.01). Discussion This candidate gene study was conducted in Pakistani asthma patients and healthy controls. This is the first study to report associations between SNPs in candidate genes and asthma in Pakistani population. In the present study, we investigated the association between 16 SNPs in 10 candidate genes (Additional file 1: Table S1) for asthma in the Pakistani population. All of the genes included in the present study were either directly or indirectly involved in pathways affecting the immunological process. Asthma is caused by interaction of multiple genes, some of which have a protective effect and others contribute to the pathogenesis of the disease, with each gene having its own tendency to be influenced by the environment. Thromboxane A2 receptor (TBXA2R) is a potent broncho- and vasoconstrictor and is associated with leukotriene synthesis. It is involved in prostaglandin and leukotriene pathways, and has diverse physiological and pathophysiological actions related to allergies, modulation of acquired immunity, atherogenesis, and neovascularization [5]. Polymorphisms in the TBXA2R gene have been associated with urticaria [6]. This study investigated associations between asthma and a TBXA2R polymorphism. We have found an association between Table 2 Genotype distribution significant among male and female gender in cases and controls and Odds Ratio with p values SNP rs2583476 rs11650680 Gene FCER1B ORMDL3 Genotype Female p-value Male Cases Controls OR(95%CI) Cases Controls OR(95%CI) T/T 42 34 1 54 22 1.99 (1.02–3.89) C/T 102 52 1.59 (0.91–2.79) 55 43 1.04 (0.57–1.89) C/C 38 32 0.96 (0.5–1.85) 35 25 1.13 (0.57–2.25) C/C 106 82 1 91 46 1.53 (0.97–2.42) C/T 65 27 1.86 (1.09–3.17) 48 39 0.95 (0.57–1.59) T/T 8 7 0.88 (0.31–2.54) 9 3 2.32 (0.61–8.85) 0.033 0.01 Saba et al. Asthma Research and Practice (2018) 4:4 rs1131882 (TBXA2R) and asthma in Pakistani population. The A allele and AA genotypes are associated with protection in the Pakistani population. In a study of other variants in this gene in Japanese subjects, TBXA2R alleles were also associated with asthma-related phenotypes [7]. Thromboxane pathways therefore play important roles in airway inflammation and remodeling in asthma patients. For all genotyped SNPs, no gender specific disease association was seen except for FCER1B gene SNP rs2583476. In this SNP the asthmatic male gender had higher TT genotype counts as compared to controls. It is clearly indicated that in our sample population of male gender TT genotype is highly prevalent in asthma patients as compared to healthy controls, whereas CC genotype is not different between males in cases and controls. Gender specific association with this SNP in asthma patients has not been reported in different world populations and this is reported in our population only. Replication in a larger study of male subjects will clarify differences between Pakistani subjects and those of other populations. In ORMDL3 gene SNP rs11650680, a significant association was found with female asthmatics. The heterozygous CT genotype is more prevalent in female asthmatics as compared to female controls. These results clearly show that CT genotype of ORMDL3 gene SNP rs11650680 is highly prevalent in female asthma cases as compared to healthy controls in our study population. A comparison was also made between male and female subjects with regards to genotype frequencies of studied SNPs under case-control model. For FCER1B gene SNP rs2583476 significant difference existed in genotype frequencies among male and female cases (OR = 1.86, 95% CI = 1.09–3.17, p = 0.01). The odds ratio results clearly indicate that male subjects are at higher risk of asthma as compared to female counter-parts. Genotype frequencies also demonstrated a high prevalence of risk alleles in males as compared to females. In the case of ORMDL3 SNP rs11650680, our results indicate that female asthmatic carriers of the CT genotype are at higher risk of disease as compared to male asthmatic subjects (OR = 1.99, 95% CI = 1.02–3.89, p = 0.03). This association with gender has not been reported in other populations and this may be due to our population genetic data is some different from other world population. This point has to be explored further in Pakistani population and needs to be taken into consideration and will be helpful in studying asthma pathogenesis in adults. This is a different and unique result we have found in this study. This may represent an ethnic difference in the genetic makeup of Pakistanis compared to other ethnicity groups. The first strong genetic evidence suggesting that genes in non-allergic, non-immune pathways may play important roles in asthma pathogenesis was the report Page 4 of 6 of ADAM33 as the first positionally-cloned asthma gene [1]. ADAM33 encodes a disintegrin and metalloprotein33 protein that participates in the bronchial remodeling process in asthma [8]. Asthma is a complex disease, but learning more about how SNP mutations in ADAM33 give rise to asthmatic conditions will provide important clues in treating asthma. The ADAM33 gene is expressed in lung fibroblasts and bronchial smooth muscle. Strong associations of rs2280091 (ADAM33) have been demonstrated in Taiwanese [9], Saudi [10], and an Asian population [11]. We also report a relatively strong association with this SNP (recessive model p = 0.005), in which the G allele has a protective effect, in contrast to the abovementioned studies in which the G allele was associated with risk. This difference may be due to different environmental exposures in our subjects or differences in the frequencies of relevant background genes in the Pakistani population and those in previous studies. It has been speculated that ADAM33 may have cytokine stimulating effects, given that other ADAM proteins (ADAM10 and ADAM17) also appear to interact with inflammatory cytokines [12]. Cysteine-rich and EGF domains of ADAM33 have been identified to have a role in cell adhesion and membrane fusion events [13]. These properties of ADAM33 suggest that it might play a role in progression of asthma [14–16]. The lack of a very strong associations in our data could be due to the relatively small size of the study sample and the fact that the subjects in our study were adults whereas most of the previous genetic studies were performed largely in children with asthma [7, 10, 17, 18]. Our findings are important in world genetic pool of asthma cases and will be helpful in further asthma pathogenesis based on these genes. These genes have to be screened fully in asthmatics in further studies instead of few SNPs and this will add information about Pakistani asthmatics in world data. Afterwards, we will be able to tell how Pakistani population genetic makeup is different from other world. Conclusions The present study suggests that variations at TBXA2R and ADAM33 genes are linked with asthma susceptibility in Pakistan. These results are same as found in Caucasians as Pakistani population is closed to it in genetic make up. FCER1B gene is associated with male and ORMDL3 in female asthmatics. Gender based association has not been seen in other studies so this suggest our population genetic data is somewhat different from rest of world. These genetic markers need to be explored fully and will be helpful in studying asthma pathogenesis in adults. These genetic markers can be important source of asthma risk in Pakistani population and needs to be studied in detail with large number of samples. Saba et al. Asthma Research and Practice (2018) 4:4 Additional file Additional file 1: Table S1. Association of Genotype and Allele Frequencies with Asthma Among Cases and Controls. (DOC 81 kb) Page 5 of 6 8. 9. Acknowledgements The authors acknowledge all the participants including cases and controls for providing their sample for research purpose. 10. Funding The research was funded by Institute of Biomedical and Genetic Engineering. 11. Availability of data and materials All data and material what so ever used in the present study is available with the corresponding author and can be reproduced whenever asked. 12. 13. Authors’ contributions NS carried out the molecular genetic studies, participated in the data analysis and drafted the manuscript. OY diagnosed the cases of asthma and helped in sample collection of cases and controls. SR participated in the study design and helped to draft the manuscript. SM participated in the design of the study and helped in molecular genetic studies. AN participated in making draft of the manuscript. MS performed the statistical analysis of data. AM participated in the design of the study and guided in the statistical analysis. GR participated in the design of the study and supervised all the work done. All authors read and approved the final manuscript. Ethics approval and consent to participate The ethical review committee of the parent organization approved this project (ERC-08-01). Written informed consent was obtained from all participants. Consent for publication Written informed consent was obtained from all participants. Competing interests The authors declare that they have no competing interests. Publisher’s Note 14. 15. 16. 17. 18. 19. 20. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 21. Author details 1 Institute of Biomedical and Genetic Engineering, G-9/1, Islamabad, Pakistan. 2 The Allergy and Asthma Institute of Pakistan, 275, Gomal Road, Islamabad E-7, Pakistan. 3Department of Biochemistry, Pir Mehar Ali Shah Arid Agriculture University Rawalpindi, Rawalpindi, Pakistan. 4Rawalpindi Medical College, Rawalpindi, Pakistan. 22. 23. Received: 31 May 2017 Accepted: 5 March 2018 24. References 1. Ober C, Yao TC. The genetics of asthma and allergic disease: a 21st century perspective. Immunol Rev. 2011;242(1):10–30. 2. Nicolae DL, Ober C. (Too) great expectations: the challenges in replicating asthma disease genes. Am J Respir Crit Care Med 2009 179: 1078–1079. 3. Holgate ST, Yang Y, Haitchi HM, Powell RM, Holloway JW, Yoshisue H, Pang YY, Cakebread J, Davies DE. The genetics of asthma ADAM33 as an example of a susceptibility gene. Proc Am Thorac Soc. 2006;3(5):440–3. 4. Sambrook J, MacCallum P, Russell D. Molecular cloning: a laboratory manual. 3rd ed. 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https://openalex.org/W2945842301	http://www.pjms.org.pk/index.php/pjms/article/download/1071/170	English	null	Usage and types of mobile medical applications amongst medical students of Pakistan and its association with their academic performance	Pakistan journal of medical sciences	2,019	cc-by	1,097	1. Dr. Mukhtiar Baig, Prof of Clinical Biochemistry, Medical Educationist, Faculty of Medicine, Rabigh, King Abdulaziz University, Jeddah, Saudi Arabia. doi: https://doi.org/10.12669/pjms.35.3.1071 2. https://statistics.laerd.com/statistical-guides/ independent-t-test-statistical-guide.php 3. Gonzalez-Chica DA, Bastos association: which one is the most appropriate for my study? An Bras Dermatol. 2015;90:523-8. 4. Bjornsen CA, Archer KJ. Relations between college students’ cell phone use during class and grades. Scholarsh Teach Learn Psychol. 2015;1:326. 5. Lepp A, Barkley JE, Karpinski AC. The relationship between cell phone use, academic performance, anxiety, and satisfaction with life in college students. Comput Hum Behav. 2014;31:343-50. * * * * * * * * * * * * 2. https://statistics.laerd.com/statistical-guides/ independent-t-test-statistical-guide.php 2. https://statistics.laerd.com/statistical-guides/ independent-t-test-statistical-guide.php I read with intrest the manuscript by Dr. Aliya Hisam et al entitled “Usage and types of mobile medical applications amongst medical students of Pakistan and its association with their academic performance.”published in March-April 2019 issue of Pakistan Journal of Medica Sciences. It’s a very interesting study and I was particularly more interested because it was very similar to the study published by me.1 p g p p 3. Gonzalez-Chica DA, Bastos association: which one is the most appropriate for my study? An Bras Dermatol. 2015;90:523-8. p g p p 3. Gonzalez-Chica DA, Bastos association: which one is the most appropriate for my study? An Bras Dermatol. 2015;90:523-8. 4. Bjornsen CA, Archer KJ. Relations between college students’ cell phone use during class and grades. Scholarsh Teach Learn Psychol. 2015;1:326. y 5. Lepp A, Barkley JE, Karpinski AC. The relationship between cell phone use, academic performance, anxiety, and satisfaction with life in college students. Comput Hum Behav. 2014;31:343-50. y I have two queries regarding this research. Firstly, the authors mentioned that “Independent sample t-test was applied to assess association between average usage of mobile medical application and academic performance and a p-value of <0.05 was taken as statistically significant.” Literature indicates, “the independent t-test, is an inferential statistical test that determines whether there is a statistically significant difference between the means in two unrelated groups.”2 So I want to ask how did the authors calculate the association by applying Independent sample t-test? For assessing association, we need different types of statistical tests.3 * * * * * * * * * * * * Response from the authors: Thank you for taking interest in the research article. Following is the response to your queries: First query: The students were divided into two groups based on mobile application usage: one group using mobile application routinely and other group not using mobile application routinely. doi: https://doi.org/10.12669/pjms.35.3.1071 Then the groups mean academic performance in the most recent examination was compared among the two groups. About 323 (72%) students whose were routinely using medical application scored 69±7% in their professional examination while 125 (28%) students, who were not using medical application scored 67±9%. The association between average usage of medical application and academic performance was statistically significant (p<0.01). yp Bjornsen et al., (2015) and Lepp et al., (2014) have calculated the association of mobile phone use and students grades by using the correlation and regression analysis.4,5 Secondly, authors have mentioned in the conclusion that “Association between average usage of mobile medical applications and academic performance of the students was statistically significant.” However, in the discussion section, they did not discuss this important finding. Surprisingly, there was not a single sentence mentioned about this. Being a reader, I was curious to know the authors’ viewpoint regarding this association. I could have applied correlation or regression in case I had both variable as continuous that is hours or minutes utilized in using medical mobile application and academic performance (marks). But instead I had two groups i.e. routinely using mobile applications and not routinely using mobile application so t test was applied. (See accompanying Table on next page.) REFERENCES: 1. Sayedalamin Z, Alshuaibi A, Almutairi O, Baghaffar M, Jameel T, Baig M. Utilization of smart phones related medical applications among medical students at King Abdulaziz University, Jeddah: A cross-sectional study. J Infect Pub Health. 2016;9(6):691-697. doi: 10.1016/j. jiph.2016.08.006. Second Query: In discussion section, it would have been good if we had discussed the association of medical mobile application with academic performance but to best of our knowledge we could not find a study to which we could relate our study results and the available ones full text was not approachable. Also because of word count limitation by the Journal we had to skip this point. Pak J Med Sci May - June 2019 Vol. 35 No. 3 www.pjms.org.pk 877 877 Mukhtiar Baig t-test and they labeled that association, which is not correct. Dr. Aliya Hisam Associate Professor, Department of Community Medicine, Army Medical College (AMC), Faculty Member National University of Medical Sciences (NUMS), Rawalpindi, Pakistan. * * * * * * * * * * * * Query 2: As the authors mentioned that “to best of our knowledge we could not find a study to which we could relate our study results and the available ones full text was not approachable,” but the unavailability of related literature is not a hurdle in discussing their finding and probable reasons behind their finding. Furthermore, it would have been better if they had mentioned this point in the discussion that related literature is not available so they cannot correlate their study results with others. * * * * * * * * * * * * Thank you for sharing the response of the authors to my queries related to their published manuscript. However, I am sorry to say their response is not scientifically sound. Authors have also mentioned the word count limitation by the journal for skipping this point. It was one of the main study outcome that was necessary to discuss because they mentioned this outcome in the title and results. Moreover, 3000- 3500 words are sufficient to describe any study results. i y Query 1: For their response to one of the query regarding the use of independent sample t-test to assess association between average usage of mobile medical application and academic performance. REFERENCES: The authors have not provided any reference for improving the knowledge of the readers that describes that independent sample t-test can be applied to assess association between two variables. Prof. Mukhtiar Baig Prof. Mukhtiar Baig pp The results authors have shown in their response letter are also showing the mean difference, not the association. Actually, in the manuscript, authors found the mean difference by applying independent The discussion on this topic is now closed - Chief Editor Pak J Med Sci May - June 2019 Vol. 35 No. 3 www.pjms.org.pk 878
https://openalex.org/W3208680848	https://nottingham-repository.worktribe.com/file/6535827/1/Main%20Document	English	null	Trends in hospital admissions for childhood fractures in England	BMJ paediatrics open	2,021	cc-by	6,195	Original research Original research Open access bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 2021 on November 22, 2021 by guest. Protec http://bmjpaedsopen.bmj.com/ bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from What this study adds? on November 22, 2021 by guest http://bmjpaedsopen.bmj.com/ ber 2021. Downloaded from ► ►Contemporary estimates of incidence for hospital admission for limb, chest, spinal and skull and facial fractures. Received 13 June 2021 Accepted 14 October 2021 ► ►The peak of upper limb fracture incidence is in 5–9 year-old children and for lower limb fractures it is in 10–15 year olds. Results During 2012–2019, 368 120 children were admitted to English NHS hospitals with a fracture. 256 008 (69.5%) were upper limb fractures, 85 737 (23.3%) were lower limb fractures and 20 939 (5.7%) were skull or facial fractures. The annual incidence of upper limb fractures was highest in children aged 5–9 (348.3 per 100 000 children) and the highest incidence of lower limb fractures was in children aged 10–15 (126.5 per 100 000 children). The incidence of skull and facial fractures in preschool (age 0–4) children has been increasing at a rate of 0.629 per 100 000 children per year. ► ►Demonstration of stable or gradually reducing trends of admissions for most fractures except preschool skull fractures. the numbers of children admitted for hospital treatment of fractures. There have been several reports of decreased numbers of children presenting to hospitals with fractures during lockdown restrictions due to the coronavirus pandemic.11 12 These studies typically report absolute numbers of children and are not able to calculate rates of fractures due to uncertainty regarding the population size or denominator number. An evaluation of injury burden and trends in a population with a known denominator is important to allow for optimal service planning both in anticipation of removal of restrictions and for future years. Implications The annual incidence of hospital admission for fractures in children has been shown to be consistent for several fracture types between 2012 and 2019. An increasing trend of admissions with preschool skull fractures was observed, though the study data do not have sufficient granularity to demonstrate if this is due to changes in practice or to accidental or non-accidental causes. © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. What this study adds? 1Orthopaedics and Trauma, University of Nottingham School of Medicine, Nottingham, UK 2Nottingham Children’s Hospital, Nottingham University Hospitals NHS Trust, Nottingham, UK 3Children and Young People Health Research, School of Health Sciences, University of Nottingham, Nottingham, UK 4Clinical Trials Unit, University of Nottingham School of Medicine, Nottingham, UK Correspondence to Mr Ben Arthur Marson; ben. marson@nottingham.ac.uk To cite: Marson BA, Manning JC, James M, et al. Trends in hospital admissions for childhood fractures in England. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/ bmjpo-2021-001187 Trends in hospital admissions for childhood fractures in England Ben Arthur Marson ‍ ‍ ,1 Joseph C Manning David J Bryson,2 Benjamin J Ollivere1 Ben Arthur Marson ‍ ‍ ,1 Joseph C Manning David J Bryson,2 Benjamin J Ollivere1 Ben Arthur Marson ‍ ‍ ,1 Joseph C Manning ‍ ‍ ,2,3 Marilyn James,4 Adeel Ikram,1 David J Bryson,2 Benjamin J Ollivere1 on November 22, 2021 by guest. Protected by copyright. http://bmjpaedsopen.bmj.com/ 0.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from To cite: Marson BA, Manning JC, James M, et al. Trends in hospital admissions for childhood fractures in England. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/ bmjpo-2021-001187 What is known about the subject? ABSTRACT Purpose Fractures to the axial and appendicular skeleton are common in children causing loss of opportunities and disability. There are relatively few studies available to quantify the number of children who have their fractures diagnosed in the emergency department and are then admitted to hospital for ongoing management. The purpose of this study is to explore trends of frequency, types and age of children sustaining fractures who were admitted for intervention to National Health Service (NHS) hospitals. Design The study uses data from the Hospital Episode Statistics and Office for National Statistics from 2012 to 2019 to calculate the annual incidence of hospital admission for limb, spine, facial and skull fractures per 100 000 children. ► ►Fractures in children are common with more hospital admissions for upper limb than lower limb fractures. ► ►Fractures in children are common with more hospital admissions for upper limb than lower limb fractures. ► ►Previous estimates of hospital admissions following childhood fractures are limited by a lack of robust denominator. ► ►Additional supplemental material is published online only. To view, please visit the journal online (http://dx.doi.org/ 10.1136/bmjpo-2021-001187). on November 22, 2021 by http://bmjpaedsopen.bmj.com/ 0 November 2021. Downloaded from METHODS Research ethics approval was not required for this study as it was a secondary analysis of publicly accessible data. Open access records for finished consultant episodes were retrieved from NHS Digital from 2012 to 2020 on 20 February 2021 (https://digital.nhs.uk). Each consul- tant episode is coded by independent hospital coders with diagnoses from the International Classification of Diseases, 10th revision classification system and interven- tions were coded using the Office of Population Services and Censuses, 5th edition code book.13 14 During the study period, 368 120 admissions were recorded with a primary diagnosis of a fracture. These included 256 008 upper limb fractures, 85 737 lower limb fractures, 20 939 skull or facial fractures, 4542 spinal frac- tures and 894 chest fractures. The breakdown of these fractures into body area is shown in table 2. Across all age groups, upper limb fractures were the most frequent fracture type accounting for 26.2%–43.6% of fractures. on November 22, 2021 http://bmjpaedsopen.bmj.com/ n 10 November 2021. Downloaded from The codes for acute fractures were classified into body regions (upper limb: clavicle, scapula, humerus, forearm, hand. Lower limb: pelvis, hip, femur, patella, lower leg and foot. Spine: cervical spine, thoracic spine, lumbar spine and sacral spine. Chest: sternum or ribs). Primary procedures were identified and classified according to type of reduction and fixation method (see online supplemental file 1). Patients without a primary diag- nosis of fracture were excluded to minimise duplications. The annual injury burden was calculated using the appropriate population estimate for different ages of children from the Office of National Statistics NOMIS service (https://www.nomisweb.co.uk/). The codes for acute fractures were classified into body regions (upper limb: clavicle, scapula, humerus, forearm, hand. Lower limb: pelvis, hip, femur, patella, lower leg and foot. Spine: cervical spine, thoracic spine, lumbar spine and sacral spine. Chest: sternum or ribs). Primary procedures were identified and classified according to type of reduction and fixation method (see online supplemental file 1). Patients without a primary diag- nosis of fracture were excluded to minimise duplications. yp g Fracture incidence was calculated per 100 000 PY. The trends for upper limb, lower limb, skull and facial frac- tures and spinal fractures are shown in figure 1. Skull and facial fractures in children aged 0–4 were demonstrated to be increasing during the study window with an annual increase of 0.629 per 100 000 PY (95% CI 0.367 to 0.891, p=0.0011). INTRODUCTION Fractures in children are a common pres- entation to the emergency department, with overall estimated annual incidence rates of 1500–3600 fractures per 100 000 children per year.1–5 Fractures in childhood cause pain and loss of opportunity.6–8 Most children contained within these series are managed in the community. However, some children require hospital admission and treatment. Admission to hospital is distressing, which is amplified if the child requires surgery,9 10 yet there is relatively little information regarding 22, 2021 by guest. Protected by copyright. The National Health Service Hospital Episode Statistics (HES) is a national dataset detailing all completed consultant episodes in England. This includes emergency care where children are admitted to hospital. The dataset does not capture details of children who are treated exclusively in the community, or for whom a fracture is identified in the emergency department and then managed in a fracture clinic. This is therefore an ideal dataset to evaluate the volume of children uest. Protected by copyright. Correspondence to Mr Ben Arthur Marson; ben. marson@nottingham.ac.uk Correspondence to Mr Ben Arthur Marson; ben. marson@nottingham.ac.uk 1 Marson BA, et al. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/bmjpo-2021-001187 Open access bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 202 Patient involvement admitted to hospitals in England as children with frac- tures are almost exclusively treated within the National Health Service (NHS). Patient involvement was not deemed to be appropriate for the design or delivery of this study as an analysis of open-source data. The aim of this study is to evaluate the trends of chil- dren admitted to English hospitals since 2012. To do this, annual incidence of fractures will be calculated and analysed to identify current trends and by evaluating the trends in operative management of paediatric fractures during this period. on November 22, 2021 by guest. Protected by copyright. http://bmjpaedsopen.bmj.com/ hed as 10.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from RESULTS The population of children living in England has increased from 12 094 205 in 2012 to 12 642 441 in 2019. The trends in population change were not uniform across all age groups. There has been an increase in the popula- tion of children aged 5–9 and 10–15 years, a decrease in the population of children aged 16–18 and no significant change in the population of children aged 0–4 (table 1). The population of children living in England has increased from 12 094 205 in 2012 to 12 642 441 in 2019. The trends in population change were not uniform across all age groups. There has been an increase in the popula- tion of children aged 5–9 and 10–15 years, a decrease in the population of children aged 16–18 and no significant change in the population of children aged 0–4 (table 1). Marson BA, et al. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/bmjpo-2021-001187 on November 22, 2021 http://bmjpaedsopen.bmj.com/ bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from was higher than the combined rate of hip and femoral fractures. The incidence of different fractures is broken down by specific body region in table 3. In all age groups, the highest incidence of fractures was experienced in the upper limb with the forearm contributing the largest proportion of fractures in children aged 0–15. The peak incidence of forearm and humeral fractures was found in younger children while hand fractures were more common in older children. The lowest mean incidence of lower limb fractures was in younger children (46.7 per 100 000 PY). Femoral fractures were the most common lower limb fracture in preschool children, whereas tibial and ankle fractures were most common in all other age groups. The incidence of hip fractures is highest in older children and pelvic fractures highest in adolescents. The incidence of admissions for skull and facial bone fractures was lowest in younger children with a mean inci- dence of 7.5 per 100 000 PY for children aged 5–9. The highest incidence was for adolescents at 50.1 per 100 000 PY. Chest fractures (rib or sternum) were rare through all age groups with a maximum mean incidence of 2.3 per 100 000 PY for adolescents. The trends in the rates for surgical management of childhood fractures and head injuries are shown in figure 2. There was a peak of manipulative manage- ment of fractures seen between 2013 and 2016 in chil- dren aged 5–15 which mirrors the peak in admissions for upper limb fracture incidence identified in the same time period. Linear regression analysis identified a statis- tically significant decrease in the rates of open reduction and fixation in younger children (annual decrease of 2.3 per 100 000 PY, 95% CI 1.6 to 2.9, p=0.0002) and adoles- cents (annual decrease 2.5 per 100 000 PY, 95% CI 1.5 to 3.6, p=0.0010) and in closed reduction±fixation of older children (annual decrease 6.6 per 100 000 PY, 95% CI 0.2 to 13.0, p=0.0446) and adolescents (annual decrease 2.5, ber 22, 2021 by guest. Protected by copyright. est. Protected by copyright. Spine fractures were rare in preschool and younger children with a mean incidence of 0.4 per 100 000 PY for children aged 0–4 and 0.9 per 100 000 PY for chil- dren aged 5–9. There was a similar number of thoracic and lumber spine fractures. on November 22, 2021 http://bmjpaedsopen.bmj.com/ bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from Table 2 Distribution of fractures admitted to hospitals in England between 2012 and 2019 according to age of child. Numbers shown are absolute numbers of consultant episodes and proportion of total fracture load Table 2 Distribution of fractures admitted to hospitals in England between 2012 and 2019 according to age of child. Numbers shown are absolute numbers of consultant episodes and proportion of total fracture load Table 2 Distribution of fractures admitted to hospitals in England between 2012 and 2019 according to age of child. Numbers shown are absolute numbers of consultant episodes and proportion of total fracture load Body region Preschool (age 0–4) Younger children (age 5–9) Older children (age 10–15) Adolescents (age 16–18) Upper limb 39 864 (32.7%) 93 648 (43.6%) 94 988 (34.8%) 27 508 (26.2%) Forearm 18 507 (15.2%) 60 950 (28.4%) 62 797 (23.0%) 8576 (8.2%) Humerus 14 820 (12.2%) 24 606 (11.5%) 8600 (3.2%) 1728 (1.6%) Hand 3809 (3.1%) 7610 (3.5%) 20 940 (7.7%) 14 475 (13.8%) Clavicle 2678 (2.2%) 399 (0.2%) 2403 (0.9%) 2544 (2.4%) Scapula 9 (0.0%) 9 (0.0%) 100 (0.0%) 126 (0.1%) Upper limb, unspecified 9 (0.0%) 58 (0.0%) 95 (0.0%) 22 (0.0%) Shoulder, unspecified 32 (0.0%) 16 (0.0%) 53 (0.0%) 37 (0.0%) Lower limb 17 292 (14.2%) 12 497 (5.8%) 37 622 (13.8%) 18 326 (17.4%) Tibia/fibula 7670 (6.3%) 8088 (3.8%) 28 470 (0.4%) 12 501 (11.9%) Femur 8382 (6.9%) 2804 (1.3%) 2991 (1.1%) 1856 (1.8%) Foot (not calcaneum or talus) 613 (0.5%) 875 (0.4%) 2418 (0.9%) 1630 (1.6%) Hip 522 (0.4%) 316 (0.1%) 1244 (0.5%) 346 (0.3%) Pelvis 56 (0.0%) 143 (0.1%) 936 (0.0%) 848 (0.8%) Patella 13 (0.0%) 166 (0.1%) 1126 (6.3%) 654 (0.6%) Talus 10 (0.0%) 42 (0.0%) 239 (0.1%) 275 (0.3%) Calcaneum 26 (0.0%) 63 (0.0%) 198 (0.1%) 216 (0.2%) Spine 112 (0.1%) 249 (0.1%) 1451 (0.0%) 2730 (2.6%) Thoracic spine 18 (0.0%) 106 (0.0%) 599 (0.2%) 911 (0.9%) Lumbar spine 11 (0.0%) 69 (0.0%) 479 (0.2%) 1058 (1.0%) Cervical spine 80 (0.1%) 56 (0.0%) 294 (0.1%) 555 (0.5%) Sacral spine 3 (0.0%) 18 (0.0%) 79 (0.0%) 206 (0.2%) Skull and facial bones 6970 (5.7%) 2025 (0.9%) 4303 (1.6%) 7641 (7.3%) Chest 256 (0.2%) 64 (0.0%) 220 (0.1%) 354 (0.3%) http://bmjpa first published as 10.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from on November 22, 2021 http://bmjpaedsopen.bmj.com/ 0 November 2021. Downloaded from demonstrate an increase or decrease in incidence rates for other fracture types. Marson BA, et al. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/bmjpo-2021-001187 METHODS The incidence was found to be decreasing for five groups of fractures. These are: (1) upper limb fractures in adolescents (annual decrease of 3.49 per 100 000 PY, 95% CI 1.944 to 5.036, p=0.0015); (2) lower limb fractures in preschool children (annual decrease 1.862 per 100 000 PY, 95% CI 1.222 to 2.502, p=0.0004); (3) lower limb fractures in younger children (annual decrease 1.773 per 100 000 PY, 95% CI 1.249 to 2.296, p=0.0002); (4) skull and facial fractures in adolescents (annual decrease 0.567 per 100 000 PY, 95% CI 0.208 to 0.926, p=0.0083) and (5) skull fractures in adolescents (annual decrease 1.03 per 100 000 PY, 95% CI 0.026 to 2.034, p=0.046). There was insufficient evidence to The annual injury burden was calculated using the appropriate population estimate for different ages of children from the Office of National Statistics NOMIS service (https://www.nomisweb.co.uk/). Incidence rates were calculated per 100 000 person years (PY) for all children and subdivided into four age groups (preschool 0–4, young children 5–9, older chil- dren 10–15 and adolescence 16–18). Annual trends were in incidence were visualised in Graphpad 7.04. Trends in injury incidence were fitted to a linear regression model with a significance level of <0.05. 2 Marson BA, et al. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/bmjpo-2021-001187 Table 1 Changes in demographics from the start and end of the study window Age group 2012 population (% total) 2019 population (% total) Annual change* (95% CI) P value Preschool (0–4) 3 393 356 (28.1) 3 299 637 (26.1) −13 533 (−27 441 to 374) 0.055 Younger children (5–9) 3 083 582 (25.5) 3 538 206 (28.0) 66 762 (52 433 to 81 092) <0.0001 Older children (10–15) 3 653 288 (30.2) 3 978 836 (31.5) 49 491 (20 402 to 78 580) 0.0059 Adolescents (16–18) 1 963 979 (16.2) 1 825 762 (14.4) −20 960 (−26 060 to −15 861) <0.0001 *Annual change calculated using a linear regression model. Table 1 Changes in demographics from the start and end of the study window 2 Open access on November 22, 2021 http://bmjpaedsopen.bmj.com/ bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from The highest incidence of cervical spine fractures was found in adolescents with a rate of 16.7 (95% CI 16.1 to 17.3) per 100 000 PY. This 3 Marson BA, et al. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/bmjpo-2021-001187 Open access Figure 1 Trends of fracture incidence for upper limb, lower limb, skull and facial fractures and spinal fractures between 2012 and 2019. Data presented separated by the age groups of children with trends and 95% CIs calculated using a linear regression model. a constant trend for most fractures, with a marginal decreasing trend in admissions for upper limb fractures in children aged 16–18, lower limb fractures in children aged 0–9 and skull fractures in children aged 10–18. g g It is not clear what has been driving this decrease in limb and skull fracture admissions in these groups. It is possible that national safety campaigns are reducing the injury burden on hospitals. The Department of Trans- port reported that 2019 had the lowest number of child and young adult casualties and fatalities following road traffic accidents which may be associated with changing demographics of road users and the influence of safety campaigns including ‘Think!’.15 16 This may also be asso- ciated with an increase in sedentary lifestyles, though more work would be required to confirm this. Unfor- tunately, in the same time period, it has been identified that there has been a steady rise in penetrating trauma caused by knife crime, which with childhood admissions for knife wounds at the highest point in 2019 compared with 2012.17 on November 22, 2021 b http://bmjpaedsopen.bmj.com/ November 2021. Downloaded from on November 22, 2021 by gu http://bmjpaedsopen.bmj.com/ vember 2021. Downloaded from The trend in upper limb fractures did not show a significant change. However, inspection of the graph shows that there was a peak in the number of admissions 2014–2015 which corresponds to a peak in the number of closed reductions. There has been an increasing aware- ness of non-surgical management of limb fractures and emergency department manipulation which avoid inpa- tient admission.18–22 Importantly, such fractures and their management would not be captured in this data. Figure 1 Trends of fracture incidence for upper limb, lower limb, skull and facial fractures and spinal fractures between 2012 and 2019. Data presented separated by the age groups of children with trends and 95% CIs calculated using a linear regression model. on November 22, 2021 http://bmjpaedsopen.bmj.com/ bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from g p The mean incidence of hospitalisation for childhood fractures was 374.8 per 100 000 children. This is lower than the 623.3 per 100 000 reported from Australia in the preceding decade (2002–2012).23 Part of this discrepancy may be that this previous study included admissions for nasal bone fractures, which contrib- uted to 30% of the included facial fractures. The study from Australia also shows a decline in the number of admissions per 100 000 during the 10 years surveyed, which may have continued into the next decade.23 We have found a higher proportion of lower limb fractures admitted to hospitals in England than in the Australian cohort. This may be due to an increasing trend in the UK of management of many upper limb fractures in the community. 95% CI 1.5 to 3.6, p=0.0010). A decrease in skull or facial fracture surgery was demonstrated for younger children with an annual decrease of 0.13 (95% CI 0.0 to 0.2) per 100 000 PY and for adolescents with an annual decrease of −1.2 (95% CI 0.8 to 1.6) per 100 000 PY. No change in operative rates was found for spinal surgery in any age group or skull and facial surgery in preschool children. Marson BA, et al. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/bmjpo-2021-001187 on November 22, 2021 by gue http://bmjpaedsopen.bmj.com/ bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from Table 3 Mean incidence of fractures per 100 000 person years (PY) admitted to hospitals in England between 2012 and 2019 according to age of child according to age of child Body region Preschool (age 0–4) Younger children (age 5–9) Older children (age 10–15) Adolescents (age 16–18) Upper limb 146.9 348.3 319.9 180.4 Forearm 68.2 226.9 211.6 56.3 Humerus 54.6 91.3 28.9 11.3 Hand 14.0 28.3 70.5 94.9 Clavicle 9.9 1.5 8.1 16.7 Scapula <0.1 <0.1 0/3 0.8 Upper limb, unspecified <0.1 0.2 0.3 0.1 Shoulder, unspecified 0.1 0.1 0.2 0.2 Lower limb 63.7 46.7 126.5 120.3 Tibia/fibula 28.2 30.2 95.7 82.1 Femur 30.9 10.4 10.1 12.2 Foot (not calcaneum or talus) 2.3 3.3 8.1 10.7 Hip 1.9 1.2 4.2 2.3 Pelvis 0.2 0.5 3.1 5.6 Patella <0.1 0.6 3.8 4.3 Talus <0.1 0.2 0.8 1.8 Calcaneum 0.1 0.2 0.7 1.4 Spine 0.4 0.9 4.9 17.9 Thoracic spine 0.1 0.4 2.0 6.0 Lumbar spine <0.1 0.3 1.6 7.0 Cervical spine 0.3 0.2 1.0 16.7 Sacral spine <0.1 0.1 0.3 1.4 Skull and facial bones 25.7 7.5 14.5 50.1 Chest 0.9 0.2 0.7 2.3 on November 22, 2021 by http://bmjpaedsopen.bmj.com/ 0 November 2021. Downloaded from many papers identifying fewer children attending emer- gency departments for treatment following fractures during the pandemic.11 12 28–30 This is likely to be due to lockdown policies limiting the availability of opportuni- ties to participate in sport and adventurous play though in one series from London a reduction in non-surgical fractures was demonstrated without a corresponding decrease in surgical cases, suggesting that some more severe injuries were still occurring.31 What is unclear is how the rates of injuries will respond to the relaxation of lockdown rules over the next 12–24 months and if the suppressed number of admissions will continue or if there will be a rebound back to (or exceeding) the rates identified in this current study. A marginal increase in the incidence of admissions for preschool head and facial fractures was observed in the data. While mathematically significant, this represents an increase of only 19 admissions per year in a preschool population of 3.3 million. In this cohort, 83.5% of the fractures sustained by preschool children were to the base of skull or the skull vault with relatively few facial fractures. Marson BA, et al. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/bmjpo-2021-001187 DISCUSSION y General practice databases have been used to develop estimates for fracture incidence in the UK. The Health Improvement Network research database from 495 of the 8228 general practitioners in the UK has been used to estimate the incidence of fractures in the under 5 popu- lation as 758 per 100 000 PY (95% CI 748–769). Moon et al used the Clinical Practice Research Datalink database from 1988 to 2012 (which covers 6.9% of the UK popula- tion) to estimate a national incidence of 1370 per 100 000 PY.24 25 Given that we have not found any major changes in the trends of fracture admissions since 2012 then it seems that approximately 30% of UK fractures result in hospital admission, however, the inpatient focus of the data for this study precludes a precise estimate. There is very little contemporary literature describing the trends of hospital admissions due to childhood frac- tures. This study has demonstrated trends in fracture patterns, distributions and treatment strategies that are valuable for exploration in future research and in plan- ning healthcare prioritisation. 22, 2021 by guest. Protected by copyright. While the dataset used has several advantages, including the national coverage of all NHS funded admissions and procedures, the most significant limita- tion is a lack of granularity within this dataset beyond number of finished consultant episodes and numbers of procedures. The data do not extend to the community rates of fractures, or for fractures treated exclusively in the emergency department. However, they demonstrate 4 Marson BA, et al. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/bmjpo-2021-001187 Open access REFERENCES this strategy has allowed us to present the primary reason for admission, accepting this limitation. As for all data- base studies, there will be an error rate associated with the input of data into the HES registry. However, the HES database is externally audited and validated by NHS Digital, with 100% of finished consultant episodes being maintained with a primary diagnosis code in the included time period.34 In this period, many of these confounders would be expected to occur at a constant rate, and while there may be an impact on the absolute values the trend results should not be grossly impacted. 1 Landin LA. Fracture patterns in children. Analysis of 8,682 fractures with special reference to incidence, etiology and secular changes in a Swedish urban population 1950-1979. Acta Orthop Scand Suppl 1983;202:1–109. 1 Landin LA. Fracture patterns in children. Analysis of 8,682 fractures with special reference to incidence, etiology and secular changes in a Swedish urban population 1950-1979. Acta Orthop Scand Suppl 1983;202:1–109. 2 Tiderius CJ, Landin L, Düppe H. Decreasing incidence of fractures in children: an epidemiological analysis of 1,673 fractures in Malmö, Sweden, 1993-1994. Acta Orthop Scand 1999;70:622–6. 3 Kang MS, Kim H-S. Characteristics and trends of traumatic injuries in children visiting emergency departments in South Korea: a retrospective serial cross-sectional study using both nationwide- sample and single-institutional data. PLoS One 2019;14:e0220798. Å 3 Kang MS, Kim H-S. Characteristics and trends of traumatic injuries in children visiting emergency departments in South Korea: a retrospective serial cross-sectional study using both nationwide- sample and single-institutional data. PLoS One 2019;14:e0220798. Å y g ample and single-institutional data. PLoS One 2019;14:e0 Å p g 4 Bergman E, Lempesis V, Nilsson Jan-Åke, et al. Time trends in pediatric fractures in a Swedish City from 1950 to 2016. Acta Orthop 2020;91:598–604. 5 Orces CH, Orces J. Trends in the U.S. childhood emergency department visits for fall-related fractures, 2001-2015. Cureus 2020;12:e11629. 5 Orces CH, Orces J. Trends in the U.S. childhood emergency department visits for fall-related fractures, 2001-2015. Cureus 2020;12:e11629. g y p Despite these limitations, this study has shown a decreasing trend of admissions for many childhood frac- tures in most age groups, except preschool skull fractures and adolescent spinal fractures, which are increasing. These trends can be used to benchmark service provi- sion for anticipated hospital volume following easing of lockdown restrictions if childhood behaviour returns to 2018–2019 activities. ORCID iDs Ben Arthur Marson http://orcid.org/0000-0002-9264-537X Joseph C Manning http://orcid.org/0000-0002-6077-4169 on November 22, 2021 by gue http://bmjpaedsopen.bmj.com/ bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from BJO contributed to study conceptualisation, analysis, editing of final draft, supervision of trainees. As guarantor, BJOaccepts full responsibility for the work and/or the conduct of the study, had access to the data, and controlled the decision to publish Funding This study was funded by Research Trainees Coordinating Centre (NIHR300240). Competing interests None declared. Patient consent for publication Not applicable. Patient consent for publication Not applicable. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data are available upon reasonable request. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. on November 22, 2021 by http://bmjpaedsopen.bmj.com/ November 2021. Downloaded from Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. Figure 2 Trends of fracture interventions for limb open or closed reductions, skull and facial fractures and spinal fractures between 2012 and 2019. Data presented separated by the age groups of children with trends and 95% CIs calculated using a linear regression model. Abbreviations: CR closed reduction. ORIF open reduction internal fixation on November 22, 2021 by gue http://bmjpaedsopen.bmj.com/ bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from While this dataset provides insufficient granu- larity to evaluate the mechanism of injury, it is possible that this increase may represent an increase in diagnosis of fractures following non-accidental injury. Also, it could reflect changes in patterns of investigations, for example, skull CT, and management. Robust evidence for trends in non-accidental injury is difficult to obtain, but a rise in non-accidental injury has been documented in the USA with 95% of children involved age <5 years26.27 Alterna- tively, there may be an increased awareness of the impact of serious head injuries in this age group and thus a lower barrier to admissions driving this change. on November 22, 2021 by guest. Protected by copyright. edsopen.bmj.com/ 2, 2021 by guest. Protected by copyright. Due to the nature of the study design, there remain some limitations. The use of finished consultant episodes has been used previously to estimate the number of admissions attributed to injuries and musculoskel- etal pathology.32 33 During the coding process, each patient episode is assigned up to 24 diagnostic codes. By analysing only primary diagnosis code, it is possible that the estimates in this study may have undercounted where patients with multiple injuries have presented. However, est. Protected by copyright. This study has not included any hospital admissions from January 2020 onwards. The international corona- virus pandemic has been extensively documented to have caused significant disruption to emergency care, with 5 Marson BA, et al. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/bmjpo-2021-001187 Open access bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 202 Figure 2 Trends of fracture interventions for limb open or closed reductions, skull and facial fractures and spinal fractures between 2012 and 2019. Data presented separated by the age groups of children with trends and 95% CIs calculated using a linear regression model. Abbreviations: CR closed reduction. ORIF open reduction internal fixation trends observed and to develop safety strategies to safe- guard infants and adolescents from these injuries. Twitter Joseph C Manning @josephcmanning Contributors BAM contributed to data acquisition, analysis, writing of first draft. JCM contributed to study conceptualisation, analysis, editing of final draft, supervision of trainee. MJ contributed to study conceptualisation, analysis, editing of final draft, supervision of trainee. AI contributed to data acquisition, analysis, editing of final draft. DB contributed to study conceptualisation, analysis, contextualisation of study, editing of final draft, supervision of trainees. Marson BA, et al. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/bmjpo-2021-001187 on November 22, 2021 http://bmjpaedsopen.bmj.com/ bmjpo: first published as 10.1136/bmjpo-2021-001187 on 10 November 2021. Downloaded from 23 Faris M, Lystad RP, Harris I, et al. Fracture-related hospitalisations and readmissions of Australian children ≤16 years: a 10-year population-based cohort study. Injury 2020;51:2172–8. 23 Faris M, Lystad RP, Harris I, et al. Fracture-related hospitalisations and readmissions of Australian children ≤16 years: a 10-year population-based cohort study. 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Available: https://datadictionary.nhs.uk/supporting_information/ opcs_classification_of_interventions_and_procedures.html [Accessed 30 Apr 2021].i ; 26 Rosenfeld EH, Johnson B, Wesson DE, et al. Understanding non- accidental trauma in the United States: a national trauma databank study. J Pediatr Surg 2020;55:693–7. 14 Organization WH. ICD-10: international statistical classification of diseases and related health problems: tenth revision. 2nd. Geneva: World Health Organization, 2004. y g 27 Thompson LW, Bass K, Agyei J. Rising incidence of non-accidental trauma in infants: a call to revisit prevention strategies. Pediatrics 2018;142:749–49. g 15 Murphy A. Reported road casualties in Great Britain: 2019 annual report. London: Department for Transport, 2020. 28 Bram JT, Johnson MA, Magee LC, et al. Where have all the fractures gone? the epidemiology of pediatric fractures during the COVID-19 pandemic. J Pediatr Orthop 2020;40:373–9.i p p p 16 Transport DF. think! 2021. Available: https://www.think.gov.uk/ [Accessed 30 Apr 2021].i p p 29 Nabian MH, Vosoughi F, Najafi F, et al. Epidemiological pattern of pediatric trauma in COVID-19 outbreak: data from a tertiary trauma center in Iran. Injury 2020;51:2811–5. 17 Allen G, Kirk-Wade E. Knife crime in England and Wales. Briefing paper. London: House of Commons Library, 2020. 18 Kurien T, Price KR, Pearson RG, et al. Marson BA, et al. BMJ Paediatrics Open 2021;5:e001187. doi:10.1136/bmjpo-2021-001187 REFERENCES There is also a need to ensure adequate provision of trained trauma staff in emergency departments to provide skilled manipulation to maintain and continue downward limb fracture admission trends. Additional work is required to evaluate the causes of the 6 Ding R, McCarthy ML, Houseknecht E, et al. The health-related quality of life of children with an extremity fracture: a one-year follow-up study. J Pediatr Orthop 2006;26:157–63. 6 Ding R, McCarthy ML, Houseknecht E, et al. 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https://openalex.org/W2128899090	https://amu.hal.science/hal-01774653/document	English	null	Multispectral texture characterization: application to computer aided diagnosis on prostatic tissue images	EURASIP Journal on Advances in Signal Processing	2,012	cc-by	9,895	Multispectral texture characterization: application to computer aided diagnosis on prostatic tissue images Riad Khelifi, Mouloud Adel, Salah Bourennane To cite this version: Riad Khelifi, Mouloud Adel, Salah Bourennane. Multispectral texture characterization: application to computer aided diagnosis on prostatic tissue images. EURASIP Journal on Advances in Signal Processing, 2012, 10.1186/1687-6180-2012-118. hal-01774653 To cite this version: Riad Khelifi, Mouloud Adel, Salah Bourennane. Multispectral texture characterization: application to computer aided diagnosis on prostatic tissue images. EURASIP Journal on Advances in Signal Processing, 2012, 10.1186/1687-6180-2012-118. hal-01774653 Multispectral texture characterization: application to computer aided diagnosis on prostatic tissue images Riad Khelifi, Mouloud Adel, Salah Bourennane Riad Khelifi, Mouloud Adel, Salah Bourennane. Multispectral texture characterization: application to computer aided diagnosis on prostatic tissue images. EURASIP Journal on Advances in Signal Processing, 2012, 10.1186/1687-6180-2012-118. hal-01774653 Distributed under a Creative Commons Attribution 4.0 International License REVIEW Open Access © 2012 Khelifi 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. Multispectral texture characterization: application to computer aided diagnosis on prostatic tissue images Riad Khelifi, Mouloud Adel and Salah Bourennane* Abstract Various approaches have been proposed in the literature for texture characterization of images. Some of them are based on statistical properties, others on fractal measures and some more on multi-resolution analysis. Basically, these approaches have been applied on mono-band images. However, most of them have been extended by including the additional information between spectral bands to deal with multi-band texture images. In this article, we investigate the problem of texture characterization for multi-band images. Therefore, we aim to add spectral information to classical texture analysis methods that only treat gray-level spatial variations. To achieve this goal, we propose a spatial and spectral gray level dependence method (SSGLDM) in order to extend the concept of gray level co-occurrence matrix (GLCM) by assuming the presence of texture joint information between spectral bands. Thus, we propose new multi-dimensional functions for estimating the second-order joint conditional probability density of spectral vectors. Theses functions can be represented in structure form which can help us to compute the occurrences while keeping the corresponding components of spectral vectors. In addition, new texture features measurements related to (SSGLDM) which define the multi-spectral image properties are proposed. Extensive experiments have been carried out on 624 textured multi-spectral images for use in prostate cancer diagnosis and quantitative results showed the efficiency of this method compared to the GLCM. The results indicate a significant improvement in terms of global accuracy rate. Thus, the proposed approach can provide clinically useful information for discriminating pathological tissue from healthy tissue. Keywords: texture characterization, multi-band texture images, spatial/spectral co-occurrence matrix, texture fea- tures, prostate cancer, classification accuracy HAL Id: hal-01774653 https://amu.hal.science/hal-01774653v1 Submitted on 25 Apr 2018 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 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 * Correspondence: salah.bourennane@fresnel.fr Groupe GSM, Institut Fresnel, Ecole Centrale Marseille D. U. de Saint Jérôme Av. Escadrille Normandie, 13397, Marseille, France 1. Introduction have been proposed with success: description through wavelet coefficient statistics [3], the Markov random field [4] or Markov chain [5] models. Nevertheless, tex- ture analysis remains a difficult problem when applied to color [6], multi or hyperspectral images; where image pixel takes its values in a multidimensional space. For this purpose, a great deal of work has been done for modeling the multispectral and hyperspectral texture analysis [7-12]. Recently, spectral imaging technology has become a topic of growing interest in color-reproduction, remote- sensing, medical imaging, and other systems. These increased research efforts are likely to propagate into other application areas such as computer vision and pat- tern recognition. Usually, texture analysis is an efficient measure to estimate the structural orientation, rough- ness, smoothness or regularity differences of diverse regions in an image scene. The main idea of extracting texture from hyperspec- tral images is the use of combined spectral and spatial information. Numerous approaches have been con- ducted on the use of Gabor filters [13], co-occurrence matrices [14-19], mathematical morphology [20-22]. The traditional popular tool used for texture-based segmentation and classification is the gray level depen- dence method (GLCM) [1,2], but some other techniques In [23], the authors used the potential of the spectral/ textural approach to improve the classification accuracy * Correspondence: salah.bourennane@fresnel.fr Groupe GSM, Institut Fresnel, Ecole Centrale Marseille D. U. de Saint Jérôme Av. Escadrille Normandie, 13397, Marseille, France Recently, it has been shown that the concept of spatial co-occurrence matrix could be generalized by assuming texture joint information between spectral bands [17,32]. 2.1. N-mode flattening matrix of a tensor 2.1. N mode flattening matrix of a tensor A multi-band image can be represented as a 3-D data array also called third order tensor X ε RI1×I2×I3, where the three entries are related to pixel localization and spectral band, and each element of X could be arranged as xi1i2i3, i1 = 1, ..., I1, i2 = 1, ..., I2, i3 = 1, ..., I3; ℝis the real manifold. Driven by classification or discrimination accuracy, one would expect that, as the number of multi or hyperspectral bands increases, the accuracy of classification should also increase. Nonetheless, this is not the case in a model- based analysis [33-35]. Redundancy in data can cause con- vergence instability of models. Furthermore, variations due to noise in redundant data propagate through a classifica- tion or discrimination model. Thus, processing a large number of multi or hyperspectral bands can result in higher classification inaccuracy than processing a subset of relevant bands without redundancy [34,35]. A tensor can be transformed into a n-mode matrix. The n-mode flattened matrix Xn of tensor X ε RI1×I2×I3 is a In × Mn matrix where: Mn = Ip × Iq, with p, q ≠n. d f Mn = Ip × Iq, with p, q ≠n. By definition, Xn is generated by the column vectors of the n-mode flattened matrix. columns are In dimen- sional vectors obtained from X by varying the index in and keeping the other indices fixed [44]. These vectors are called “n-mode vectors”. The three n-mode flatten- ing of a third-order tensor are shown in Figure 1. One way of overcoming this problem is to adopt a proper selection band method before applying classifica- tion task. The reason is that, in a selection band proce- dure, the amount of data is reduced into a lower dimensional subspace without practically losing relevant information [36]. In addition, computational require- ments for processing large hyperspectral data sets might be prohibitive and a method for selecting a data subset is therefore sought [33]. 2. Spatial and spectral gray level dependence method (SSGLDM) However, the main problem of texture analysis of multi-band images is related to the high dimension of the data and its high correlation. Page 2 of 13 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 been used in feature bands selection [42,43], it consists of analyzing the amount of information in a subset of features (bands), measuring the degree of independence between image bands as a relevance criterion. of intra-urban land cover types; Claussi [24] studied the effect of gray quantization on the ability of co-occur- rence probability statistics; Kiema [25] examined the gray-level co-occurrence based texture image fused to thematic mapper (TM) imagery to expand the object feature base to include both spectral and spatial features; while Bau and Healey [26] used a bank of rotation scale invariant Gabor feature vectors to represent the spec- tral/spatial properties of a region. In this article, we investigate the problem of the analy- sis of multispectral image textures allowing a joint spec- tral and spatial analysis of texture. Therefore, we aim to add spectral information to classical texture analysis methods that only treat gray-level spatial variations. We try to apply the proposed method on prostate cancer textured multispectral images. These images have been chosen to reflect different grades of malignancy in pro- static tissues, and they correspond to different structural patterns as well as apparent textures. Besides, color opponent features were first introduced in color texture characterization with fairly good perfor- mance [27] and later extended to deal with multi-band texture images [28]. Other methods combine color and texture information for the segmentation of color images [29]. Moreover, several researches studied the spatial interaction within each channel and interaction between spectral channels, applying gray level texture techniques to each channel independently [30], or using 3D colour histograms as a way to combine information from all colour channels [31]. The remainder of this article is organized as follows: Section 2 describes a new method for multi and hyper- spectral texture analysis, based on joint spatial and spec- tral information; while Section 3 is concerned with some comparative results. Finally, Section 4 gives a con- clusion of the article. Recently, it has been shown that the concept of spatial co-occurrence matrix could be generalized by assuming texture joint information between spectral bands [17,32]. However, the main problem of texture analysis of multi-band images is related to the high dimension of the data and its high correlation. 2.2. SSGLDM algorithm The idea of the SSGLDM is based on the estimation of the second-order joint conditional probability density multi-dimensional functions P (V, VΔ\|d, θ). Each P (V, VΔ\|d, θ) is the probability that two spectral vectors V and VΔ with components (i1, ..., ik, ..., in), and (j1, ..., jl, ..., jn) respectively, occur for a given distance d and direction θ (see Figure 2), where n is the number of spectral bands, and (i(k), j(l)) ε [1, Ng]2, Ng is the number of gray levels. Several approaches have been investigated by looking into how to remove information re-dundancy resulting from highly correlated bands [33,37-40]. Most of the methods usually involve two separate tasks: (a) selecting the bands that can indicate the particular material well, feature bands selection; and (b) removing the feature bands contributing redundant information, redundancy reduction [41]. Recently, Information theory has also In the whole article, we consider multi-spectral data as a third-order tensor denoted by X, in which the entries are accessed via three index. Page 3 of 13 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Figure 3 Structure form representation of P (V, VΔ\|d, θ ). Figure 1 Flattening matrices of a third order tensor [52]. Figure 3 Structure form representation of P (V, VΔ\|d, θ ). A3, B3. Let T denotes the matrix that horizontally con- catenates At 3 and Bt 3, and vertically concatenates Bt 3 and At 3, whereAt 3, Bt 3 denote the transposed matrices of A3, B3 respectively. T can be represented as follows: Figure 1 Flattening matrices of a third order tensor [52]. Unlike the spatial gray level dependence method in which the estimated second-order joint conditional den- sity functions can be described in a matrix, we propose to represent P (V, VΔ\|d, θ) in a structure form of an array Ioccu which is the vector of occurrences and a matrix MV,V which is the matrix of V and VΔ compo- nents. Each row in the matrix MV,V corresponds to V and VΔ components. The use of this structure can help us to compute the occurrences while keeping the corre- sponding components vectors of V and VΔ. 2.2. SSGLDM algorithm For example if we take the first value of Ioccu (i.e Ioccu(1)), the corre- sponding components vectors of V and VΔ are in the first row of MV,V (see Figure 3), so Ioccu(1\|d, θ) = P(V(1), V(1) \|d, θ) where 3 B3 T = At 3 Bt 3 Bt 3 At 3 (1) T = At 3 Bt 3 Bt 3 At 3 (1) (1) The use of this procedure yields us to calculate the spatial and spectral gray level dependence for both θ and (θ + π). The use of this procedure yields us to calculate the spatial and spectral gray level dependence for both θ and (θ + π). Finally, the matrix MV,V is obtained by keeping only the rows of T with no repetitions, however, the number of repetitions for each row is represented in an array of occurrences called Ioccu. At the end of the process, Ioccu is normalized with respect to the size of MV,V, so that each component represents the probability of occur- rence of a given combination (V, VΔ). The spatial and spectral gray level dependence algorithm can be given as follows: V(1) = MV,V(1, 1 : n) and V(1) = MV,V(1, n + 1 : 2 × n). (1: n): designs columns 1 to n. In order to compute the matrix MV,V, we define two sub-tensors A, B extracted from the tensor data X for each direction θ as shown in Figure 4. Using the 3- mode flattening matrix of A, B, we obtain respectively STEP 1: For a given distance d and angle θ, extract the two sub-tensors A and B from the tensor data X. STEP 2: Compute the 3-mode flattening matrices A3, B3. STEP 3: Build the matrix T. Figure 2 3D representation of the two vectors V and VΔ for a given distance d = 1 and θ = 45°. STEP 4: Compute MV,V by keeping only the rows T with no repetitions. STEP 4: Compute MV,V by keeping only the rows T with no repetitions. STEP 5: Compute Ioccu. 2.3. Proposed generalized textures features related to SSGLDM Haralick texture features comprise 14 features as sum- marized in [1]. However, the most used in literature are energy, entropy, inertia, local homogeneity and correla- tion. In this article we propose seven new generalized texture features. Suppose that V(k) = (i(k) 1 , i(k) 2 , . . . , i(k) n ), and V(k) = (j(k) 1 , j(k) 2 , . . . , j(k) n ), where 1 ≤k ≤m, and 1 ≤m ≤N2n g is the length of the vector of occurrences, Ng is the number of gray levels and n is the number of bands. Suppose that V(k) = (i(k) 1 , i(k) 2 , . . . , i(k) n ), and V(k) = (j(k) 1 , j(k) 2 , . . . , j(k) n ), where 1 ≤k ≤m, and 1 ≤m ≤N2n g is the length of the vector of occurrences, Suppose that V(k) = (i(k) 1 , i(k) 2 , . . . , i(k) n ), and V(k) = (j(k) 1 , j(k) 2 , . . . , j(k) n ), where 1 ≤k ≤m, and 1 ≤m ≤N2n g is the length of the vector of occurrences, Figure 2 3D representation of the two vectors V and VΔ for a given distance d = 1 and θ = 45°. Figure 2 3D representation of the two vectors V and VΔ for a given distance d = 1 and θ = 45°. g Ng is the number of gray levels and n is the number of bands. Page 4 of 13 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 (a) (b) (c) (d) Figure 4 The two sub-tensors A, Bextracted from the tensor data Xfor each direction θ. (a) 0°, (b) 45°, (c) 90°, (d) 135°, d = 1. (b) (a) (b) (d) (c) (d) (c) ensors A, Bextracted from the tensor data Xfor each direction θ. (a) 0°, (b) 45°, (c) 90°, (d) 135°, d = 1. Figure 4 The two sub-tensors A, Bextracted from the tensor data Xfor each direction θ. (a) 0°, (b) 45°, (c) 90°, (d) 135°, d = 1. Figure 4 The two sub-tensors A, Bextracted from the tensor data Xfor each direction θ. (a) 0°, (b) 45°, (c) 90°, (d) 135°, d = 1. For convenience, P(V(k), V(k) \|d, θ) will be written as P(V(k), V(k) )in the following equations. Energy: Entropy: E = − k=m k=1 P(V(k), V(k) )log(P(V(k), V(k) )) (3) (3) H = k=m k=1 P2(V(k), V(k) ) (2) (2) The entropy is the counterpart measure of energy. It measures randomness and is smaller for a smooth image than for a coarse image. This descriptor is also known as angular second moment or uniformity. In our case, it measures the spa- tial/spectral image homogeneity. ces in Signal Processing 2012, 2012:118 /2012/1/118 Page 5 of 13 Page 5 of 13 Page 5 of 13 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Inertia: ⎧ ⎪⎪⎨ ⎪⎪⎩ I = k=m k=1 [V(k) −V(k) ] 2P(V(k), V(k) ) where : V(k) −V(k) 2 = (i(k) 1 −j(k) 1 ) 2 + (i(k) 2 −j(k) 2 ) 2 + · · · + (i(k) n −j(k) n ) 2 (4) This descriptor is also known as contrast or difference moment. It is a measure of image intensity contrast or he spatial/spectral variations present in an image to σ 2 i = k=m k=1 (V(k) −Vx) 2P(V(k), V(k) ) σ 2 j = k=m k=1 (V(k) −Vy) 2P(V(k), V(k) ) ⎧ ⎪⎪⎨ ⎪⎪⎩ I = k=m k=1 [V(k) −V(k) ] 2P(V(k), V(k) ) where : V(k) −V(k) 2 = (i(k) 1 −j(k) 1 ) 2 + (i(k) 2 −j(k) 2 ) 2 + · · · + (i(k) n −j(k) n ) 2 (4) (4) This descriptor is also known as contrast or difference moment. It is a measure of image intensity contrast or the spatial/spectral variations present in an image to show the texture fineness. The correlation descriptor measures the linear depen- dency of spectral vectors in the co-occurrence matrix. ensors A, Bextracted from the tensor data Xfor each direction θ. (a) 0°, (b) 45°, (c) 90°, (d) 135°, d = 1. The inertia value is high when similar spectral vectors are adjacent to each other in the input image and pro- vides a measure of coarseness (similarity). In practice, as the number of bands increases, the joint probability P(V(k), V(k) ) decreases. Consequently, the values of P(V(k), V(k) )become nonsignificant which implies a poor overall classification [17,32]. Local homogeneity: In = k=m k=1 1 1 + V(k) −V(k) 2 P(V(k), V(k) ) (5) To overcome this issue, we suggest to apply the pro- posed SSGLDM on a subset of bands in the multi-spec- tral image, instead of processing the whole set of bands. The subset of bands can be chosen to remove informa- tion redundancy resulting from highly correlated bands. (5) The local homogeneity or inverse difference moment is a counterpart measure to the contrast descriptor. Asymmetry: Asymmetry: 2.4. Band selection by minimization of dependent information I = k=m k=1 [(V(k) −Vx) + (V(k) −Vy)] 3P(V(k), V(k) ) (6) In [43], the extraction of selected subsets of spectral image bands can be obtained by means of a criterion based on the minimization of the dependent information (MDI), which consists of a relation between the joint entropy and the union of the conditional entropies of the considered set of image bands. This criterion could be defined by the following expression: where: (V(k) −Vx) = (i(k) 1 −x1) 2 + · · · (i(k) n −xn) 2 (V(k) −Vy) = (j(k) 1 −y1) 2 + · · · (j(k) n −yn) 2 Vx = (x1, x2, . . . , xn) = k=m k=1 V(k)P(V(k), V(k) ) Vy = (y1, y2, ..., yn) = k=m k=1 V(k) P(V(k), V(k) ) DI = H(A1, . . . , An) − n i1=1 H(Ai1\|Ai2, . . . , Ain) (9) (9) Where Ai1 is a random variable representing the image band i1. Ai2, ..., Ain are the complementary variables of Ai1; H(A1, ..., An) is the joint entropy, which represents the total amount of joint information of set of variables A1, ..., An; and H(Ai1\|Ai2, . . . , Ain) is the conditional entropy that represents the amount of independent information in image band Ai1 having measured the rest image bands Ai2, ..., Ain. This descriptor measures the distribution of spectral vectors values around the vectors average using third order statistics. Proeminence: I = k=m k=1 [(V(k) −Vx) + (V(k) −Vy)] 4P(V(k), V(k) ) (7) This technique behaves as an unsupervised feature selection criterion, providing very satisfactory results with respect to classification accuracy when using the selected bands, even outperforming the other supervised methods used in the comparison in most situations [43]. These performances justify our choice of using MDI [43] for selecting the best relevant bands in the second part of our experiments. This descriptor measures the distribution of spectral vectors values around the vectors average using fourth order statistics. Correlation: I = 1 σiσj k=m k=1 (V(k) −Vx)(V(k) −Vy)P(V(k), V(k) ) (8) (8) 3. Experimental results The first part of this section is concerned with multi- spectral texture characterization. For this purpose, an Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Page 6 of 13 The data set consists of 624 textured multi-spectral images, of 128 by 128, with each image taken with 16 spectral channels (from 500 to 650 nm) with a 5 nm step [45]. The images were captured using a classical microscope and CCD camera. A liquid crystal tunable filter (LCTF) was inserted in the the optical path between the light source and the chilled CCD camera. The LCTF has a bandwidth accuracy of 5 nm. extensive experiments are carried out on many multi- spectral images for use in prostate cancer diagnosis. Furthermore, the results of proposed method (SSLGMD) are compared with GLCM, followed by per- formance comparative study and discussion. The second part deals with the curse-of-dimensionality problem. Thus, we extract the best relevant bands of multispectral prostate cancer using MDI technique proposed by Sotoca et al. [43]. Thereafter, we provide some experi- mental results, in order, to see the impact of the selected bands using MDI criterion on the classification accuracy performance using proposed method and GLCM respectively. The captured images have been chosen to reflect differ- ent grades of malignancy in prostatic tissues and they are labeled into four classes: 176 cases of cancer (Pca), 160 cases of BPH, 144 cases of PIN and 144 cases of Stroma (STR).a The samples were routinely viewed at prostatic section seen at low power (40x objective magnification) by two highly experimented independent pathologists. The pathologist initially views slides at low power, thereby enabling the location of potentially abnormal regions. Subsequent analysis of these regions at high power enables the histological grading of these areas. 3.1. Data set Over the last decade, multi-spectral imagery in pros- tate cancer has become a very useful tool for analyzing and diagnosing pathologies. The prostate cancer is the second most common cancer in men after the skin cancer, and it is also the second in the list of causes for cancer death after the lung cancer. However, the most known method for prostate cancer diagnosis is the prostate-specific antigen (PSA) blood test. In the case of a positive diagnosis, the urologist will often advise a needle biopsy, in which a tiny piece of a tissue is taken from the prostate for analysis [45]. From the analysis, different grades of malignancy correspond to different structural patterns as well as to apparent tex- tures. After the analysis, an experienced pathologist selects the images to reflect the different patterns in prostatic tissues, four major groups usually should be identified (see Figure 5). The X-Y resolution depends on the magnification cho- sen, which is usually high for visualization purposes. In this study, the images of 128 by 128 pixels were cap- tured at an X-Y resolution of 0.12 µ/pixel. Usually, when a biopsy is submitted for analysis, it is very rare that the pathologist finds that the sample is per- fectly normal. There must be at least some benign condi- tion that would explain the high levels of PSA that usually justify needle biopsy. So the main issue is to iden- tify benign from malignant and premalignant conditions. For this purpose, Figure 6 summarizes the different steps used in this article for classification process. The proposed methodology is very important task in prostate cancer diagnosis and could be viewed as a computer- aided system to automatically classify pathological pros- tate images, since each image can be classified into an appropriate class of prostatic tissue. ●Stroma: STR (normal muscular tissue) ●Benign prostatic hyperplasia: BPH (a benign condition) ●Prostatic intraepithelial neoplasia: PIN (a precursor state for cancer). 3.3. Features selection for prediction procedure (Step 2) p p p Feature selection technique is applied to reduce the number of features before applying the classification task. Irrelevant features may have negative effects on a prediction procedure. Moreover, the computational complexity of a classification algorithm may suffer from the curse of dimensionality caused by several features. Features can be selected in many different ways. One 3.2. Features extraction (Step 1) ●Prostatic carcinoma: PCa (abnormal tissue devel- opment corresponding to cancer). The goal of this step of analysis is to characterize the four different groups of multi-spectral images by (a) (b) (c) (d) Figure 5 Images showing representative samples of the four classes. (a) Stroma, (b) BPH, (c) PIN, (d) PCa. (a) (d) (a) (b) (b) (c) (b) (d) (a) Figure 5 Images showing representative samples of the four classes. (a) Stroma, (b) BPH, (c) PIN, (d) PCa. Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Page 7 of 13 Figure 6 Classification procedure. Figure 6 Classification procedure. extracting features in order to make classification possible. scheme is to select features that correlate strongest to the classification variable. All generalized and traditional features mentioned in Sections 3.4 and 3.5 have been used for our experi- ments, and the best performances have been achieved by choosing (d = 1) and the four principal directions (θ = 0°, θ = 45°, θ = 90°, θ = 135°). This has been called maximum-relevance selection. Many heuristic algorithms can be used, such as the sequential forward, backward, or floating selections [42]. On the other hand, features can be selected to be mutually far away from each other, while they still have high correlation to the classification variable. This scheme, termed as minimum-redundancy-maximum- relevance selection [42], has been found to be more powerful than the maximum relevance selection. This justifies the use of this technique in our experiments. We note that, the problem of spatial selection rela- tionships (i.e d and θ) for defining co-occurrence matrices is addressed in [46]. These matrices are maxi- mally reflecting the structure of the underlying texture. We note that, this technique is not applied when the number of feature is small. 3.5. Results and discussion To illustrate the classification of different classes of prostate cancer, we show the confusion matrices obtained by using SSGLDM for different choices of S. Tables 2a,b depict the results using GLCM (S = 1 band) where Tables 2c,d give the corresponding results using SSGLDM for (S = 16 bands). As can be seen from these tables, the use of SSGLDM with (S = 16) yields worst results in terms of global accuracy rate. However, Tables 2e,f show the achieved improvements when using SSGLDM with (S = 3 bands). Note that in all the cases, BPH and Stroma classes present the highest error rate in terms of classification due to the similarities between two classes; however the use of SSGLDM with (S = 3 bands) reduces significantly the error rate in these classes. The assessment of the classification performance was made using 3-fold cross-validation. Thus, data were randomly splited into 3-sets of a roughly equal size. Splitting was carried out such that the proportion of samples per class was roughly equal across the sets. Each run of the 3-fold cross-validation algorithm con- sisted of a classifier designed on two data subsets (train- ing) while testing was performed on the remaining subset; this is repeated three times. The SVM optimiza- tion was implemented using LIBSVM library through its Matlab interface [48]. The penalty term C of Gaussian kernel, was fixed to 200 and s2 was selected by using a fivefold cross validation [48]. So that, fivefold cross vali- dation was applied on training data in order to estimate s2 which gives the highest classification accuracy rate. On the other hand, tests of the computation time were performed for the both SSGLDM and GLCM. For this purpose, we used a PC Intel® Core(TM) 2 CPU, 2.66 GHz and 3.58 GB Ram. The two methods were imple- mented using Matlab 7.1. Times are given for the com- putation of the co-occurrence matrices and all the features of each method (SSGLDM and GLCM), on images 128 × 128 mentioned in Section 3.1. As shown in on Table 3 The execution time using SSGLDM with (S = 2) is almost the same as GLCM. Firstly, we compared the performance of SSGLDM with GLCM by the overall classification rate as a criter- ion of the comparison, with reference to a set of images manually classified by experts. 3.4. Classification process (Step 3) A classification process usually involves training and testing data which consist of some data instances. Each instance in the training set contains one “target value” (class labels) and several “attributes” (features). Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Page 8 of 13 3.4.1. Using SVM for classification Table 1 shows the classification results obtained by using SSGLDM, for different subsets of bands (S) for both 32 and 64 gray levels quantification. In [47], several neural networks were compared to the SVM for the classification of hyper-spectral data. The robustness of SVMs was demonstrated and the best results were obtained using a non-linear SVM. In addi- tion, [47] studied Radial Basis Functions (RBF) classifiers and SVM and they noted the favorable behavior of the SVM, from both a theoretical and a practical point of view (see Appendix). It can be clearly observed from Table 1 that the SSGLDM yields better results than the GLCM when S is less than five bands. Best results are obtained with S = 3 bands, for both 32 and 64 gray levels. However, with 16 bands we obtained a classification accuracy rate around 93%. This may derive from the high correlation of the data. Moreover, as the set of bands increases, the joint probability P (V, VΔ\|d, θ) decreases. Consequently, the values of P (V, VΔ\|d, θ) become nonsignificant which implies a poor overall classification. Visually, the images illustrated in Figure 5 are very similar, so the use of SVM for classification issue could be suitable for our applications. 3.5. Results and discussion Like in the GLCM, in which the co-occurrence matrix is computed for each band, we can apply SSGLDM for each subset of bands in the image. Let S be a subset of a bands in the image (a ≤Nb) where Nb is the number of image bands. For example, if S = (Bl, Bl+1) is a subset of two bands; the SSGLDM can be applied Nb −1 times in the image by varying l from 1 to Nb −1. Thus, GLCM can be seen as a special case of SSGLDM when S contains only one band. Note also that the time keeps growing, when increas- ing the set of bands during the process. However, it decreases for (S = 16), because the SSGLDM was applied just one time for the whole set of spectral bands. Thus, one can conclude that the use SSGLDM with (S = 2) is a quit good compromise between classifi- cation results and computation. The number of features is computed for each band in the GLCM, so the total number of features is 7 × (4 directions) × 16 bands = 448. However in the case of SSGLDM, the number of features depends on the choice of subset of bands (S). For example, if S contains two bands, the number of features is 7 × (4 directions) × (Nb −1) bands = 420, where Nb = 16. The minimum redundancy-maximum relevance selection algorithm [42] was used to ensure good classification accuracy by selecting 40 better features from all features generated by SSGLDM. Table 1 Classification results obtained by using the SSGLDM Subset of bands (S) 1(GLCM) 2 3 4 5 16 Overall classification (%) (32 gray levels) 94.87 97.44 97.76 97.11 96.31 93.91 Overall classification (%) (64 gray levels) 95.51 97.12 97.44 96.96 96.79 93.27 Table 1 Classification results obtained by using the SSGLDM Subset of bands (S) 1(GLCM) 2 3 4 5 16 Overall classification (%) (32 gray levels) 94.87 97.44 97.76 97.11 96.31 93.91 Overall classification (%) (64 gray levels) 95.51 97.12 97.44 96.96 96.79 93.27 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Page 9 of 13 3.5.1. 3.5.1. Receiver operating characteristic (ROC) In order to plot ROC curves, the classifier should be tested using different parameters re-sulting from differ- ent values for false alarm (false positive rate FPR) and sensitivity (true positive rate TPR) rates. 3.5. Results and discussion Since there are two classes, prior probabilities are linked by the relation: πpositive = 1 −πnegative (10) πpositive = 1 −πnegative (10) (10) ROC curves could be plotted by varying the positive values πpositive. ROC curves could be plotted by varying the positive values πpositive. Figure 7 shows a comparative study of the ROC curves using GLCM and SSGLDM respectively, for both 32 and 64 gray levels. Dark, blue and red curves indicate the ROC curves of classified image using SSGLDM (S = 3 bands), GLCM and SSGLDM (S = 16 bands) respec- tively. These results indicate that for all possible values of prior probability πpositive the SSGLDM features with (S = 3) perform better when compared with that derived from GLCM for both 32 and 64 gray levels. The results demonstrate also that our new proposed technique improves ability to distinguish cancer prostate tissues from healthy ones. However, one can note, that the major issue arise from our proposed method is related to the high dimen- sion of the data and its high correlation. This fact is clearly seen from Table 1 and 2 where classification accuracy is degraded while using SSGLDM with 16 bands. Thus, the main question to be solved is: does the band selection procedure improve the overall classifica- tion rate when using SSGLDM? How would one obtain the optimal number that maximizes classification accu- racy and minimize computational requirements? In the remainder of this section, we show the influ- ence of selected band method using MDI criterion (Sec- tion 2.4) on the overall classification rate by applying the SSGLDM. 3.5. Results and discussion Receiver operating characteristic (ROC) Table 2 Confusion matrices obtained by using (a) GLCM (32 gray levels), (b) GLCM (64 gray level), (c) SSGLDM (S = 16 bands, 32 gray level), (d) SSGLDM (S = 16 bands, 64 gray level), (e) SSGLDM (S = 3 bands, 32 gray levels), (f) SSGLDM (S = 3 bands, 64 gray levels) Classified as: BPH PCa PIN Stroma Error (%) (a) BPH 135 1 24 0 15.62 Pca 0 175 0 1 0.57 PIN 2 0 141 1 2.08 Stroma 0 0 3 141 2.08 Overall 5.13 (b) BPH 138 0 22 0 13.75 Pca 0 176 0 0 0 PIN 3 0 141 0 2.08 Stroma 0 0 3 141 2.08 Overall 4.49 (c) BPH 149 0 11 0 6.88 Pca 0 173 0 3 1.70 PIN 0 0 136 8 5.56 Stroma 0 11 5 128 11.11 Overall 6.09 (d) BPH 145 0 15 0 9.38 Pca 0 174 0 2 1.14 PIN 0 0 135 9 6.25 Stroma 5 4 7 128 11.11 Overall 6.73 (e) BPH 147 0 13 0 8.13 Pca 0 175 0 1 0.57 PIN 0 0 144 0 0 Stroma 0 0 0 144 0 Overall 2.24 (f) BPH 147 0 13 0 7.5 Pca 0 175 0 1 0.57 PIN 0 0 143 1 0.69 Stroma 0 0 1 143 0.69 Overall 2.56 Table 2 Confusion matrices obtained by using (a) GLCM (32 gray levels), (b) GLCM (64 gray level), (c) SSGLDM (S = 16 bands, 32 gray level), (d) SSGLDM (S = 16 bands, 64 gray level), (e) SSGLDM (S = 3 bands, 32 gray levels), (f) SSGLDM (S = 3 bands, 64 gray levels) Let πi be the prior probabilities in each class i. In the case of uniform prior probabilities, πi can be written as: ∀i πi = 1 N, where: N is the number of classes. Let us suppose a two-class prediction problem (binary classification), in which the outcomes are labeled either as positive (p) or negative (n) class. This is achieved by considering that BPH as the negative diagnosis while PIN and PCa together form the positive diagnosis out- come (the classification of Stroma is relatively simple because of its homogeneous nature at low resolution) [49,50]. 3.6. SSGLDM using band selection In order to exploit inter-band correlation for reducing the multi-spectral band representation, the unsupervised band selection technique by Sotoca et al. [43] has been used to extract the best relevant bands of multispectral prostate cancer database. Table 3 Consuming time Subset of bands 1 (GLCM) 2 3 4 5 16 Time per image (sec) 0.63 0.67 1.3 1.62 3.13 1.56 All classification rates shown in this section were computed by using 3-fold cross-validation, such as described before. Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Page 10 of 13 N t th t th l ifi ti d d i (a) (b) Figure 7 The comparative ROC curves. (a) 32 gray levels (b) 64 gray levels. (a) (a) (b) Figure 8 Behavior of the overall classification accuracy versus the selected bands. (a) 32 gray levels (b) 64 gray levels. (a) (b) Figure 7 The comparative ROC curves. (a) 32 gray levels (b) 64 gray levels. (a) (a) (b) (b) Figure 8 Behavior of the overall classification accuracy versus the selected bands (a) 32 gray levels (b) 64 gray levels Figure 7 The comparative ROC curves. (a) 32 gray levels (b) 64 gray levels. Note that the same classification procedure used in Section 5.4 was applied for each data cube constructed by the selected bands. However, in the case of SSGLDM the features selection technique [42] was not used because of the small number of features (Figure 6). (b) Figure 8 Behavior of the overall classification accuracy versus the selected bands. (a) 32 gray levels (b) 64 gray levels. To see the impact of the selected bands using MDI criterion on the classification accuracy performance, we tested the SSGLDM with a variable number of selected bands of multispectral images. Therefore, the plot of Figure 8 shows the result when using 2, 3, 4, 5, 6 and 16 selected bands as input data. A clear improvement of the classification accuracy can be observed by using five selected bands. However, the SSGLDM yields worst results, when more than six selected bands are used. Thus, processing a large number of multispectral bands can result in higher classification inaccuracy than pro- cessing a subset of relevant bands without redundancy. To gain an insight into the classification of different classes of prostate cancer, the confusion matrices of both SSGLDM and GLCM using five selected bands are also given in Table 4. 3.6. SSGLDM using band selection As can be seen from this table, the use of band selection procedure before applying SSGLDM reduces significantly the error rate in classes and insures a good discrimination power between differ- ent types of tissues. Figure 9 illustrates the ROC curves obtained with the two methods GLCM and SSGLDM using five selected bands as input data. This clearly demonstrates that our new proposed SSGLDM results in an improved ability to distinguish cancer prostate tissues from healthy ones. On the other hand, the classical GLCM provides lower classification accuracy than SSGLDM. However, when more bands selected are used, GLCM performs better. This is mainly due to the use of minimum redundancy- maximum-relevance selection [42] that reduces the number of features and ensure a good classification accuracy. Finally, Table 5 summarizes the processing time for both GLCM and SSGLDM (implemented using Matlab 7.1) versus selected bands. This experiment is conducted on an Intel® Core(TM) 2 CPU, 2.66 GHz and 3.58 GB Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Page 11 of 13 Table 4 Confusion matrices obtained by using (a) GLCM (5 selected bands, 32 gray levels), (b) SSGLDM (5 selected bands, 32 gray levels), (c) GLCM (5 selected bands, 64 gray levels), (d) SSGLDM (5 selected bands, 64 gray levels) Classified as: BPH PCa PIN Stroma Error (%) (a) BPH 147 0 13 0 8.13 Pca 0 174 2 0 1.14 PIN 2 12 129 1 10.42 Stroma 9 0 1 134 6.94 Overall 6.41 (b) BPH 148 0 12 0 7.5 Pca 0 174 2 0 1.14 PIN 2 11 130 1 9.72 Stroma 1 0 1 142 1.39 Overall 4.81 (c) BPH 141 0 15 4 11.88 Pca 0 175 1 0 0.57 PIN 0 13 130 1 9.72 Stroma 17 0 1 126 12.5 Overall 8.33 (d) BPH 149 0 11 0 6.88 Pca 0 176 0 0 0 PIN 2 10 131 1 9.03 Stroma 0 0 1 143 0.69 Overall 4.01 (a) (b) Figure 9 The comparative ROC curves using five selected bands as input data. (a) 32 gray levels (b) 64 gray levels. 3.6. SSGLDM using band selection Table 4 Confusion matrices obtained by using (a) GLCM (5 selected bands, 32 gray levels), (b) SSGLDM (5 selected bands, 32 gray levels), (c) GLCM (5 selected bands, 64 gray levels), (d) SSGLDM (5 selected bands, 64 l l ) (a) (b) (b) Figure 9 The comparative ROC curves using five selected bands as input data. (a) 32 gray levels (b) 64 gray levels. carried out on many multi-spectral images for use in prostate cancer diagnosis and quantitative results showed the efficiency of this method compared to the Gray GLCM. SSGLDM has also pro-vided better perfor- mances in terms of classification accuracy and computa- tional complexity. Finally, due to the aspect of this area of research, many issues could be suggested. Open pro- blems that can be investigated in the future include the following: Ram. As mentioned before, times are given for the com- putation of co-occurrence matrices and all features for each method. It’s clear from the table that the use of band selection technique reduces significantly the time computation. It’s clear from the table that the use of band selection technique reduces significantly the time computation. On the other hand, comparing the results of two meth- ods, SSGLDM ran much faster than the GLCM mainly because SSGLDM features are extracted from the whole data cube of selected bands, unlike the GLDM features which are computed from each selected band. (1) The new texture characterization method described in this article focuses on second order statis- tics. Therefore, the way forward could be to investigate alternative methods using higher-order statistics. 4. Conclusion This article describes a new method to generalize the concept of spatial gray level dependence method by assuming the presence of texture joint information between spectral bands. Two ways have been suggested to implement the proposed spatial and spectral gray level dependence method (SSGLDM): (a) applying SSGLDM for each subset of bands in the multi-spectral image; and (b) making a connection between band selection and SSGLDM by using MDI criterion before applying SSGLDM. Extensive experiments have been (2) In this work, the most time consuming task was, by far, the computation of the generalized co-occurrence matrix, which mainly depends on spectral vector-pairs distances and a large numbers of spectral bands. The (2) In this work, the most time consuming task was, by far, the computation of the generalized co-occurrence matrix, which mainly depends on spectral vector-pairs distances and a large numbers of spectral bands. The Table 5 Computation time Number of selected bands 2 3 4 5 6 GLCM (Time per image (sec)) 0.08 0.12 0.16 0.2 0.24 SSGLDM (Time per image (sec)) 0.05 0.09 0.12 0.17 0.21 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Khelifi et al. EURASIP Journal on Advances in Signal Processing 2012, 2012:118 http://asp.eurasipjournals.com/content/2012/1/118 Page 12 of 13 Page 12 of 13 where ai are the Lagrange coefficients. It is worth not- ing that all mappings used in the SVM learning occur in the forme of inner product. This allows us to define the classical Gaussian kernel Ks given by this formula: nature of the calculation makes it suitable for parallel processing because the same calculations are performed on successive image blocks. (3) The generalized multi-band texture method is pro- posed in this article to solve the characterization of multi-band texture images problem. Medical data sets that use multi-spectral data have been used to evaluate our proposed algorithm. In future, to apply our pro- posed algorithms to other applications such as hyper- spectral satellite imagery or skin cancer detection. (3) The generalized multi-band texture method is pro- posed in this article to solve the characterization of multi-band texture images problem. Medical data sets that use multi-spectral data have been used to evaluate our proposed algorithm. References In the case of a nonlinear classification of samples, the training vectors xi are mapped into a higher (maybe infi- nite) dimensional space by the function , w is the vec- tor of hyperplane coefficients (orientation), b is a bias term. The regularization parameter C controls the gen- eralization capabilities of the classifier and it must be selected by the user, and ξi are positive slack variables enabling to deal with permitted errors. The decision function is found by solving the convex optimization problem 1. RM Haralick, K Shanmugam, IH Dinstein, Textural features for image classification. IEEE Trans Syst Man Cybern. SMC-3(6), 610–621 (1973) 1. RM Haralick, K Shanmugam, IH Dinstein, Textural features for image classification. IEEE Trans Syst Man Cybern. SMC-3(6), 610–621 (1973) 2. LK Soh, C Tsatsoulis, Texture analysis of SAR sea ice imagery using gray level co-occurrence matrices. IEEE Trans Geosci Remote Sens. GeoRS-37(2), 780–795 (1999) 3. M Do, M Vetterli, Wavelet-based texture retrieval using generalized gaussian density and kullback-leibler distance. IEEE Trans Image Process. IP 11(2), 146–158 (2002) 4. G Hazel, Multivariate gaussian MRF for multispectral scene segmentation and anomaly detection. 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C Munzenmayer, H Volk, C Kublbeck, K Spinnler, T Wittenberg, Multispectral texture analysis using interplane sum and difference histograms, Procs DAGM Symp, Zurich, Switzerland, (Springer, 2002), pp. 25–31 y p p g pp 7. JA Benediktsson, M Pesaresi, K Arnason, Classification and feature extraction for remote sensing images from urban areas based on morphological transformations. IEEE Trans Geosci Remote Sens. 41(9), 1940–1949 (2003). Support vector machines (SVM) The SVMs are mainly a nonparametric method, yet some parameters need to be tuned before the optimiza- tion. In the case of Gaussian kernel, there are two para- meters: C, which is the penalty term, and s, which is the width of the exponential. The aim of SVM is to produce a model (based on the training data) that predicts target value of data instances in the testing set which are given only by the attributes. Given a labeled training data set {(x1, y1), ..., (xn, yn)}, where xi Î ℝn and yi Î {−1, 1}, the SVM [47,51] require the solution of the following optimization problem: min w,ξi,b 1 2∥w∥2 + C i ξi (:1) Received: 8 September 2011 Accepted: 29 May 2012 Published: 29 May 2012 where 〈.,.〉: is the inner product. where 〈.,.〉: is the inner product. 4. Conclusion In future, to apply our pro- posed algorithms to other applications such as hyper- spectral satellite imagery or skin cancer detection. kσ(xi, xj) = ⟨φ(xi), φ(xj)⟩ = exp − xi −xj 2 2σ 2 (:5) (:5) where the norm is the Euclidean norm and s Î ℝ+ tunes the flexibility of the kernel. The classification of a sample × is achieved by looking to which side of the hyperplane it belongs Appendix Where b can be used for computing ai. Where b can be used for computing ai. Acknowledgements (:1) g I would to acknowledge contribution from Dr. MA Roula and the Pathology department team at the Queen’s university of Belfast under the direction of Prof. Hamilton for kindly providing maging data used in this study. constrained to: Endnote aThe data set used in this study were provided from Pathology department team at Queen’s university of Bel- fast under the direction of Prof. Hamilton. f(x) = sgn n i=1 yiαikσ(xi, x) + b (:6) (:6) Competing interests The authors declare that they have no competing interests. yi(⟨φ(xi), w⟩+ b) ≥1 −ξi ∀i = 1, . . . , n ξi ≥0 ∀i = 1, . . . , n (:2) (:2) Received: 8 September 2011 Accepted: 29 May 2012 Published: 29 May 2012 Received: 8 September 2011 Accepted: 29 May 2012 Published: 29 May 2012 References 19(1), 17–28 (2002). doi:10.1109/79.974718 37. EM Bassettm, SS Shen, Information theory-based band selection for multispectral systems. Proc SPIE. 3118, 28–35 (1997) 15. 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https://openalex.org/W4385064705	https://air.unimi.it/bitstream/2434/994110/2/Exploring%20the%20role%20of%20retinal%20fluid%20as%20a%20biomarker%20for%20the%20management%20of%20diabetic%20macular%20oedema.pdf	English	null	Exploring the role of retinal fluid as a biomarker for the management of diabetic macular oedema	Eye	2,023	cc-by	7,505	1The David J. Apple International Laboratory for Ocular Pathology, Department of Ophthalmology, University of Heidelberg, Heidelberg, Germany. 2Byers Eye Institute, Stanford University, Palo Alto, CA, USA. 3John and Liz Tory Eye Centre, Sunnybrook Health Sciences Centre, Toronto, ON, Canada. 4Department of Ophthalmology and Vision Sciences, University of Toronto, Toronto, ON, Canada. 5Hospital General de Catalunya, Barcelona, Spain. 6Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy. 7Eye Clinic, IRCCS MultiMedica, Milan, Italy. 8Novartis Pharma AG, Basel, Switzerland. 9Department of Ophthalmology, University of Münster Medical Center, Münster, Germany. 10Present address: OcuTerra Therapeutics, Basel, Switzerland. ✉email: ramin.khoramnia@med.uni-heidelberg.de www.nature.com/eye REVIEW ARTICLE OPEN Exploring the role of retinal ﬂuid as a biomarker for the management of diabetic macular oedema Ramin Khoramnia 1✉, Quan Dong Nguyen 2, Peter J. Kertes 3,4, Laura Sararols Ramsay5, Stela Vujosevic 6,7, Majid Anderesi8,10, Franklin Igwe8 and Nicole Eter9 © The Author(s) 2023 Anti-VEGF therapies are associated with signiﬁcant gains in visual acuity and ﬂuid resolution in the treatment of diabetic macular oedema (DMO) and have become the standard of care. However, despite their efﬁcacy, outcomes can be unpredictable, vary widely between individual eyes, and a large proportion of patients have persistent ﬂuid following initial treatment, with a negative impact on visual outcomes. Anatomical parameters measured by optical coherence tomography (OCT), in addition to visual acuity, are key to monitoring treatment effectiveness and guiding retreatment decisions; however, existing guidelines on the management of DMO lack clear recommendations for interpretation of OCT parameters, or proposed thresholds of various markers to guide retreatment decisions. Although central subﬁeld thickness (CSFT) has been widely used as a marker for retreatment decisions in clinical trials in DMO, and a reduction in CSFT has generally been shown to accompany improvements in best-corrected visual acuity with treatment, analyses of the relationship between these parameters show that the correlation is small to moderate. A more direct relationship can be seen between an increased magnitude of CSFT ﬂuctuations over time and poorer visual acuity, suggesting that control of CSFT could be important in maximising visual outcomes. The relationship between visual outcomes and qualitatively assessed intraretinal ﬂuid and subretinal ﬂuid is also unclear, although quantitative assessments of ﬂuid parameters suggest that untreated intraretinal ﬂuid and subretinal ﬂuid negatively impact visual outcomes. These ﬁndings highlight a need for clearer guidelines on the management of retinal ﬂuid to improve visual outcomes for patients with DMO. Eye; https://doi.org/10.1038/s41433-023-02637-2 Received: 23 January 2023 Revised: 16 May 2023 Accepted: 13 June 2023 INTRODUCTION thickness or ﬂuid on optical coherence tomography (OCT) images, resulting in variation in disease management between countries, regions, and private or public healthcare settings. This review aims to summarise existing knowledge on the role of retinal ﬂuid in the pathophysiology of DMO and the short- and long-term impact on functional outcomes of unresolved ﬂuid following treatment. The prevalence of diabetic macular oedema (DMO) was estimated to be 4% among people with diabetes in 2020, accounting for 18.83 million adults worldwide, with a projected increase to 28.61 million adults by 2045 [1]. DMO is a leading cause of visual impairment in patients with diabetes, particularly type 2 diabetes. Although DMO can occur at any age, a relatively high proportion of those affected come from a young, working-age population who are diagnosed on average at 50 years of age [2–5]. PATHOPHYSIOLOGY OF DMO DMO In a post hoc analysis of Protocol I, eyes receiving ranibizumab plus prompt or deferred laser with higher average levels of oedema (calculated as excess CRT [≥250 µm] averaged over 52 weeks) gained 9.3 fewer Early Treatment Diabetic Retinopathy Study (ETDRS) letters than those with lower levels at the end of Year 3. Patients with the longest duration of persistent oedema (calculated as the number of study visits with CRT ≥250 µm during the ﬁrst 52 weeks of treatment) gained 4.4 fewer letters than those with the least persistent oedema [45]. The negative correlation between duration and extent of oedema and visual outcomes was suggested to be due to photoreceptor degeneration [45, 46]. Fig. 1 Pathophysiology of diabetic macular oedema. A Colour fundus photograph showing exudative diabetic macular oedema and moderate non-proliferative diabetic retinopathy. B Optical coherence tomography image showing diabetic macular oedema with sub-foveal neuroretina detachment (subretinal ﬂuid), intrar- etinal cysts (intraretinal ﬂuid) and hyperreﬂective spots showing activated microglial cells (indicated with arrows). Fig. 1 Pathophysiology of diabetic macular oedema. A Colour fundus photograph showing exudative diabetic macular oedema and moderate non-proliferative diabetic retinopathy. B Optical coherence tomography image showing diabetic macular oedema with sub-foveal neuroretina detachment (subretinal ﬂuid), intrar- etinal cysts (intraretinal ﬂuid) and hyperreﬂective spots showing activated microglial cells (indicated with arrows). Although both IRF and SRF are markers of DMO, IRF tends to be present in almost all patients at treatment initiation, while SRF is present in less than half [20–23]. SRF is generally considered to be associated with worse visual acuity than IRF at baseline and indicative of more severe disease, although it usually responds quickly to treatment [22–26]. Untreated ﬂuid, irrespective of compartment, results in a gradual loss of vision over time and a delay in therapy initiation can lead to poorer treatment outcomes [27, 28]. Rates of persistent DMO in clinical studies (based on CSFT ≥ 250 µm) can be in the range of 20–60% after 2 years of treatment [47–49]. A secondary analysis of Protocol T showed rates of persistent DMO at Week 24 (deﬁned as CSFT ≥250 µm at each completed study visit through Week 24) of 31.6% for aﬂibercept, 41.5% for ranibizumab and 65.6% for bevacizumab. CURRENT DMO MANAGEMENT AND IMPACT OF PERSISTENT OEDEMA Historically, the mainstay of DMO treatment was focal or grid laser photocoagulation; however, this has largely been supplanted with the availability of anti-VEGF therapies as a ﬁrst-line treatment as, although laser can be effective in preventing DMO progression, the rate of vision improvements is low [29]. Certain indications remain for laser, however, including the vasogenic subform of DMO, eyes with DMO and central retinal thickness (CRT) less than 300 μm, or eyes with persisting vitreomacular adhesion. Subthres- hold grid laser treatment can be useful in eyes with higher visual acuity affected by early diffuse DMO [30]. Anti-VEGF therapies are associated with signiﬁcant gains in visual acuity and ﬂuid resolution and have become the standard of care for DMO treatment [31–33]. Intravitreal corticosteroids are another, less common treatment option for patients who are non- or incompletely responsive to anti-VEGFs [34–36]. In clinical practice, where anti-VEGF administration tends to be less frequent than in clinical trials, response to treatment can be even lower. A retrospective chart review of patients receiving anti- VEGF therapy at 10 sites in the US assessed the proportion of patients with best-corrected visual acuity (BCVA) of 20/40 or better combined with CRT ≤250 µm on time domain (TD)-OCT or ≤300 µm on spectral domain (SD)-OCT over 12 anti-VEGF injections. For injections 1–9, BCVA of 20/40 or better was achieved by 52–62% of patients and the deﬁned CRT threshold was achieved by 26–34% of patients. The proportion of patients achieving both BCVA and CRT endpoints ranged from only ~20–40% over all 12 injections [51]. A post hoc analysis of eyes with persistent DMO from Protocol I (approximately 40% of patients in that study) showed that eyes receiving ranibizumab with prompt or deferred laser with chronic persistent DMO (failure to achieve CSFT <250 μm and a ≥10% reduction from the 24-week visit on ≥2 consecutive study visits) at 3 years had worse visual acuity than those without, highlighting the importance of optimising anatomical outcomes for these patients [47]. Despite the efﬁcacy of available treatments for DMO, outcomes can be unpredictable and vary widely between individual eyes. In addition to visual acuity, anatomical parameters measured on OCT are key to monitoring treatment effectiveness and guiding treatment decisions; current guidelines on DMO management recommend that retreatment decisions should be based on a combination of visual acuity and OCT ﬁndings [30, 37, 38]. PATHOPHYSIOLOGY OF DMO DMO Patients with diabetes are at increased risk of several comorbidities, which can be complex to manage, and DMO is associated with a substantial treatment burden for patients, caregivers, and healthcare systems. The overall visit burden, time investment and complexity of therapy may limit capacity for anti- vascular endothelial growth factor (VEGF) treatment and reduce adherence [2, 6–9]. As a result, in real-world settings, the frequency of treatment received by patients can be lower than in clinical trials, which may lead to sub-optimal visual outcomes [10–13]. DMO can occur at any stage of diabetic retinopathy and is characterised by thickening of the macula leading to impairment of central visual function [14, 15]. Leakage of ﬂuid and lipid-rich exudate into the retina, due to breakdown of the blood-retinal barrier, or focal leakage of microaneurysms distorts the retinal architecture, leading to thickening and reduced visual acuity if the centre of the macula is affected [14–17]. In addition to an increase in central subﬁeld thickness (CSFT), morphologic hallmarks of DMO visualised by OCT are the accumulation of intraretinal ﬂuid (IRF) and subretinal ﬂuid (SRF), decreased reﬂectivity in the outer retinal layers, hyperreﬂective foci, and vitreomacular traction (Fig. 1) [14, 15, 18, 19]. y p Another potential contributor to undertreatment in DMO is a lack of clear recommendations on retreatment in current treatment guidelines, particularly on the interpretation of retinal R. Khoramnia et al. 2 Fig. 1 Pathophysiology of diabetic macular oedema. A Colour fundus photograph showing exudative diabetic macular oedema and moderate non-proliferative diabetic retinopathy. B Optical coherence tomography image showing diabetic macular oedema with sub-foveal neuroretina detachment (subretinal ﬂuid), intrar- etinal cysts (intraretinal ﬂuid) and hyperreﬂective spots showing activated microglial cells (indicated with arrows). However, the association between anatomic parameters and visual outcomes is not fully deﬁned and there is a lack of clear guidance on the interpretation of OCT parameters in relation to treatment decisions or on a threshold for OCT markers to denote treatment response. In a clinical setting, retreatment decisions are often qualitative rather than quantitative and consider factors such as the pattern of DMO, involvement of the centre of the fovea, integrity of the inner and outer retinal layers, presence and quantity of hyperreﬂective foci, and extension/reﬂectivity of retinal cysts. Other considerations are visual acuity of the contralateral eye, the general systemic condition of the patient, and the ability of the patient to attend frequent appointments. PATHOPHYSIOLOGY OF DMO DMO Among these patients, rates of chronic persistent DMO (deﬁned as failure to achieve CSFT < 250 µm and a reduction in CSFT of at least 10% relative to the Week 24 visit on at least two consecutive visits) at 2 years were 44.2% with aﬂibercept, 54.5% with ranibizumab and 68.2% with bevacizumab [50]. PATHOPHYSIOLOGY OF DMO DMO Examples of response to anti-VEGF treatment are shown in Fig. 2. Examples of response to anti-VEGF treatment are shown in Fig. 2. Although CSFT has been widely used as a marker for retreatment decisions in clinical trials in DMO, often in combina- tion with visual acuity criteria, there is a large degree of variation in re-treatment thresholds applied. In trials of adaptive treatment regimens, such as pro re nata or treat-and-extend, retinal thickness thresholds for retreatment ranged from 225 to 325 µm, with or without additional criteria such as a change of >10% from the previous visit, the absence of stable measurements over consecutive visits or associated SRF and/or IRF [28, 39–44]. A lack of response to treatment is associated with poor visual outcomes for patients with DMO. In a post hoc analysis of Protocol I, eyes receiving ranibizumab plus prompt or deferred laser with higher average levels of oedema (calculated as excess CRT [≥250 µm] averaged over 52 weeks) gained 9.3 fewer Early Treatment Diabetic Retinopathy Study (ETDRS) letters than those with lower levels at the end of Year 3. Patients with the longest duration of persistent oedema (calculated as the number of study visits with CRT ≥250 µm during the ﬁrst 52 weeks of treatment) gained 4.4 fewer letters than those with the least persistent oedema [45]. The negative correlation between duration and extent of oedema and visual outcomes was suggested to be due to photoreceptor degeneration [45, 46]. p p g Although CSFT has been widely used as a marker for retreatment decisions in clinical trials in DMO, often in combina- tion with visual acuity criteria, there is a large degree of variation in re-treatment thresholds applied. In trials of adaptive treatment regimens, such as pro re nata or treat-and-extend, retinal thickness thresholds for retreatment ranged from 225 to 325 µm, with or without additional criteria such as a change of >10% from the previous visit, the absence of stable measurements over consecutive visits or associated SRF and/or IRF [28, 39–44]. o e co secut e s ts o assoc ated S a d/o [ 8, 39 ]. A lack of response to treatment is associated with poor visual outcomes for patients with DMO. CURRENT DMO MANAGEMENT AND IMPACT OF PERSISTENT OEDEMA Factors associated with an increased likelihood of persistent DMO, including high baseline CSFT and limited early visual and Eye R. Khoramnia et al. 3 ig. 2 Optical coherence tomography images showing response to anti-vascular endothelial growth factor treatment in patients with diabetic macular oedema. A An example of ‘good’ response with resolution of retinal ﬂuid after 4 monthly anti-vascular endothelial growth actor injections. B Delayed response to monthly anti-vascular endothelial growth factor treatment. wing response to anti-vascular endothelial growth factor treatment in patients with esponse with resolution of retinal ﬂuid after 4 monthly anti-vascular endothelial growth ti-vascular endothelial growth factor treatment. 3 Fig. 2 Optical coherence tomography images showing response to anti-vascular endothelial growth factor treatment in patients with diabetic macular oedema. A An example of ‘good’ response with resolution of retinal ﬂuid after 4 monthly anti-vascular endothelial growth factor injections. B Delayed response to monthly anti-vascular endothelial growth factor treatment. morphologic responses, have been shown to be predictive of long-term outcomes; therefore, the identiﬁcation of a simple biomarker of treatment response would further support the management of patients with DMO [52–55]. Furthermore, the correlation increased in groups with higher CSFT when stratiﬁed by baseline levels. However, the correlations were small to moderate at best, with changes in CSFT accounting for a small proportion of the total changes in visual acuity, leading the authors to conclude that the ﬁndings did not support CSFT as a surrogate for BCVA [60–63]. A further post hoc analysis of patients receiving ranibizumab and/or laser photocoagulation in the RESTORE/RESTORE-extension studies showed a low correlation between CSFT and BCVA at baseline, which was lost over time [64]. Low to moderate correlations were also seen in a number of smaller retrospective cohort analyses or consecutive case series, although further small studies showed a lack of signiﬁcant correlation [23, 65–71]. RELATIONSHIP BETWEEN OCT MARKERS OF RETINAL FLUID AND VISUAL OUTCOMES CSFT is reﬂective of a number of parameters and pathophysio- logical processes, which is assumed to be based to a large degree on the contribution of retinal ﬂuid. However, a study using a deep- learning approach to assess the correlation between CSFT and IRF or SRF volume showed that CSFT is only partly driven by IRF, and not SRF, volume at baseline and during anti-VEGF treatment and is therefore not a direct measure of exudative activity. Using SD-OCT images from 656 patients from Protocol T, a moderate correlation was seen between CSFT at baseline and IRF alone (0.688) or IRF and SRF combined (0.753), whereas the correlation between CSFT and SRF was low (0.408). Under anti-VEGF therapy, the correlation between CSFT and IRF alone (0.797) and IRF and SRF combined (0.805) increased to high, whereas the correlation between CSFT and SRF alone decreased (0.082) [24]. This and other recent studies using artiﬁcial intelligence approaches suggest that retinal ﬂuid volume may be a more reliable biomarker for the monitoring of DMO than CSFT [22, 24, 26]. In terms of the impact of individual ﬂuid types on visual outcomes, a post hoc analysis of the RESTORE study showed that patients treated with ranibizumab, laser photocoagulation, or both, with baseline intraretinal cystoid ﬂuid (IRC) height ≤380 µm had better BCVA than those with IRC > 380 µm at both baseline and Month 12; IRC height at baseline was also a better predictor of outcomes than CSFT. However, in patients followed up through the RESTORE-extension study, there was no signiﬁcant difference between IRC groups at Month 36 [64]. A moderate negative correlation between IRC height and BCVA was also seen in a retrospective case series of 66 patients that had not received treatment with anti-VEGFs in the prior 3 months or steroids in the prior 6 months [69]. Conversely, a retrospective cohort study of 119 patients receiving ranibizumab showed no signiﬁcant correlation between IRC and BCVA [68], while a study of 159 patients receiving bevacizumab showed that baseline ‘severe IRF’ (≥50% of the linear scan of the horizontal raster scan of the fovea) was signiﬁcantly more likely in eyes gaining 3 or more lines of BCVA compared with eyes that lost 3 or more lines. Similar ﬁndings were also seen in patients with more moderate vision changes (gain or loss of ≥1 line of vision). RELATIONSHIP BETWEEN OCT MARKERS OF RETINAL FLUID AND VISUAL OUTCOMES In the Cleveland Clinic study of eyes receiving anti-VEGF therapy, with the same measure of ﬂuctuation, there was a mean difference of 6.87 ETDRS letters over 12 months per 100 µm CSFT SD and the difference between the quartiles with the highest and lowest ﬂuctuations was 9.7 ETDRS letters after 12 months [73]. These ﬁndings suggest that CSFT ﬂuctuations over time may be prognostic of visual outcomes in patients with DMO treated with anti-VEGFs. g p Many analyses on the association between ﬂuid and visual outcomes rely on qualitative rather than quantitative assessment of ﬂuid parameters, which may not provide a complete reﬂection of this relationship e.g. measures of IRC height may only take the highest cyst into account and only a moderate correlation exists between SRF ﬂuid volume and ﬂuid height at baseline and during treatment [24]. A volumetric analysis of SD-OCT images from eyes receiving anti-VEGF treatment in the Protocol T trial using a deep- learning algorithm showed signiﬁcantly higher IRF and SRF in eyes with worse BCVA at baseline, and for IRF after a year of treatment. SRF had a stronger association with BCVA than IRF, with every 10 nL reduction in ﬂuid in the central fovea translating to an improvement in ETDRS letter score of 0.34 and 0.15, respectively, during Year 1. Although the presence of SRF was associated with worse BCVA and higher IRF volume at baseline, and with greater improvements in BCVA at each assessment through 12 months of treatment, there was no difference in BCVA or IRF between eyes with or without SRF after 12 months [22]. A retrospective cohort study using a similar approach to quantify IRF, SRF, and total retinal ﬂuid showed that presence of IRF or SRF after 12 months of anti-VEGF treatment was associated with signiﬁcantly lower BCVA. In a comparison of ﬂuid volume quartiles (quartile 1 having the lowest volume), IRF alone was associated with a signiﬁcant difference in BCVA for the second, third, and fourth ﬂuid quartiles of −2.23, −4.41, and −8.63 letters, respectively, at 1 year; SRF was associated with a signiﬁcant difference in the fourth quartile only (of −5.38 letters); a combination of the two was associated with signiﬁcant differences in the third and fourth quartile, of −4.79 and −8.85 letters, respectively [26]. RELATIONSHIP BETWEEN OCT MARKERS OF RETINAL FLUID AND VISUAL OUTCOMES Generally, in clinical studies in DMO, including treatment with anti-VEGF, laser, or corticosteroids, BCVA improvement in response to treatment is accompanied by a reduction in retinal thickness [32, 39, 49, 56–58]. However, the nature of the relationship between visual outcomes and retinal thickness and whether a direct association exists is unclear. CSFT is the most used OCT biomarker in DMO management based on the association between central involvement of DMO and visual acuity, and greater reproducibility compared with other measures of retinal thickness such as foveal centre point thickness [18, 59]. Post hoc analyses of multiple clinical trials (Protocol T, TREX- DME, and DAVE trials using anti-VEGF therapies; TYBEE and HULK trials of corticosteroids; and the Protocol I trial of focal/grid laser) showed a correlation between CSFT and BCVA at baseline and following treatment, or in change in CSFT and BCVA over time. Generally, in clinical studies in DMO, including treatment with anti-VEGF, laser, or corticosteroids, BCVA improvement in response to treatment is accompanied by a reduction in retinal thickness [32, 39, 49, 56–58]. However, the nature of the relationship between visual outcomes and retinal thickness and whether a direct association exists is unclear. CSFT is the most used OCT biomarker in DMO management based on the association between central involvement of DMO and visual acuity, and greater reproducibility compared with other measures of retinal thickness such as foveal centre point thickness [18, 59]. f While a strong correlation may not exist between CSFT and BCVA at discrete timepoints or based on a speciﬁc difference between two timepoints, a more recent post hoc analysis of data from the Protocol T and Protocol V studies and a retrospective cohort study at the Cleveland Clinic (Cleveland, OH, USA) have shown a signiﬁcant correlation between increased ﬂuctuations in CSFT over the course of anti-VEGF treatment and worse visual outcomes [72, 73]. Based on data from the Protocol T and V trials (in eyes receiving anti-VEGF therapy or focal/grid laser), there was a difference of 1.61 and 3.04 ETDRS letters, respectively, between Eye R. Khoramnia et al. 4 BCVA outcomes than those without and patients receiving combination treatment of ranibizumab plus laser had similar outcomes regardless of SRF presence or absence [64]. patients in quartiles with the highest and lowest CSFT ﬂuctuations (measured as the standard deviation [SD] of a patient’s CSFT over treatment) after 12 months [72]. RELATIONSHIP BETWEEN OCT MARKERS OF RETINAL FLUID AND VISUAL OUTCOMES The authors suggested that eyes with less IRF have more baseline macular ischaemia and thus less room for improvement, or that greater IRF at baseline may simply allow for a more signiﬁcant reduction with anti-VEGF therapy, resulting in improved BCVA [23]. p y In addition to CSFT and retinal ﬂuid, ellipsoid zone integrity and, in particular, the relative ellipsoid zone reﬂectivity ratio has been identiﬁed as a potential biomarker for therapy surveillance and prediction of visual acuity outcomes. A longitudinal study showed a correlation between elipsoid zone integrity and visual acuity from baseline to Year 5, demonstrating the relationship beyond 1 year of therapy [74]. Another study assessed semi-automated quantiﬁcation of retinal and choroidal biomarkers on OCT in patients with diabetic retinopathy complicated by macular oedema. All three OCT biomarkers evaluated—number of hyperreﬂective foci, ellipsoid zone reﬂectivity ratio, and choroidal vascularity index—have been suggested to correlate with visual acuity change or treatment outcomes. The study demonstrated excellent reproducibility of these biomarkers on SD-OCT with and without enhanced depth imaging mode, regardless of the presence of macular oedema [75]. A further retrospective review of visual outcomes in DMO patients receiving anti-VEGF therapy showed the extent of ellipsoid zone and external limiting membrane disruption at 12 months is negatively correlated with the area and number of intraretinal cysts at baseline [76]. py g p In a sub-analysis of the RISE and RIDE trials, SRF at baseline was predictive of better visual outcomes following treatment with ranibizumab [25]. A retrospective cohort study of eyes treated with an intravitreal dexamethasone implant also showed that SRF at baseline was predictive of better visual outcomes following treatment with dexamethasone implants, with treatment-naïve eyes showing a better response than refractory eyes [71]. The VIVID-DME and VISTA-DME studies, however, showed similar BCVA gains at Weeks 52 and 100 for patients treated with aﬂibercept irrespective of baseline SRF but BCVA loss of approximately 2 letters for those with baseline SRF treated with laser compared with a gain of greater than 2 letters for those without baseline SRF [21]. A post hoc analysis of the RESTORE/RESTORE-extension studies also showed worse BCVA outcomes in patients with baseline SRF at 12 months following laser treatment, while patients treated with ranibizumab with baseline SRF had better REFERENCES Rose MA, Vukicevic M, Koklanis K, Rees G, Sandhu S, Itsiopoulos C. Experiences and perceptions of patients undergoing treatment and quality of life impact of diabetic macular edema: a systematic review. Psychol Health Med. 2019;24:383–401. 30. Schmidt-Erfurth U, Garcia-Arumi J, Bandello F, Berg K, Chakravarthy U, Gerendas BS, et al. 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Injection frequency and response to bevacizumab monotherapy for diabetic macular oedema (BOLT Report 5). Br J Ophthalmol. 2013;97:1177–80. AUTHOR CONTRIBUTIONS All authors made substantial contributions to the conception and design of this review article and interpreting the relevant literature, contributed to the writing and review of the article and approved the ﬁnal manuscript. Correspondence and requests for materials should be addressed to Ramin Khoramnia. Reprints and permission information is available at http://www.nature.com/ reprints ACKNOWLEDGEMENTS 56. Brown DM, Emanuelli A, Bandello F, Barranco JJE, Figueira J, Souied E, et al. KESTREL and KITE: 52-week results from two phase III pivotal trials of broluci- zumab for diabetic macular edema. Am J Ophthalmol. 2022;238:157–72. Medical writing and editorial support under the guidance of the authors was provided by Susan Browne, PhD, of Novartis Ireland, Ltd, in accordance with Good Publication Practice (GPP 2022) guidelines (http://www.ismpp.org/gpp-2022). Med- ical writing support was funded by Novartis Pharma AG. zumab for diabetic macular edema. Am J Ophthalmol. 2022;238 57. Diabetic Retinopathy Clinical Research Network, Elman MJ, Aiello LP, Beck RW, Bressler NM, Bressler SB, et al. Randomized trial evaluating ranibizumab plus Eye R. Khoramnia et al. 7 COMPETING INTERESTS Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. RK reports grants from Chengdu Kanghong; grants, personal fees, and nonﬁnancial support from Alimera, Bayer, Novartis, Roche, and Zeiss; and personal fees and nonﬁnancial support from Allergan, Apellis, and Heidelberg Engineering. QDN reports consulting fees from Boehringer Ingelheim, Genentech, Kriya, and Regeneron. PJK reports research support from Bayer, Novartis, RenegxBio, and Roche; honoraria for advisory boards from Apellis, Bayer, Boehringer Ingelheim, Biogen, Novartis, Roche, and Viatris; lecture fees from Bayer, Pﬁzer, and Zeiss; support for meetings/travel from Apellis, Bayer, and Roche; advisory board membership for Apellis and Novelty Nobility; equity ownership in ArcticDx; and medical writiting support from Novartis. LSR reports payment/honoraria from Bayer and Novartis; support for meetings/travel from Bayer, Novartis, and Roche; and participation in advisory boards for Bayer, Novartis, and Roche. SV reports consulting fees from Abbvie, Alimera, Bayer, Boehringer Ingelheim, Novartis, Roche, and Zeiss; payments/honoraria from Abbvie, Alimera, Apellis, Bayer, Novartis, Roche, and Zeiss; and participation in advisory boards for Abbvie, Alimera, Apellis, Bayer, Boehringer Ingelheim, Novartis, and Roche. MA is an employee of OcuTerra Therapeutics and was an employee of Novartis at the time the manuscript was initiated. FI is an employee and shareholder of Novartis Pharma AG. NE reports grants to her department from Bayer and Novartis; consulting fees from Alcon, Allergan, Apellis, Bayer, Novartis, and Roche; payments/honoraria from Allergan, Apellis, Bayer, Novartis, and Roche; and support for meetings/travel from Roche. 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https://openalex.org/W2982824610	http://deepblue.lib.umich.edu/bitstream/2027.42/149976/1/abhw_1.pdf	English	null	MNCs, NTBs and "New Protectionism": Trade Barriers in an Era of Global Capital	Deep Blue (University of Michigan)	2,019	cc-by	56,890	Doctoral Committee: Professor William R. Clark, Texas A&M University, Co-Chair Assistant Professor Iain G. Osgood, Co-Chair Professor Alan V. Deardor Dr. Andrew M. Kerner, Michigan State Univ. & Center for Political Studies Associate Professor Robert W. Mickey This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. by Alton B.H. Worthington A dissertation submitted in partial fulllment of the requirements for the degree of Doctor of Philosophy (Political Science) in The University of Michigan 2019 Alton B.H. Worthington abhw@umich.edu 0000-0002-0535-812X This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. DEDICATION This dissertation is dedicated to: Prof. D. Michael Shafer, who saw something in me and set me on this path, and to whom I owe immeasurable thanks for a lifetime of inquiry, Prof. D. Michael Shafer, who saw something in me and set me on this path, and to whom I owe immeasurable thanks for a lifetime of inquiry, George Wesley Hale, likely my family's rst quantitative social scientist, whose work under much tougher conditions inspired me, and whom I would have loved to meet, and George Wesley Hale, likely my family's rst quantitative social scientist, whose work under much tougher conditions inspired me, and whom I would have loved to meet, and Alton Homer Hale, my rst social science instructor, whom I miss dearly. I hope he would have enjoyed reading this work as much as I would have enjoyed arguing about it with him. Alton Homer Hale, my rst social science instructor, whom I miss dearly. I hope he would have enjoyed reading this work as much as I would have enjoyed arguing about it with him. ii ii ACKNOWLEDGEMENTS One of the consequences of a project taking as long to complete as this dissertation has is that you accumulate a large community of people who helped you along the way. I must confess that, at times, I was worried the acknowledgments would take longer to write than the dissertation. Throughout the creation, development, and completion of this work, I've been lucky to have the support of a group of people too expansive to include in totality here. However, I'd like to do my best to acknowledge the support I've received, despite the risk of leaving some individuals out. This section should be considered incomplete at best, and I oer profound apologies to friends, colleagues, partners of friends and colleagues, and others who I've failed to include as I rush to complete this. You have all changed me in profound ways, and for that I am immeasurably grateful. In my time at Michigan, I've had the privilege to share space with spectacular colleagues, including former students. The community at Michigan was one of the things that kept me going. Without the support of the communities in the Polit- ical Economy Workshop, the Interdisciplinary Workshop in American Politics, the Conict and Peace Research and Development seminar, and the former Nameless Internal Seminar, I wouldn't have learned the craft of collaboration as quickly. I was lucky enough to have Paul Poast as a peer mentor when I started at Michigan. Paul's superlative character has been discussed elsewhere and by others, so what meager words I could add about his teaching, his mentorship, or his collegiality seem unnec- essary. He brings out the absolute best in those around him, and I was lucky to be iii around him. It's part of why I wished so hard that while was a young professor at my alma mater (Rutgers) that he would also coach the football team. (Needless to say, he'd be disappointed if I so thoroughly forgot about comparative advantage that I'd consider that a good idea.) It's no exaggeration to say I would run through brick walls for Paul. Alongside Paul, other more senior grad students taught me the ropes and remained good friends. William MacMillan taught me stats and skepticism. David Smith taught me intellectual integrity and the true meaning of archival research. Neill Mohammad taught me perseverance and how to be a better teacher and course administrator. ACKNOWLEDGEMENTS Without Cassandra Graftröm's friendship and help, navigating grad school would have been much less fun and much less intellectually stimulating. I also had the joy of watching former students become colleagues and successful in their own lives. Among them, Geo Lorenz, Meredith Blank, Dustin Gamza, Jason Davis (who provided feedback on various parts of this project and to whom I owe a great debt), Hakeem Jeerson, and Chris Skovron remained close, and I owe them my gratitude. I met Jule Krüger rst when she visited Michigan (at the Center for Political Studies) from Essex. Later, she returned to Ann Arbor in a variety of research roles, and her steadfast dedication to truth, openness, and research fundamentals taught me a great deal. I'm lucky to know her and all of these other wonderful scholars. Among my other grad student peers, I owe particular thanks to a few others. Vincent Arel-Bundock, who always pushed me to do better, was always present for an intense conversation about political economy or programming in R. To this day, I cherish our conversations, which always leave me feeling better and full of new research ideas. I was especially lucky to share time with amazing peers. I shared many hours in the classroom and outside it with Matthew Wells, Tim Ryan, and Kristyn Karl. Having them in my life made it better. I'd also be remiss if I didn't thank Laura Seago, Elizabeth Mann, and Diana Greenwald for their support. While they arrived more recently, Logan Woods, Erin Cikanek, and Alexander Furnas have iv also been great friends and colleagues. I particularly owe Erin and Alexander thanks for their support in the nal stretches of my dissertation. Each of them pushed me at the end, and provided help in ways large and small. Having them there at the end has been essential. But I would not have made it through the rst years of grad school, nor beyond it, without Jessica Steinberg, Richard Anderson, Trevor Johnston, or Logan Casey. Each of them is an exceptional person. They are brilliant, but more importantly they are good people. Each of them changed my life in ways I could not have imagined, and I have grown as a person in their presence. When I miss the long evenings working on problem sets or grading papers, it is because I miss their company. ACKNOWLEDGEMENTS Having all of these wonderful people as colleagues is a large part of what made my experience so rich. Even among all these great people, I must specically acknowledge the support and friendship of Shaun C. McGirr. I'm not exactly sure why we clicked in such a way early on in grad school, but Shaun became like a brother to me. We shared some incredible travel experiences, long evenings at the oce, and he carpooled back to the East Coast with me when I was traveling for holidays. All along, we enjoyed conversations about politics and research and the philosophy of science which were exactly what I hoped to experience from grad school. More importantly, we talked about life. At times when I considered throwing in the towel, Shaun pushed me to keep going, and was always there to read a draft or workshop an idea. Perhaps no other person outside my committee saw as much of this project as Shaun. I owe him immensely for his support on the dissertation and beyond it, which just about makes up for the gas money and bridge tolls he owes me. Outside of the department, I was supported by a community of people who kept me centered and gave me perspective. One of the people who was there from the beginning to the end was Jimmy Curtiss. He kept me caeinated, kept me company, and at times, kept me sane. Conversations with him were always lively and always v v important, even if they weren't on heavy topics. I only wish I was well-read enough to keep up with him at times. The rst new person I met in Ann Arbor, my roommate and friend Tyler Brown, gave me perspective on the later stages of grad school, although I took my time in using the knowledge. Although he was excellent at explaining his groundbreaking research in Kinesology to a political scientist, I fear I never reciprocated to the same degree. He welcomed me into his home and made my rst years in Ann Arbor much nicer. Marcus Wolverton has been a great friend, and the time we've shared has given me the energy to continue. I must also thank Anthony Musci and Greg Mercier for their constant support and valuable perspective. ACKNOWLEDGEMENTS They both always knew when to bug me about the work and when to invite me to work on projects outside the work as a reset and refresh. I only wish I had gained their skills at project management. Although we're separated by distance, Dina Arèvalo has been a constant companion. Her determination and sheer writing output inspires me, and her curiosity draws out the best in others. Our conversations have kept me going, even when it felt like projects were dead-ends. In recent years, Bryan Hart has been a great friend and a source of insight on topics of intellectual property. His cocktail making skills would be reason enough to spend time with him, but he also provided me with invaluable advice and perspective when I felt unable to nish. For teaching me about data visualization, for always oering help, and for oering space for coworking which helped me stay on track in the phase of revisions, I can't thank Amy Cesal enough. I regret not using the tools she helped me hone in this project, but I look forward to using what she's shown me in the future. For his support as the disseration and the degree neared the nish, I also owe Ryan Echlin my gratitude. He has also been an invaluable help in navigating resources, on campus at Michigan and beyond. But most importantly, he has been my wrenching buddy for the motorcycling hobby which kept me going when work got tough. All of these people have given me help, support, and advice which helped me continue moving forward, even when I felt vi a bit rudderless. While colleagues and advisors have been great, I couldn't have done it without these people. Many faculty I met at Michigan shaped my thinking in ways great and small. Without meeting Jana Von Stein, Scott Page, and Anna Grzymala-Busse, my in- tellectual world would have been much smaller and much less interesting. Robert Franzese was an important force in shaping my thinking and my exibility, both in political economy and empirical research. The times I spent teaching for and inter- acting with James Morrow made me a better scholar. Christian Davenport welcomed me, a weird political economist, into a growing community of conict scholars, a com- munity I cherish, despite the dierences in our inquiry. ACKNOWLEDGEMENTS I've learned important lessons about how to be a scholar in the world from him, and for that I am thankful. Allan Stam, before his departure to lead the Batten School at Virginia, was an advisor who pushed me to be better in the classroom and in my own research. He oered me opportunities to work on projects I couldn't have imagined before grad school, and that led me to learn in ways which made me intellectually exible. Although he couldn't remain with this dissertation project to its completion, his input during the early days was extensive and critical. Maybe now I can write the book on the politics of the automobile that he always thought I should. I could not complete these acknowledgements without recognizing faculty with whom I've worked on the projects which were not my dissertation. Working for Anne Pitcher gave me a great appreciation for the diculties of survey research and the need to remain persistent in one's work. Her perspective and support have made my life richer. Working with Edie Goldenberg reinvigorated me and taught me the skills of navigating the university which I would have otherwise missed. That she played a role in my early days of grad school (teaching our How to be an Academic rst-year seminar, an eye-opener for a student with few such touchstones in his own life) and the later days (working on her amazing student voter mobilization project) vii seems both poetic and lucky. To Ken Kollman, co-author and advisor, I can only say: Thank you for everything, and I'm sorry my revisions on our work were so late. I've worked with Ken on two projects which challenged me in ways my other work had not, and his mentorship within and beyond the projects has been essential. Without the help of all of these faculty, I would have been unable to make it to the completion of this project. The members of my committee, who have shaped my intellectual life, deserve special acknowledgement. I arrived at Michigan with a lot of ideas but not necessarily the skills or knowledge to develop them. Clearly, one comes to grad school to work with faculty on projects and intellectual growth. But I never imagined the advisors I'd have or the growth I'd experience. Rob Mickey has been a collaborator, an employer, and an advisor. ACKNOWLEDGEMENTS As I leave Michigan's program, he is the Director of Graduate Studies, and I can imagine no better person for that role. His enthusiasm, his broad knowledge, his interdisciplinarity and, most importantly, his empathy, make him a great advisor and a great advocate. His support and help, especially in the closing stages of the project, were invaluable to me. I only hope we can return to our collaborative work soon, which at times was much more exciting than my own. Alan Deardor's teaching (in a trade economics class where I mostly kept my head down) drove me down the path of my dissertation. Although I didn't return to burden him with my work for many years, his own research on non-tari barriers, his clear way of explaining things, and his detailed and generous feedback have been essential to the project reaching any kind of conclusion. I hope that we can collaborate in the future to produce even better work. Andrew Kerner has been a key part of my intellectual growth. I will never forget how, in every conversation we've had, I've always come away both more condent and more hopeful about progress. He humored me early on in grad school when I had wild ideas about ination targeting, and helped to ground me when those wild ideas weren't tractable. My rst independent project from start viii to nish happened with Andrew's help, and only hope there is more to come. I can say, without qualication, that Andrew is a person without whose presence my life would have been less rich. From his arrival at Michigan, Iain Osgood has been a critical part of my academic life. Beyond the fact that I nally had someone to talk about rm-centric theories of trade with, his presence has made Michigan an altogether better place. He is a commensurate scholar, but more importantly, he is a outstanding person. Without his kind, but persistent, pressure, I doubt my work would have been as good, or as complete. Most of all, I have to acknowledge the contributions of my chief advisor and co- chair, Bill Clark. Bill and I share a common advisor - D. Michael Shafer - from our respective times at Rutgers, and it was my great luck in life that Michael connected us. ACKNOWLEDGEMENTS I can say without hesitation that no single person changed the way I look at the world like Bill has. From his way of framing research questions and papers to his unique skill at creating analogies which lend insight, Bill has given me many lessons. My own teaching and research is lled with little reminders of his eect on my life and my mind. In the time I spent being taught by Bill, teaching alongside Bill, working with Bill, and just conversing with Bill, I was always growing. I know of no greater joy. Finally, I must recognize the love and support which my family gave to me. My aunt Cindy and uncle Don Readlinger have been persistent cheerleaders. They have always been generous with their time, their prayers, and, when a conference ight delay or archival work trapped me in New Jersey, a last-minute place to stay back in the home state I miss so much. Without them, this whole process would have been more dicult, and more lonely. My aunt Sharon, whose lab research and Masters degree experience gave her particular insight into my grad school stress, has always been there for me. Even before I took on this grad school thing, she has been like another parent to me. I cannot thank her enough for everything. But most of all, I ix have to acknowledge the contributions of my mother, Ellen Hale. I can't imagine it's easy putting up with a kid like me, but she did, and she was steadfast in her support. I'm sure that, at times, she thought I would never nish, but she never hinted at it. At other times, when I thought I would never nish, she kept me going. She's one of the smartest people I know, and I would be nowhere in life were it not for the sacrices she made and the lessons she taught me. A portion of everything I do comes from her support. However, all mistakes in the work that follows are solely my responsibility. have to acknowledge the contributions of my mother, Ellen Hale. I can't imagine it's easy putting up with a kid like me, but she did, and she was steadfast in her support. I'm sure that, at times, she thought I would never nish, but she never hinted at it. TABLE OF CONTENTS DEDICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv LIST OF APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi CHAPTER I. Introduction: A Global Economy with National Policies . . . 1 II. A Typology of Trade Barriers: Understanding the Eects of New Protectionism . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.2 Existing Research on Measurement, Eects, and Politics of NTMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.3 Conceptualizing Non-tari Measures . . . . . . . . . . . . . . 18 2.4 Motivating a New Typology of Trade Barriers . . . . . . . . . 20 2.5 A Typology of Barriers to Trade . . . . . . . . . . . . . . . . 25 2.5.1 Location-discriminating Costs: Where it's Made . . 27 2.5.2 Characteristic-discriminating Costs: How it's Made 29 2.5.3 Firm-discriminating Costs: Who Makes It . . . . . . 32 2.5.4 Indiscriminate Costs . . . . . . . . . . . . . . . ACKNOWLEDGEMENTS At other times, when I thought I would never nish, she kept me going. She's one of the smartest people I know, and I would be nowhere in life were it not for the sacrices she made and the lessons she taught me. A portion of everything I do comes from her support. However, all mistakes in the work that follows are solely my responsibility. To all these people and many more, I can only say: Thank you. x x TABLE OF CONTENTS . . . 33 2.5.5 Limitations of this Typology . . . . . . . . . . . . . 35 2.6 Considering Winners and Losers from Market Access Barriers - Single-Country/Unilateral Policy Change . . . . . . . . . . . 36 2.6.1 Location-discriminating Costs . . . . . . . . . . . . 37 2.6.2 Characteristic-discriminating Costs . . . . . . . . . 37 2.6.3 Firm-discriminating Costs . . . . . . . . . . . . . . 38 . Introduction: A Global Economy with National Policies . . . 1 . A Typology of Trade Barriers: Understanding the Eects of New Protectionism . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.2 Existing Research on Measurement, Eects, and Politics of NTMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.3 Conceptualizing Non-tari Measures . . . . . . . . . . . . . . 18 2.4 Motivating a New Typology of Trade Barriers . . . . . . . . . 20 2.5 A Typology of Barriers to Trade . . . . . . . . . . . . . . . . 25 2.5.1 Location-discriminating Costs: Where it's Made . . 27 2.5.2 Characteristic-discriminating Costs: How it's Made 29 2.5.3 Firm-discriminating Costs: Who Makes It . . . . . . 32 2.5.4 Indiscriminate Costs . . . . . . . . . . . . . . . . . . 33 2.5.5 Limitations of this Typology . . . . . . . . . . . . . 35 2.6 Considering Winners and Losers from Market Access Barriers - Single-Country/Unilateral Policy Change . . . . . . . . . . . 36 2.6.1 Location-discriminating Costs . . . . . . . . . . . . 37 2.6.2 Characteristic-discriminating Costs . . . . . . . . TABLE OF CONTENTS . 37 2.6.3 Firm-discriminating Costs . . . . . . . . . . . . . . 38 xi 2.6.4 Indiscriminate Costs . . . . . . . . . . . . . . . . . . 39 2.7 Considering Winners and Losers from Market Access Barriers - Two/Multi-country Policy Changes . . . . . . . . . . . . . . 39 2.7.1 Location-specic Costs . . . . . . . . . . . . . . . . 40 2.7.2 Characteristic-specic Costs . . . . . . . . . . . . . 42 2.7.3 Firm-specic Costs . . . . . . . . . . . . . . . . . . 47 2.7.4 Indiscriminate Costs . . . . . . . . . . . . . . . . . . 51 2.8 Summarizing Winners and Losers . . . . . . . . . . . . . . . 54 2.9 Conclusions and Moving Forward . . . . . . . . . . . . . . . . 55 III. Market Access Barriers and Formation of Protection-Seeking Coalitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 3.1 Considering NTMs and Existing Explanations for Trade Barriers 60 3.2 Motivations for Demanding and Supplying Protection . . . . 63 3.2.1 Who Can Lobby for Protection? . . . . . . . . . . . 65 3.2.2 Revisiting A New Typology of Market Access Barriers 67 3.2.3 Consumers, Government, and Firms . . . . . . . . . 69 3.3 Lobbying for Protection . . . . . . . . . . . . . . . . . . . . . 71 3.3.1 On location-discriminating policies . . . . . . . . . . 72 3.3.2 On characteristic-discriminating policies . . . . . . . 75 3.3.3 On rm-discriminating policies . . . . . . . . . . . . 79 3.3.4 On policies that add costs indiscriminately . . . . . 84 3.3.5 Considering all policies at once, and government in- centives . . TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . 86 3.3.6 Hypotheses for unilateral policy creation . . . . . . 90 3.4 Discussion and Conclusions . . . . . . . . . . . . . . . . . . . 92 IV. Industry Characteristics and the Contours of Market Access Restrictions in the US . . . . . . . . . . . . . . . . . . . . . . . . 95 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 4.2 Some Existing Discussions of Trade Barriers and Causes of Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 4.3 Hypothesized Patterns of Market Access Barriers . . . . . . . 100 4.4 UNCTAD's TRAINS Database and Market Access Barriers . 102 4.4.1 Translating NTMs to Market Access Barriers . . . . 104 4.5 Data and Analysis . . . . . . . . . . . . . . . . . . . . . . . . 106 4.5.1 Market Access Barriers . . . . . . . . . . . . . . . . 107 4.5.2 Industry Characteristics . . . . . . . . . . . . . . . . 109 4.5.3 Product/Industry Coding and Concordance . . . . . 112 4.5.4 Data Summary . . . . . . . . . . . . . . . . . . . . 114 4.5.5 Model Design . . . . . . . . . . . . . . . . . . . . . 116 4.6 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 4.6.1 Location-discriminating Costs . . . . . . . . . . . . 117 xii 4.6.2 Characteristic-discriminating Costs . . . . . . . . . TABLE OF CONTENTS 122 4.6.3 Firm-discriminating Costs . . . . . . . . . . . . . . 126 4.6.4 Indiscriminate Costs . . . . . . . . . . . . . . . . . . 130 4.6.5 Summary of Results . . . . . . . . . . . . . . . . . . 134 4.6.6 Areas for Future Research . . . . . . . . . . . . . . 135 4.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 xiii LIST OF TABLES Table 4.1 Summary Statistics, Full Sample . . . . . . . . . . . . . . . . . . . . 115 4.2 Correlation of Barrier Count Across Measures . . . . . . . . . . . . 115 4.3 Summary of model results . . . . . . . . . . . . . . . . . . . . . . . 118 4.4 OLS with clustered standard errors . . . . . . . . . . . . . . . . . . 119 4.5 Log-linear model with clustered standard errors . . . . . . . . . . . 120 4.6 Negative Binomial Model with clustered standard errors . . . . . . . 121 4.7 OLS with clustered standard errors . . . . . . . . . . . . . . . . . . 123 4.8 Log-linear model with clustered standard errors . . . . . . . . . . . 124 4.9 Negative Binomial Model with clustered standard errors . . . . . . . 125 4.10 OLS with clustered standard errors . . . . . . . . . . . . . . . . . . 127 4.11 Log-linear model with clustered standard errors . . . . . . . . . . . 128 4.12 Negative Binomial Model with clustered standard errors . . . . . . . 129 4.13 OLS with clustered standard errors . . . . . . . . . . . . . . . . . . 131 4.14 Log-linear model with clustered standard errors . . . . . . . . . . . 132 4.15 Negative Binomial Model with clustered standard errors . . . . . . . 133 A.1 Overdispersion Assumption Test . . . . . . . . . . . . . . . . . . . . 140 B.1 Recoding of TRAINS NTM Measures . . . . . . . . . . . . . . . . . 141 xiv A. Testing the Negative Binomial Model's Overdispersion Assumption . . 139 B. Coding of Market Access Barriers, UNCTAD TRAINS NTM Codebook 141 Appendix A. Testing the Negative Binomial Model's Overdispersion Assumption . . 139 B. Coding of Market Access Barriers, UNCTAD TRAINS NTM Codebook 141 xv ABSTRACT The post-World War II global economy is characterized by two broad phenomena: liberalization of tari barriers that restricted trade across a broad swath of industries and countries, and the expansion of global direct investment and the rise of supply chains in goods trade. However, the secular decline of taris has not meant universal liberalization. In some industries, taris and tari-like policies still restrict trade. In others, measures that are unlike taris have either become more prominent as taris fell or have risen to provide alternate protection. Work in political science and economics has advanced a wide variety of explana- tions for the transition from taris to non-tari measures generally, or for the presence or absence of specic non-tari trade instruments in particular. A more limited body of work has examined the substitution across policy instruments, but has not at- tempted to generalize beyond the policies under consideration. We assert that part of the limitation in this work arises from the traditional dichotomy of taris versus non-tari measures. To resolve this shortcoming and advance the discussion of trade politics and trade agreements, this dissertation advances a new framework for considering trade- distorting policies that apply both at- and behind-the-border. Policies are categorized according to how they apply costs - according to the location of a good's production, the content in or process of production, or the rm that produces it. Policies that raise costs indiscriminately are also considered. We explain the logic of this typology and the distributive consequences of each policy. Then, we explain the distributive consequences of imposing these policies in xvi a single-country and two-country interaction. From these distributive outcomes, we introduce a theory of protection-seeking where rms in a given industry lobby for lev- els and varieties of protection that serve their interests in light of the preferences of other politically-salient rms. Industry characteristics like foreign investment, prod- uct dierentiation, industry concentration, and rm eciency all work to shape the types of trade-distorting instruments which industries may obtain from responsive governments. With this lobbying logic established, we test that theory against data from the United States in 2012. Comparing industry characteristics against the pres- ence or absence of the four types of policies across more than 3000 types of products, we nd support for some implications of the lobbying theory, but also nd areas for further inquiry. ABSTRACT This dissertation contributes to the wider discussion of evolving protectionism in political economy through clarication, by advancing a new logic of protection- or liberalization-seeking coalitions that considers multinational rms, and through an investigation of the contours of protection in the United States, a critical power in the global goods and investment market. In doing so, it moves the discipline closer to understanding the deep links between global investment ows and the politics of global trade and trade policy. xvii Introduction: A Global Economy with National Introduction: A Global Economy with National Policies Policies The second era of globalization, following the Second World War, has been char- acterized both by an increase in global trade, due in no small part to reductions in taris, and an increase in global capital ows as rms have built global supply chains and distributed production across national borders. There has also been a signicant increase in intraindustry trade cars from Europe being exported to the US while cars from the US are exported to Europe, for instance and intrarm trade companies acting as exporter on one side of a transaction and importer on the other that has made the global goods trade look somewhat dierent than the wine for cloth of classical trade theory. To the same extent, as the nature of trade has changed, so too has the nature of trade protection. As taris across many products have been reduced to zero or near zero, the focus of many recent trade agreements have turned to the non-tari measures that distort or restrict trade. These policies, which have risen as a consequence of greater domestic regulation and alternative attempts to protect rms have distributive consequences that may be like or unlike those of taris. The work that follows is comprised of three papers that examine the issue of non-tari barriers and their comparison to taris. These papers introduce a new framework for considering trade-distorting policies, 1 develop a new theory of industrial demand for dierent kinds of policies, and then test that theory using a database on non-tari measures. In Chapter II, we introduce a new way of classifying policies based not on whether the policy is a tari or not, or though identifying each trade-distorting policy as unique, but rather by how additional costs imposed by the policy apply to a product. This new typology considers policies as one of four types: location-discriminating, characteristic-discriminating, rm-discriminating, and indiscriminate. Location-discriminating policies are those, like taris, which impose additional costs or restrictions on a product based on where it is produced. These kinds of policies drive price wedges between goods that are produced in dierent places. Characteristic-discriminating policies are applied based on how something is made (process) or some element of that good's content. The product standards, environ- mental standards, and labor rules that are becoming more frequent components of trade agreements, are some examples of this type of policy. Policies These policies drive price wedges between dierent varieties of good within the same broad category, or restrict sale of some varieties altogether. Firm-discriminating policies are those that add cost on the basis of who makes a product. Targeted subsidies, licensing laws, preferential purchasing rules, and the like, which give benets to certain rms, add costs to all other rms' products on the basis of the rms' identity. These policies can be persis- tent costs or can apply in such a way that rms must pay the cost upon entry to a market, regardless of where or how they produce a good. These policies drive price wedges between dierent rms' goods, regardless of how or where they are produced. The nal kind, indiscriminate cost policies, raise the costs for all producers. While these policies may have dierent eects across products due to dierences in rm size, eciency, or output, these costs apply to all. Policies that require product testing, or specic labeling, or some kinds of broader economic policy like consumption taxes, can have the eect of distorting trade. By moving beyond a discussion of taris and 2 not-taris, the dierences in the trade-distorting eects of dierent policies, and similarities of policies within the same category, is made clearer. From this typology, we then consider the distributional consequences when these policies are implemented. First, we consider a single-country case where a government raises a hypothetical policy that applies costs of only one type of cost. For a location- discriminating cost, the winners and losers from the policy divide on where a product is made. For a characteristic-discriminating one, it is the variety of good produced by a rm, and whether or not it is targeted by the policy, which determines whether it benets or loses from imposition of the policy. For rm-discriminating costs, being targeted for additional cost makes a rm a loser from the policy, but other rms are winners. Indiscriminate cost policies divide industries on how well a rm can absorb the additional costs. Some rms may be forced to exit by the extra costs, while others may bear it. The more interesting outcomes come from considering a two-country case, where policies of each type are raised by one or both countries. Policies While for some policies, like location-discriminating ones, familiar patterns of winners and losers arise, dividing on exporting versus importing rms, for others there are novel implications. In some cases, when governments raise costs on some varieties or some rms, the winners and losers divide within industries and across national borders. For considering the implications of trade agreements that harmonize standards, or that confer reciprocal access to specic rms, these intraindustry divisions are important. When raising indiscriminate costs, in one or both countries, the eects expected in the single- country case are intensied. By identifying what policies apply dierent kinds of costs, and what the eects on dierent rms within and beyond a country should be, we move one step closer to understanding the politics of trade barriers in this era of global capital. In Chapter III, we use the typology introduced in Chapter II to derive a theory In Chapter III, we use the typology introduced in Chapter II to derive a theory 3 3 of protection seeking by rms. A signicant portion of existing theory on protection- seeking has considered only taris or tari-like policies. More recent work looking to non-tari measures has usually considered them an additional xed cost of market entry. This theory instead considers how rms may choose to lobby for protection across a variety of policies. While each rm would prefer policies that make it an eect monopolist, that is not likely to occur. So, rms consider what policies to demand in light of what other rms in their industry might demand or oppose. Building o familiar endogenous tari theory logic, we consider how demands for location- discriminating, characteristic-discriminating, rm-discriminating, or indiscriminate cost policies will change in light of industry characteristics. Dierent features of an industry will shape demands for policy, such that dier- ent rms should demand dierent kinds of policies, especially in light of competition from outside rms. We assume that a variety of rms domestic rms produc- ing for the domestic market, domestic exporters, and foreign rms with a domestic presence all have the opportunity to lobby for policy. This lobbying will reect the preferences of these rms over the set of potential policies. As a result, when an industry has deeper integration with the global economy, through foreign direct investment, that industry should demand (and obtain) fewer location-discriminating policies. Policies When industries produce homogeneous products, they are unlikely to de- mand characteristic-discriminating policies, but when goods produced are heteroge- neous, politically-salient rms will demand policies that privilege the varieties they produce. Industries where a few rms are large highly concentrated industries are likely to be distorted by rm-discriminating policies, and where concentration is low policies may arise to block new entrants. The presence or absence of indiscrimi- nate costs is determined in part by the eciency of rms in the industry. When the most ecient rms in an industry might benet overall from these kinds of policies, they will lobby for them. However, when there is not a sucient mass of compara- 4 tively more-ecient rms, these sorts of costs should not be demanded, which makes them less likely. Finally, all of these policies, by adding costs to a good, should limit the use of others, to some degree. While they are not substitutes to the same degree that policies within the same type are, governments are constrained such that rms must consider which policy they prefer most (rather than demanding all) when lob- bying. This logic reects some existing theory, but also builds on the new framework to advance new hypotheses for when protection arises and what form it takes. Chapter IV tests the theory of Chapter III using data from the United States in 2012. We adapt existing data on non-tari measures from UNCTAD's TRAINS database and examine which industries obtain dierent kinds of trade-distorting poli- cies. We also use disaggregated data on industry characteristics to identify when producers of dierent products are more or less likely to demand dierent policies. Across more than 3000 product-lines, we compare industry characteristics of the pro- ducing sector and the number of policies of each type that aect those products. The results from these empirical models suggest some support for the protection- seeking politics discussed in Chapter III. As expected, the presence or absence of FDI is associated with greater or fewer location-discriminating costs. However, FDI also appears to have signicant relationships with other types of costs. Industries that produce heterogeneous goods appears to obtain dierent forms of protection than do those producing homogeneous goods. Overall industry size also covaries with the number of policies in place. A Typology of Trade Barriers: Understanding the Eects of New Protectionism Policies There appears to be consistent evidence that the presence or absence of one form of policy is associated with more or fewer policies of other types. While this was not expected by theory, it does open avenues for further inquiry. These results provide a rst picture of the relationship between the characteristics of industries and the kinds of protection they obtain. Taken together, these papers identify the more complex politics that underlie agreement on reducing trade barriers beyond taris. They also clarify when some 5 policies act as substitutes for taris, while others act as complements or imperfect substitutes. As trade agreements like TPP and TTIP focus more on policies that are not taris and policies that apply behind the border, a deeper focus on these sorts of policies, and the more complex distributional consequences thereof, is warranted. This new framework, then, helps to explain some of the reasons why protectionism in the global economy has taken the form it has, and what areas for liberalization should be possible going forward. policies act as substitutes for taris, while others act as complements or imperfect substitutes. As trade agreements like TPP and TTIP focus more on policies that are not taris and policies that apply behind the border, a deeper focus on these sorts of policies, and the more complex distributional consequences thereof, is warranted. This new framework, then, helps to explain some of the reasons why protectionism in the global economy has taken the form it has, and what areas for liberalization should be possible going forward. 6 6 2.1 Introduction Taris today are at or near zero across many products and countries. With the exception of agricultural and textile products generally and some specic products or industries in each country, import duties no longer pose a signicant barrier to trade. However, several stylized facts about international trade suggest that the progress made in liberalizing trade through reduced tari barriers has been oset by new forms of protection. These non-tari barriers distort global goods ows in a variety of ways. Some of this protection appears to be quite similar to taris, while other barriers work quite dierently. The change in protection from taris to non-taris has meant liberalization in some industries, or for some products, and not for others. In explaining the pattern of imperfect liberalization, it is necessary to understand when these policies are substitutes for taris and when they are not. The presence or absence of trade protection is motivated by the distributive consequences of policy. Policies that distort markets may divert benets from consumers towards a protected group of producers, and may redistribute among groups of producers, along factor, sector, or intra-industry lines. Comparing unlike polices or discriminating between 7 7 policies with similar consequences obscures the changes in benets that accompany the shift from taris to NTBs that has characterized much of global trade politics since World War II. The need to consider how domestic policy distorts trade ows is even more impor- tant in light of a few general facts about the global goods market today. Trade theory has evolved in light of these facts, which has led to the new trade theories of Krug- man and Melitz. These theories aim to explain the most striking fact about trade ows in the global economy: signicant ows of intra-industry trade. Rather than the wine-for-cloth trade of classical (and neoclassical) trade theories, some trade appears to be like-for-like. The United States sends Boeing wide-body jet airliners to European carriers, while Europe sends Airbus wide-body jet airliners in the opposite direction to carriers based in the US, for instance. This intra-industry trade has been a feature of the global economy throughout the past 60 years, rising in volume to its recent levels. 2.1 Introduction In 2011, intra-industry trade was between 25% and 75% of trade ows (in aggregate, not bilateral ows) in various regions of the global economy, highest among the most developed economies (Trade Analysis Branch 2013). The other strik- ing feature of global trade in goods is the degree of intra-rm trade. This kind of trade, where one economic agent is both the exporter from the sender country and the importer in the recipient country, has become an important part of the global economy. These rms sometimes produce multiple versions of similar products for dierent markets, as well. Global supply chains are a dening feature of multinational rms, and the rise of multinationals has also meant a rise in intra-rm trade. Traditionally, the main distinction made in the study of trade barriers has been between taris and non-tari measures. Given the historical dominance of taris as a means of shaping trade ows (and raising revenues), this distinction may seem ap- propriate. However, placing all other measures that distort trade ows into a single conceptual category hides important dierences among non-tari barriers and ob- 8 scures similarities between taris and some non-tari alternatives. Existing research often refers to non-tari measures in general while looking at only one policy in particular, or attempts to create a synthetic measure for non-tari barriers that computes a single at-the-border cost equivalent for the variety of policies that may distort trade beyond taris. If we seek to explain the persistence of protection across countries and industries, even in the face of secular tari declines, we must reframe how we consider trade-distorting policies to clarify, both theoretically and empirically, when policies are substitutes and when they are not. This paper introduces an alter- native framework for considering trade-distorting measures that exist at and behind national boundaries. This framework recategorizes policies as similar or dierent with regards to how costs apply to goods entering a market. In this framework, policies may add cost based on location of production, the process or content of a good's production, the rm which produced it, or indiscriminately across all varieties of a good. After reviewing existing discussions of taris and NTMs, both in measurement and eect, we outline the Market Access Costs framework and explain distributive implications of policies and liberalization in a single-country and trading-partner con- text. 2.1 Introduction In introducing this new framework and highlighting some of the dierences in distributive consequences of dierent types of market access costs, we aim to advance our understanding and discussion of trade-related policies in the present era of global markets and national policies. 1. In general, these early surveys focused on quantitative and at-the-border measures, but col- lected information on a wide variety of NTMs as individual producer responses. The collection of these measures in 1968 and 1969 produced around 800 NTM notications, broken into ve cate- gories. This collection preceded the creation of the NTM-related codes in the Tokyo Round (General Agreement on Taris and Trade 1969, 1970). 2.2 Existing Research on Measurement, Eects, and Politics of NTMs There is a large and well-established literature on the eects of taris both in economics and political science. There is also a robust literature on the economics 9 and politics of non-tari measures (NTMs), but on the whole it is less prominent than that of taris. This is likely in part a consequence of the relative recency of NTMs' importance, but also likely due to NTMs being more dicult to measure and categorize, as well as theorize about, than taris are. Even before NTMs became part of negotiations in large, multilateral trade agree- ments, these kinds of policies were known to be distortionary. When, in the years preceding the Tokyo Round of GATT, researchers began collecting information - through surveys of member states - on other policies that hampered trade ows, poli- cies were considered individually and grouped into large summary categories that were largely descriptive.1 Various classications, with inclusion or exclusion of var- ious policy types, have been used by individual researchers and intergovernmental bodies as data collection and theory have improved. As non-tari measures are primarily dened by what they are not, there is a wide variety of ways to measure and categorize NTMs. The precise denition of what a non-tari barrier (or measure) is has generally converged on key components dened by Baldwin (1970). Walter (1972) and Walter and Chung (1972) expand on this with a renement of Baldwin that focuses on the intent of policy as distortionary, noting that some policies will be distortionary as an unavoidable spillover eect. While these denitions, generally taking the form of non-tari barriers are any policy that distorts the natural ows of goods across national borders also generally focused on at-the-border measures, more recent interpretations have included behind the border measures as distortionary and as NTMs (Laird 1997; Staiger 2012; De Melo and Nicita 2018). However, as Deardor and Stern (1998) note, any taxonomy of NTMs will be incomplete precisely because NTMs are dened by what they are not. 10 In discussing the measurement of specic NTMs or NTMs in general, the com- parison has been to the eects of taris, either in price or quantity distortions. ( ) 3. This requires use of import elasticities, which are estimated in Kee, Nicita, and Olarreaga (2008b), where the overall restrictiveness index is also computed. 2. A more recent update and review of Deardor and Stern (1998) can be found in Bora, Kuwa- hara, and Laird (2002), as well. 2.2 Existing Research on Measurement, Eects, and Politics of NTMs A general summary of measurement methods is outlined in Deardor and Stern (1998), and summarized briey here.2 As NTMs often work dierently than taris, one common method of evaluating restrictiveness of NTMs is to count the number of policies and/or the degree of product coverage of dierent measures. By counting the number of policies, initially through producer reporting of these barriers, but later through more comprehensive surveys of regulatory and policy regimes, it is possible to evaluate the degree to which market entry is hampered by policy barriers. However, not every policy acts in the same fashion, and mapping these counts to measures of restrictiveness can be dicult. Other research has attempted to calculate ad-valorem tari equivalents of NTM restrictiveness. In general, these measures use a model of trade ows based on models of international trade and/or direct measurements of prices in the protected market versus global prices. Measurement of trade volume distortions, rather than price distortions, are an alternative measure of the eects of NTMs. Kee, Nicita, and Olar- reaga (2008a) does this as a rst stage for eventual computation of price eects, and later a total trade restrictiveness index across a variety of countries.3 However, Kee, Nicita, and Olarreaga (2008b) looks only to core NTMs (quantitative restrictions and tari-like NTMs) and domestic agricultural support. This is motivated by com- parability to taris, but also highlights how other NTMs function dierently, both theoretically and empirically. A more general method for evaluating the restrictiveness of NTMs is calculation of a trade restrictiveness index, which may or may not include taris in the index. This method focuses on the total distortions of trade for a particular product or across a 11 whole economy, not necessarily the form this protection takes or the specic policies that restrict trade. These are done in one of two ways, generally. One, building o of the work of Anderson and Neary (1994, 1996, 2003) and Feenstra (1995), estimates simplied barrier estimates motivated by the logic of general equilibrium models of trade, but with the general equilibrium feedbacks ignored (Kee, Nicita, and Olarreaga 2008a). Alternatively, a gravity model of trade can be used to estimate expected goods ows across borders, with taris, when known, included in the model and the non-tari barriers derived from model residuals (Hiscox and Lastner 2008; Mayer and Zignago 2005). Each of these measurement strategies has strengths and drawbacks. 2.2 Existing Research on Measurement, Eects, and Politics of NTMs Counts are relatively easy to compose, although categorization and comparison can be dicult. However, simple counts of measures do not suggest the intensity of protection around particular products within an economy. The calculation of ad-valorem equivalents eases comparison to existing measures of protection (that are largely based on tari rates, either bound or eective), but require stronger assumptions and sometimes are dicult to compute for NTM regimes that work in ways dierent from core, tari-like NTMs. The measurement challenges above have meant that, compared to taris, the pic- ture of non-tari barriers' eects on the global economy is somewhat less clear. Still, there has been signicant research on the political economy of protection through non-tari means. Some research has focused on one particular kind of NTM, or has used one policy instrument as a proxy for NTMs generally. Other work has looked to substitution across multiple instruments, or from taris to non-tari instruments. An overarching theme of this literature is that the question of whether NTMs, or even individual non-tari measures, are substitutes for taris or not remains unresolved. Both at the theory stage, involving assumptions or arguments about actors' prefer- ences, and at the point of measurement, examining the level or frequency of NTMs 12 across countries and industries, there remains a lively debate as to who demands protection in the form of NTMs, what form that demanded protection takes, when governments provide that protection, and what eect that protection has. In focusing on a single kind of NTM, or comparing NTM politics to that of tari politics, some progress has been made in understanding how these policies persist or are eliminated. Core to these arguments are assumptions of preferences of dierent groups over these policies, or ways in which these policies work similarly to or dif- ferently from taris. What follows is a sample of the literature discussing specic non-tari trade instruments and NTMs in general. Much early research on NTMs focused on comparing quantitative restrictions to taris. A signicant portion of this discussion focused on the equivalence (or not) of taris and quotas, in response to seminal work by Bhagwati (Bhagwati 1965, 1968). In responding to some of these arguments about the comparison of quotas and taris, Deardor (1987) introduces the idea that quantitative restrictions are used when actors don't believe taris will work. 2.2 Existing Research on Measurement, Eects, and Politics of NTMs By using quantitative, rather than price, measures, NTMs (quotas) may mitigate some of the short-run and longer-run concerns that arise with price measures like taris. A more recent return to the discussion of quotas and antidumping suggests that shift from taris to quotas to antidumping measures is a natural progression in some industries, and is driven in part by restrictions placed on trade distorting measures (Anderson and Schmitt 2003). The use of subsidies to support domestic industry is another means by which governments can distort trade. Rickard (2012) looks to subsidies as an alternative to taris for domestic industrial support within democracies. In testing the protec- tionist bias in majoritarian politics of Grossman and Helpman (2005) beyond taris, Rickard nds that a similar logic holds for this NTM policy. Looking to the specicity of subsidies within EU countries, Park (2012) argues that the sectoral targeting of subsidies follows a dierent logic than general state aid, and both are associated with 13 the degree of labor and capital centralization.4 This issue of targetability in industrial support is discussed and demonstrated elsewhere in broader discussions of industrial protection and political incentives related to political geography (McGillivray 2004). The role of geographic concentration in trade politics is another area where com- parisons of taris and non-tari measures have been considered. In an eort to examine what industries receive protection and adjudicate a long-running debate in the endogenous protection literature, Busch and Reinhardt (1999, 2000) turn to hard core NTM measures across industries in the US, which suggest that geographically- concentrated but politically diverse industries (industries that cover many electoral districts, but are geographically concentrated) obtain protection more than other industries. This nding is corroborated, albeit with additional complexity from con- siderations of electoral institutions and party strength, by McGillivray (2004) using European country industries. More general discussions of NTMs as protection have focused on ways in which NTMs in general are similar to or dierent from taris. The interaction of institu- tional settings and the ways in which NTMs may work dierently (or are observed dierently) than taris is one avenue of inquiry. Two large cross-national studies of poltical institutions and the provision of protectionism engaged directly with NTMs, but treated them as related (not necessarily substitutes or compliments) to taris. 4. Specically, Park nds that sectoral, targeted aid is lowest when centralization is very high or low, but general aid increases with centralization overall, supporting the idea that the logics of targeted vs general support are dierent. 2.2 Existing Research on Measurement, Eects, and Politics of NTMs In looking to democracies alone, Manseld and Busch (1995) suggests that NTBs are substitutes for taris, and are used when sectoral interests and societal interests align. However, a look beyond just democracies to compare protectionist policy in nondemocracies suggests that overall, taris are lower, but NTMs (especially qual- ity NTMs like product standards) are higher in democracies, suggesting that the Optimal Obfuscation of Magee, Brock, and Young (1989) may have some empirical 14 support (Kono 2006). support (Kono 2006). In a broader discussion of the anti-protectionist pressure that accompanied in- creased economic interdependence, Milner (1988) highlights the rising importance of NTMs, although NTMs are not discussed independently of broader anti-protectionist pressure that comes from rms with export or multinational interests. However, the arguments therein are consistent with some of the more recent arguments on anti-NTM pressure among multinational rms. Specically, Milner highlights intra- industry divisions over protectionism, which have become an important part of the NTM discussion as well. This rm-centric logic of NTM preferences also arises out of political economy models based on new-new trade theory, which highlight how industries divide over barriers to trade, both xed and variable costs (Osgood 2016). Although they are not always as clearly perceived as barriers to trade, intellectual property rights (IPR) policies have become part of the NTM discussion. (Shadlen, Schrank, and Kurtz 2005) Osgood and Feng (2017) build o of existing literature on the role of IPR harmonization in trade agreements in the US and the economics of IPR in trade. These policies generally change standards in US partner countries. Industries in the US, where rms are generally producers of new IP, are in support of or indierent to new IPR policies. There is some evidence that consumers of IP- intensive goods abroad may oppose these policies, but these appear to have dierent distributional consequences (and thus politial consequences) than some other NTMs. Technical standards can also act as barriers to trade. Evidence from the use of standards as protection in agriculture suggests that these regulatory rules function as substitutes for other forms of protection, appearing more prevalent when other forms of protection are lower (Long, Kastner, and Kassatly 2013). Another way of viewing technical standards is as xed costs of market entry. If these policies act as a xed cost of market entry, one might expect them to redistribute across rms. 2.2 Existing Research on Measurement, Eects, and Politics of NTMs Recent research, expanding on models of trade with heterogenous rms from Melitz 15 (2003), suggests that it should be the most productive rms that prefer this form of protection (Gulotty 2014; Abel-Koch 2013). The use of policies that privilege domestic producers, sometimes called buy na- tional policies, also distort trade. D.-H. Kim (2010) suggests that, compared to tari barriers, where intra-industry trade may lead to lower trade barriers, the demands for protection in public procurement are higher in cases of intra-industry trade, as rms have fewer free-riding incentives in lobbying. In the context of international agree- ments and trade liberalization, it also appears that these public procurement policies are commonly used, in part because they are more opaque, making enforcement of in- ternational agreements more dicult (Rickard and Kono 2014). This relative opacity also appears to make these policies more prevalent in democracies (Kono and Rickard 2014). Others have used the topic of NTMs to examine questions in other areas of political science. Grieco (1990) uses the negotiations over NTMs in, and evidence of subsequent compliance following, the Tokyo Round of GATT as evidence of neorealist politics in the international system. In focusing on each of the six codes on NTMs introduced and examining the relative national gains or losses from dierent potential outcomes (and eventual compliance), this at once treats NTMs much like one might consider taris - as barriers at the border for protection of national interest - and very dierent than taris, as each type of policy (national preference, subsidies, pricing rules, etc.) was used to protect vital interest groups within the respective countries in dierent ways. The dierences in compliance (and the logic for complying) across the codes suggests that not all NTMs worked the same ways, and states complied with those that suited their national interest (Grieco 1990). More recent research has focused on the dierences between dierent kinds of NTMs and the politics related to instrument selection. Evidence from Japanese trade policy suggests that electoral competition and electoral incentives drive the choice of 16 trade instruments among subsidies, voluntary export restraints, and GATT/WTO legal measures. This look to multiple measures, protectionism by other means, focuses on the politics behind using unilateral versus bilateral or international le- gal protectionism. 5. This imperfection recalls Bhagwati's Law of Constant Protection and discussions in the lit- erature cited above (Bhagwati 1988). 2.2 Existing Research on Measurement, Eects, and Politics of NTMs Naoi (2009) takes particular note of the issues that arise when looking to only one measure, framing it as a selection bias, suggesting a degree of substitutability. In particular, Naoi argues that import-injured rms are indierent to the method of protection among the 3 discussed options, but that exporters dis- prefer subsidies because of retaliation risks. The dierence among the three is in the institutions that shape their implementation, and the political incentives that arise therefrom. Existing research on NTMs has opened many avenues of inquiry. In examining this literature, it is clear that there are some NTMs in particular that appear to be substitutes for taris. But, there are also ways in which they are not. The work discussed above makes a variety of dierent assumptions and arguments about how NTMs divide winners and losers. There are also disagreements over whether NTMs represent public or private goods for those seeking protection. In some cases, it appears to be the most competitive industries that seek out protection through NTMs. In other cases, it is the less competitive producers who seek support. In considering NTMs as a whole versus taris, it seems sensible to consider NTMs as imperfect substitutes for taris.5 With this in mind, it may be time to change the framing of the discussion and move beyond a logic of taris and everything that is not taris. 17 2.3 Conceptualizing Non-tari Measures While considering matters of measurement is critical for empirical analysis of trade and trade politics, measurement issues alone do not motivate a rethinking of trade policies. The state of current theory on non-tari measures highlights the more important motivation for looking at NTMs in a new way: attempts to use the existing tari/non-tari framework require assumptions that are imprecise and that distort our understanding of the political dynamics behind the new debates in trade liberalization. Discussions of xed versus variable cost, particular idiosyncrasies of how an specic NTM is implemented, at-the-border versus behind-the-border costs, and how NTMs cover or are observed skirt a real problem: some measures that aect trade ows are very much like taris, while other measures are clearly not. Theories about trade-distorting measures rely upon assumptions about how poli- cies apply costs to certain goods within a market. Therefore, we must consider how these assumptions drive our theory towards or away from a better model of trade politics. For the study of taris, the assumption that import taxes applied some additional cost to imported varieties, while domestically-produced varieties are ex- empted, is quite reasonable. Taris are clearly a policy that adds costs to a good once it crosses a border. But, the same logic does not clearly apply to other poli- cies that are considered to be trade-distorting measures. The compromises to our assumptions that must be made to accommodate both regulatory barriers, like safety standards, and targeted policies, like export subsidies to certain rms, obscure our understanding of both. The ways in which supporters and opponents of each policy should divide within industries will be dierent, and furthermore will be dierent from how industries divide on tari measures. Improvement of our theories of trade politics requires a rethinking of how gov- ernments can distort markets, and therefore can inuence trade ows. Policies that appear to be barriers in some cases, but not in others, may be so because real-world 18 markets, which are structured dierently in dierent economies, do not neatly reect the assumptions of our models.6 One knows a tari is a trade-distorting measure be- cause aects a foreign producer's access to a market, but does not apply to domestic production. One need only look to tari schedules to know that a good, even one that does not actually cross the border, would have restricted access to a given market. 6. This is not to say that models do not have an important place in our understanding of trade politics. Simplications are necessary. However, we must take care to consider which simplications clarify and which obscure. 2.3 Conceptualizing Non-tari Measures There is no line in a tari schedule or roughly-analogous metric that applies for many other measures. The ad-valorem equivalent cost of a requirement to pasteurize milk that is used to produce cheese, or government policy that privileges domestic rms in the distribution of market licenses is not always clear. Yet, dierences in pasteur- ization rules, or limits on which individuals or rms obtain licenses for sale in a given market still distort cross-border ows of goods. It will alter both the composition of - eliminating import of some goods - and volumes of - reducing imports of prohibited varieties with incomplete substitution - trade between two, or among many, countries. They also distort production and sale of goods within that market. Attempts to force all other measures at once into a like taris or not like taris framework ignores important distinctions among non-tari measures. Assumptions about other market-distorting policies that frame costs of all other policies in the same fashion, be it as xed market entry costs for all producers, xed entry costs for foreign producers, variable costs for all producers, or variable costs for only foreign producers, lead to theories of trade, and thus theories of trade politics, that are critically imprecise. As domestic and international trade politics reects the new reality of protectionism, that taris are generally in decline while a whole host of other policies appear to distort trade, this inaccuracy prevents productive discussion of the issues at hand. 19 7. This may be considered an extreme interpretation of monopolistic competition, in a sen 7. This may be considered an extreme interpretation of monopolistic competition, in a sense. 2.4 Motivating a New Typology of Trade Barriers Each product in the market is dened by a variety of characteristics. Individual products can be dened by size, aspects of quality, what materials were used in their manufacture, country or region of origin, the rm that produced them, how they are packaged, and other intrinsic features. Taken together, these characteristics are what make an orange grown by Tropicana-aliated growers in Florida dierent from ones grown by Tropicana-aliated growers in Brazil, and also dierent from oranges grown by independent growers in either Florida or Brazil. Although these oranges may be comparable, they are dierent from each other in location of production or rm associated with that production. In the eyes of some laws, and in the eyes of consumers, these dierences may matter. Taken to the limit, no two products on the market are identical in every way.7 Dierent theories of trade and trade politics consider some of these product char- acteristics more or less relevant to understanding goods ows. For most discussion of trade barriers to date, the salient characteristic has been country of origin: whether a good is produced locally or beyond a territorial boundary. In most models of trade, trade barriers have been costs applied at the border only as a market entry cost imposed on products produced outside the border. Consistent with that at the border logic, the primary mode of comparison be- tween taris and non-tari measures has been through the use of ad-valorem tari equivalents. This works well for trade-distorting measures that function in a similar manner to taris, restricting net imports in favor of domestic alternatives or sub- stitute goods by creating price wedges between imports and domestic alternatives. However, for other kinds of measures, especially those that create dierences within categories of goods, this comparison misses essential within-industry redistributions. For instance, while restrictions on the sale of consumer electronics that create certain 20 kinds of electromagnetic interference8 may raise prices or reduce trade by a certain level for the industry as a whole they do not aect all products in the market evenly. If some varieties in the global market already meet the standard, they would enter with eectively zero additional cost.9 Other products not meeting that global standard would require costly modication before sale. Existing measurement of trade barriers does allow for ne-grained analysis of tar- is. 8. For instance, in the US and EU, consumer electronics must not generate more radio frequency noise than a specied standard. The frequency ranges and maximum noise levels dier in each market, which requires manufacturers to change the characteristics of RF shielding to suit each market's rules or surpass both standards. Further, it prevents some products from entering the markets altogether, as modication is not economically feasible. For example, for the production of IT equipment power supplies, products sold in the EU must conform with CISPR 32/EN 55032, while US products must comply with 47 CFR 15.109. Both regulations concern RF emissions, but have dierent frequency ranges - CISPR 32 has two ranges, Part 15 has 3 - and emissions levels as specied at dierent distances across product classes (Hegarty 2018). A product may be designed to exceed the noise limits of both standards, but those standards would restrict dierent non-compliant products. 10. The full description of this 10-digit HS code is: Telephone sets, including telephones for cellular networks or for other wireless networks; other apparatus for the transmission or reception of voice, images or other data, including apparatus for communication in a wired or wireless network (such as a local or wide area network), other than transmission or reception apparatus of heading 8443, 8525, 8527 or 8528; parts thereof: Telephone sets, including telephones for cellular networks or for other wireless networks: Machines for the reception, conversion and transmission or regeneration of voice, images or other data, including switching and routing apparatus: Other p 9. Additional testing to demonstrate this compliance to local authorities may add cost to imported varieties, but it is likely this is a cost faced by locally-produced varieties, as well. 8. For instance, in the US and EU, consumer electronics must not generate more radio frequency noise than a specied standard. The frequency ranges and maximum noise levels dier in each market, which requires manufacturers to change the characteristics of RF shielding to suit each market's rules or surpass both standards. Further, it prevents some products from entering the markets altogether, as modication is not economically feasible. For example, for the production of IT equipment power supplies, products sold in the EU must conform with CISPR 32/EN 55032, while US products must comply with 47 CFR 15.109. Both regulations concern RF emissions, but have dierent frequency ranges - CISPR 32 has two ranges, Part 15 has 3 - and emissions levels as specied at dierent distances across product classes (Hegarty 2018). A product may be designed to exceed the noise limits of both standards, but those standards would restrict dierent non-compliant products. 9. Additional testing to demonstrate this compliance to local authorities may add cost to imported varieties, but it is likely this is a cost faced by locally-produced varieties, as well. 10. The full description of this 10-digit HS code is: Telephone sets, including telephones for cellular networks or for other wireless networks; other apparatus for the transmission or reception of voice, images or other data, including apparatus for communication in a wired or wireless network (such as a local or wide area network), other than transmission or reception apparatus of heading 8443, 8525, 8527 or 8528; parts thereof: Telephone sets, including telephones for cellular networks or for other wireless networks: Machines for the reception, conversion and transmission or regeneration of voice, images or other data, including switching and routing apparatus: Other 2.4 Motivating a New Typology of Trade Barriers The Harmonized System, the common baseline for many countries' tari sched- ules, contains narrow categories (USITC 2018; World Customs Organization 2017). For home internet routers (wired and wireless), the specic tari line 8517.62.00.9010 covers a wide variety of products that handle the transmission of digital data. Cer- tainly it would be possible to further divide the category into Wireless Routers, then Transmitting and Receiving in the 5 GHz band, then Producing RF Emis- sions Above FCC Standards or Producing RF Emissions Below FCC Standards, but the logical limit of this exercise is uniquely coding every potential variety of every potential product that may be included in a tari schedule. However, these hypo- thetical additional classications are based on another policy: the FCC standards. The way that RF emissions rules aect the relationship between the US (or EU) 21 markets and the global market for goods is complex. The requirements eectively prohibit the sale of some (non-compliant) varieties. This prohibition reduces trade by the volume of modems not imported because of non-compliance. Some of that is oset by increased imports of compliant modems, even beyond what might have been imported in the absence of the regulations.11 While in the aggregate, this may be equivalent to the eect of a modest ad-valorem tari, two issues arise. First, an increase in some imports is not generally consistent with the logic of how taris aect markets. Increases in cost of market entry for imported goods should shift demand towards alternatives unaected by that additional cost. Second, the ad-valorem tari equivalent would not necessarily divide the market in the same way as the regulation does. Even when considering trade theory that focuses on the dierential eects of trade barriers on rms of dierent size or heterogenous productivity, the distortions in the market for goods aect rms based on the characteristics of the rm, not of the product.12 This illustrates a problem with the division of all trade-distorting measures into two groups: taris and not. In some cases, non-tari alternatives have the same kinds of market-distorting eects as import taris do, and so computing an equivalent tari is a reasonable exercise. 12. In these models, the heterogenity comes from dierences in productivity of the rms - their ability to turn the labor stock into the goods they produce, which they observe after an initial investment. Melitz 2003 11. This is dependent on the degree to which consumers substitute between the non-compliant alternatives and the compliant ones, given price, etc. 2.4 Motivating a New Typology of Trade Barriers When onerous import rules on certain goods slow time-to-market, or require importers to pay additional costs (either directly in customs fees or indirectly to sta or agents to administer the customs process), the way it distorts markets works the same way as would an import tax of the same magnitude. But for many of the non-tari barriers to trade used in the global goods market today, measurement on the basis of a tari equivalent is at best imprecise and at worst obscures important distributional outcomes, especially within industries. Attempts to compare trends in non tari barriers to trade to trends in taris Attempts to compare trends in non-tari barriers to trade to trends in taris 22 have been sidetracked by the simple fact that non-tari measures are neither pure substitutes nor pure complements to import duties. Some alternatives function in much the same way as taris, cleanly dividing winners and losers along geographic lines. Others, however, like the RF standards mentioned above, do not necessarily have that eect. There are wireless routers produced in China or Malaysia that meet the standard and others that do not. It is not the fact that those routers are produced in China or Malaysia that makes them subject to the standard. If they were produced within the US or EU, they would still need to meet requirements. What follows is an attempt to refocus the discussion of the political economy of trade- distorting measures on the politics. To better understand the politics of demands for dierent kinds of policies, one must rst understand how dierent kinds of rules distort markets dierently. The extensive and mature political economy literature on trade politics has built o economic theory to explain interactions between groups, the eects of institutions, and the role that global forces have played in shaping demands for and supply of trade policy. Rogowski's seminal work on the factor-based political cleavages that arise when exposure to trade changes relies on the economic theory of Stolper and Samuelson13 (Rogowski 1987; Stolper and Samuelson 1941). Later work by Gilligan (1997a) brought the Ricardo-Viner specic-factors model to bear on questions of po- litical coalitions around RTAA. Other signicant work in economics, including on endogenous tari theory and some work on non-tari barriers, also uses the specic- factors framework to generate hypotheses about when protection should arise. 13. Stolper and Samuelson's work is, in turn an extension of the Heckscher-Ohlin model, discussed widely and by many. See Leamer et al. (1995) for a widely-read treatment and review. 14. See also Alt et al. (1996) for a discussion of S-S and R-V models in political economy and an introduction to the economics of, but not the politcal models using, Krugman's Increasing Returns to Scale models. 2.4 Motivating a New Typology of Trade Barriers (Ray 1981a, 1981b; Magee, Brock, and Young 1989; Grossman and Helpman 1994) Subse- quent work by Gilligan (1997b) turned to models of intra-industry trade, building o the model of intraindustry trade under monopolistic competition introduced in Help- man (1981) and Krugman (1979) to explain how lobbying for intraindustry protection 23 is a private good, signicantly changing the dynamics of rm-level trade preferences. A recent wave of research in both economics and political science has looked to the- ories of rm behavior over trade policy when rms are themselves not identical, and the subsequent intra-industry cleavages over trade policy. Related work has focused on the stark heterogeneities in tari protection across products of a single industry or sector (Bombardini 2008; Bombardini and Trebbi 2012; Osgood 2016; I. S. Kim 2017). This work builds o the work of Melitz and others and models of rm heterogeneity and gains from trade (Melitz 2003; Melitz and Ottaviano 2008). Indeed, as Rodrik (1995) suggest, any political-economic model of trade policy must explicitly specify individuals' (or actors') preferences over policy options.14 Looking to models of trade for these preferences is natural. However, it was not always so. Schattschneider (1935) focuses directly on interest group politics at the in- dustry and rm level and explains the pressure politics and coalition-building around the Smoot-Hawley taris, laying the groundwork for future study of interest group politics in trade. However, the work above has largely considered only taris, or tari setting. As existing work has suggested the politics of NTBs is more complex, with more complex distributional considerations, there is value in considering whether adopting existing trade logic is the best avenue for considering the politics of NTMs, of NTMs and taris together, of both in the presence of a complex, interconnected global economy. Perhaps it is time to take a fresh look at trade-distorting policies and derive assumptions about actors' preferences from there. Acknowledging the progress made in political economy to date, but also the dif- culties in developing theory on NTMs in the same consistent way as with taris, it seems one way forward is to reconsider how we codify barriers to trade. 2.4 Motivating a New Typology of Trade Barriers Instead of the dichotomy of taris and not taris, or specic theories (with attendant preference 24 assumptions) for each particular kind of non-tari policy, it may be more productive to focus on how dierent kinds of trade distorting policies divide industries, putting the cleavages at the center of the discussion.15 15. It is also possible that this change in perspective may help with measurement and modeling of trade ows, by indicating how trade ows may be distorted, either in volume or composition. 2.5 A Typology of Barriers to Trade Governments use many dierent policies to inuence trade ows and shape access to their markets, often for the benet of groups of domestic actors. Referring to these as either taris or not taris ignores key dierences among the latter group, and ignores what all such policies have in common: they are all barriers to trade. Whether their primary intent is distortion of trade ows, or the diminution of trade is a secondary outcome, policies that prevent trade in goods that might otherwise have owed between markets are a barrier. This section introduces and describes a typology of trade barriers based on the manner in which they discriminate, rather than whether they are import duties or not. Some non-tari policies work very much like taris, imposing at-the-border costs on a product that generate revenues for the imposing government. Even policies that do not yield similar rents to governments work in the same manner as taris do, by creating a price wedge between imports and domestically-produced alternatives. The winners are dened by the location in which they choose to locate production of their good. But, not all policies work in this manner. Trade can be distorted by policies that restrict market access on any basis, as long as those goods cross borders to enter the market. Taris and tari-like policies are the most straightforward kind of trade distorting policy, but they are by no means the only policies that can, intentionally or incidentally, distort trade ows. To simplify comparison of policies and place taris within a single framework of 25 trade-distorting policies, it is necessary to focus instead on how the policy adds costs to the production of a good. Recall from above that each variety of good in a market can be dened by where it is made, how it is made (or what it contains), and who makes it. These three dimensions cover the ways in which policy can be used to raise the price of some goods (but not others) on a market. This increase can come about because of increased cost of production, to comply with a policy or absorb fees, or increased price to market because of policies which directly aect nal prices. 16. The degree to which the winners and losers are politically salient depends on a number of factors, including whether a producer is a rm located either as a producer or with headquarters within political boundaries. p 17. For instance, a ban on the sale of all electronics containing lead-based solder. 2.5 A Typology of Barriers to Trade There are four types of barriers: those that impose additional costs based on the location of production of a good, those that impose costs on a good based on its innate characteristics or methods of production, those that impose additional costs for some producers (rms) of a good, and those policies that impose additional costs on all goods sold in a market. In the subsequent sections, we introduce the types of barriers with examples. we then explain some of the consequences of each type of barrier when they are imposed (or removed) as unilateral changes, then as changes in the context of a bilateral relationship. In the subsequent sections, we introduce the types of barriers with examples. we then explain some of the consequences of each type of barrier when they are imposed (or removed) as unilateral changes, then as changes in the context of a bilateral relationship. 2.5 A Typology of Barriers to Trade By raising the price of some varieties, inducing consumers to change their consumption behavior, governments can improve the fortunes of some producers, at the expense of others.16 When those policies privilege varieties produced domestically, the eect is the expected decline in trade, specically imports. Compared to a free-trade scenario, the overall volume of trade is lower. Some goods may be more or less aected by the policy, but the policy is clearly a trade barrier. However, trade can also be distorted through changes in the composition of trade ows. Policies that privilege certain varieties of goods17 can lead to patterns of trade where overall volumes are not signicantly distorted, but where the variety of goods traded (or, more specically, imported) is reduced. Compared to that same free-trade scenario, the goods that enter that market are qualitatively dierent. Some varieties are blocked, in the same manner that a tari or an import quota might block imports. All of these policies can be trade barriers, if the additional costs prohibit trade (goods ows across borders) that might otherwise have occurred. The winners and losers from barriers in each of these categories can be more dicult to identify than in the simple tari (or tari-like) case, but they do exist. Despite the additional complexity, placing trade barriers in this larger framework has some advantages. The 26 distributive consequences (and thus the distributive politics) of trade barriers and other market-distorting policies have the same logic: the prots and losses of aected rms. Firms are not swayed by aggregate, economy-wide gains and losses in welfare when making decisions about political pressure. It is the gains or losses to that rm that are salient, and that drive those rms to pressure governments for relief through policy changes. Competitors in a market are still competitors, regardless of where their product is produced or how similar it is to a rm's own.18 This new typology divides trade barriers into groups based on how they raise the market price of goods. 18. While these characteristics may aect competitors' costs, there is no reason to suspect that rms have a particular preference for domestic competitors over foreign ones. 19. It is also possible that producers may accept lower prot margins on each unit, such that imported varieties sell at the same price, but this is not necessarily the case for all producers. At the margins, some producers will have prot margins too small to absorb the additional costs imposed by the tari. 2.5.1 Location-discriminating Costs: Where it's Made The most clear example of a trade distorting policy is one that imposes additional costs that depend on the location of production of a good. By driving a wedge between international and domestic market prices, these policies divert some consumption 27 away from foreign-produced goods towards domestic alternatives, when they exist. Location-discriminating costs create a protected market within the boundaries of a geographic area, and goods that are traded across that border arrive at a market at a higher cost than they would otherwise. Taris are clearly location-specic barriers, as they impose an additional cost on each imported good. Whether calculated as a portion of an import's stated value or as a specic cost on each imported unit, taris raise the cost of the good for consumers.19 There are a variety of tari-like policies that have been used in place of taris, often when tari protection has been prohibited by international agreement. Quotas, by restricting access to markets and, in some cases, charging importers for quota licenses, also increase the cost of a product traded across a border. Voluntary export restrictions work in the same way, but are administered by the government of the exporting market. Other policies look quite dierent, but also discriminate based on the location of production. Policies requiring a minimum of local (within the customs area) content discriminate between products based on location of origin. Onerous customs procedures, or special inspections for imported products only, are also ways of imposing costs on only some products in a market: those produced outside the borders. Similarly, policies that indirectly raise the cost of goods originating outside na- tional borders, such as currency manipulation, can generate location-discriminating costs that act as barriers to trade. If, by distorting the local-market price of a prod- uct, these sorts of manipulations make goods manufactured abroad more costly for consumers, currency manipulation can impose location-discriminating costs, and thus shape trade ows. Other currency and capital controls, such as limited currency con- vertibility, restrictions on repatriation of prots, or measures that impose additional 28 costs on the conversion of one currency into another, are another form of location- discriminating cost. To access a market that is behind a barrier of this kind, produc- ers who manufacture outside that currency area (and therefore in terms of a dierent currency) face additional costs that local competitors do not. 20. These characteristics can be both the content of the product or the methods used in its pro- duction. In the same way that a prohibition on a chemical in a particular product may the basis for a market access cost, so too may the use of a particular technology, or laws on the labor used to produce a good add cost based on how it's made. 2.5.1 Location-discriminating Costs: Where it's Made In addition to costs imposed by policy, other natural barriers to trade, such as transportation costs, are a location-discriminating cost. In the same manner as tar- is, import regulations, quotas, or location-specic import restrictions, transportation costs can prevent goods from entering markets where they might otherwise nd buy- ers. Although these are not costs that governments can impose on goods, they can act as natural barriers, reducing the need for policy-based protection of local producers. In all of these cases, market access is restricted is through increased product costs applied dierentially based on the location of origin. In thinking about trade barriers this way, taris and some other non-tari barriers are clearly substitutes. A tari can be replaced by a quota, or an import inspection, or currency manipulation that generates similar costs on imported goods. In this case, the net eect of the change in policy should be minimal, as long as the magnitude of the cost increase on foreign- produced products is largely the same. As will be explained in the next section, the producers who benet from the protection aorded by the tari will be the same who benet from these location-discriminating alternatives. 21. Put slightly dierently, dierences in production technology matter in these cas 2.5.2 Characteristic-discriminating Costs: How it's Made A second manner in which policy can discriminate is on the basis of a prod- uct's characteristics.20 These types of policies raise the market entry cost of some varieties and not others, depending on how a product is made. Also, characteristic- discriminating barriers impose costs that apply to varieties of goods with certain 29 dening features. Characteristics-discriminating barriers can aect only products considered to be low-quality varieties, only those considered to be high-quality varieties, or a set of goods where quality ranking is not obvious. Although canonical examples of these sorts of barriers take the form of safety standards (and thus, exclude what might be considered low-quality varieties by some), the dening feature of these kinds of policies is that they separate market access on the basis of a good's characteristics. Prohibiting the sale of certain kinds of cheeses made with unpasteurized milk on safety grounds, for instance, may eliminate varieties that are considered by some consumers to be high-quality varieties. It is not the case that quality and characteristics are the same concept. Other characteristic-discriminating policies may relate to the factors used in pro- duction or the externalities generated in production. Labor standards, which are regularly part of trade agreements, create restrictions on how a product is produced. These policies, which often require standards in an exporting country to meet those of the importing country, divide markets based on the process of production, and the inputs used in production. Environmental standards, whether legislated or adopted as an industry code, have similar eects. When products produced in a manner in- consistent with the standard face additional barriers, it is that manner of production, not the location of production or who produced it, which matters. Usually, these standards are implanted with the expressed aim of mitigating harmful behavior, but the way they divide industries is primarily along lines of how the product is made.21 As these policies restrict market access based on product characteristics, they ap- ply equally to locally- and foreign-produced goods. For example, a variety of reproof door that does not meet a country's minimum safety standards will not be permissible for sale whether it is produced within that country's borders or abroad. 23. This is not to suggest that these dierences do not matter, but rather to suggest that the content of a nal good may be produced in more than one way. GATT does consider standards which regulate content dierently than those which regulate processes, with respect to considering trade barriers. However, in this framework, the process and the content both make up the characteristics of a good. 22. There are some regulations that are characteristic-discriminating, but directed towards imports only, or imports of only some countries. These policies impose location-discriminating costs in addition to characteristic-discriminating costs. 2.5.2 Characteristic-discriminating Costs: How it's Made Regulations that require that a food product be refrigerated from harvest to market apply to all 30 varieties of that product, not just to those produced outside the country's borders.22 Characteristic-discriminating costs often take the form of nished-product standards, but can also include policies restricting the sources of a product or the manner in which it is produced, even if that has little or no eect on the content of the nal good.23 In addition, some forms of labeling standards for instance, restricting the use of the label ice cream to only desserts made with cream from cows milk create dierences between products in consumers' minds on the basis of quality and com- parability, eectively changing the value (or, inversely, the price) of the good to the consumer. Characteristic-discriminating costs distort markets by either raising the nal cost of some varieties of a product (the non-compliant ones), making them less appeal- ing than other varieties, or blocking some varieties from reaching market (eectively, raising costs suciently to ensure no consumer would ever purchase it) altogether. When these varieties are unavailable, consumers will substitute among whatever al- ternatives are available to them. Alternatives includes products within the same category of goods (but of a dierent variety) and all other goods and services. The eect of these kinds of policies, and the characteristic-discriminating costs they impose, is somewhat more complex. When some varieties of a product bear a policy-related cost before entering the market, the total cost of that variety increases. Facing that increased price, some consumers will shift their consumption to other, less costly varieties. Indeed, this very consumption-shifting behavior is sometimes the goal of policy. If policymakers wish to discourage behavior, targeting products (or varieties of products) with dierential costs is one way of changing consumer 31 behavior. However, not all producers (if each producer makes only one or a few varieties) will be aected in the same way by these costs. This division will arise within industries and regardless of location of production. Producers facing characteristic-discriminating costs in a market cannot avoid them by changing the location of production. Instead, it is the product itself that must change. If rms can alter their production to meet local-market requirements, and thus avoid these characteristic-discriminating costs, then they are likely to do so. 2.5.2 Characteristic-discriminating Costs: How it's Made However, the natural characteristics of the market (homogeneity of the product, pos- sibilities for technological/product innovation), the legal framework around adapting to new varieties (intellectual property law/patents, etc.), and the rms own ability to change (capital necessary to change production, sunk costs, etc.) all contribute to determine whether a rm can eectively adapt to avoid characteristic-discriminating costs. If they cannot, they may still attempt to enter a market with a non-compliant good, and bear the costs thereof. However, that good's market cost will reect the non-compliance, and is likely to make the variety less attractive to consumers. 24. A simple example of this is US military aircraft purchasing, where the supply chain for some aircraft produced by US rms include foreign suppliers and subcontractors, while aircraft produced by foreign competitors may include US rms in their supply chain. Some policies also require domestic production, although this varies from case to case. 2.5.3 Firm-discriminating Costs: Who Makes It Other characteristics of the policy environment create costs that apply only to goods produced by certain rms or costs that apply to all producers except some ex- cluded rms. These policies create dierences in market access based on who produced a good. Alternatively, these policies may impose costs, or exclude from additional costs, products that are sold by or marketed by certain rms. Firm-discriminating costs can take a variety of forms. Policies on import licensing that provide specic importers with authorization to import, or to sell, are a common one. Were two dierent rms to attempt import of otherwise-identical products (for instance, shoes made from man-made materials in China), the rm with an existing import license would have an advantage over their competitor. Obtaining import 32 licenses can be a costly and time-consuming process, which creates additional costs for new entrants. Government procurement policies that require that the contractor be a domestic rm, or that privilege the bids of domestic rms, set a price wedge between dierent producers. This kind of policy works dierently than a location- discriminating cost, as it is the rm, not the location of production, to which the privilege or cost is tied.24 Alternatively, policies to support national winners or to provide nancial assistance to certain rms (in the form of loans or subsidies) also create rm-specic costs by lowering the eective price-to-market for one rm. In some cases, intellectual property laws can create rm-discriminating costs, restricting market access on a particular variety of good to one or a small group of producers, forcing others to either pay fees to license the IP or nd ways to produce a dierent variety which is not aected by the IP rights. When costs apply only to the goods of certain producers, the eect on sales and prots is as expected: consumers will change their purchasing behavior to reect the cost dierences, or rms will absorb some of the costs in the form of reduced prots. In either case, rm-discriminating costs can create dierences in market competitiveness between two rms producing identical (or nearly-identical) products in the same place. The rm facing the additional costs cannot escape them by changing the location of production or the characteristics of their goods. The costs of rm-discriminating policies are tied to the identity of the producer. 25. With xed costs, scale eects are an important consideration, as a single, indiscriminately applied testing cost adds a smaller cost to each of 1000 units sold than to a single unit sold. 2.5.4 Indiscriminate Costs The nal category of costs are those that apply to all products within a given category. These are costs, either xed or variable, that apply to any good enter- ing a market. These can be considered indiscriminate costs because they impose 33 additional requirements on producers without conditions on location, the quality or characteristics of the good produced, or the identity of the producer. Labeling requirements, where goods sold must include additional documentation on the packaging to inform the customer of the content and characteristics, are one example of this kind of market access cost. If all varieties and producers must un- dertake the same labeling process, it is a cost applies without discrimination. Cer- tications/inspections requirements that apply to all products within a category of goods work similarly. If the certication process is the same for all potential entrants, then the cost of testing and certication is an indiscriminate cost. Other policies, like consumption taxes, can act as an indiscriminate cost, as long as they are applied equivalently across all permutations of producer, quality, and location of origin within a given category of goods. Consumption taxes make goods more costly for consumers by raising the eective cost of all goods (usually, in proportion to their sale price or value-added). These indiscriminate costs can take the form of a single, xed cost of market entry - such as a testing requirement - or a per-unit cost paid by all varieties, like an excise tax. In either of these cases, it is the application of the cost to all varieties that matters. Indiscriminate costs of market access cannot be avoided. They apply to all rms and varieties, and without regard to location of production. While the exact nature of the requirements that generate the costs (testing, labeling, etc.) may vary from market to market, for all producers seeking to enter a given market, those costs must be paid. Whether the cost applies before the good reaches market (testing, certication, labeling) or at the time of sale (consumption taxes), the eect on the price of the good to the consumer is the same. Indiscriminate costs raise the price for all consumers of all varieties, although not necessarily equally across varieties, and aect their consumption decisions accordingly.25 34 2.5.5 Limitations of this Typology The four categories above span the variety of manners in which costs can be imposed on goods that might enter a market. The typology does, however, have some limitations. Some policies appear to impose costs in a variety of ways, or impose costs that are conditioned on more than one aspect of a product's characteristics. For instance, a temporary import restriction for sanitary and phytosanitary reasons may be considered to apply both a location-discriminating (based on where something is made) and a characteristic-discriminating (based on the process used to produce the goods) cost. While this makes sense if conceiving of these costs as orthogonal dimensions of a policy's total cost prole, for simple categorization it can lead to disputes or uncertainty about how a policy should be considered. Similarly, this typology is generally agnostic with respect to two important features of policy-induced costs: whether costs are a xed or variable cost and whether the costs are revenue generating or not. For some models of trade, the distinction between xed and variable costs of a given policy matter. If a cost applies only at market entry, and not on a per-unit basis, then there will be important scale or productivity eects that are ignored here. A one-time cost of obtaining an import license or adjusting a product's characteristics to meet safety or content standards is a relatively greater per-unit cost when expected sales are low. In contrast, a variable cost will have the same eect on prices no matter the size of the rm or the volume of sales. Similarly, for the study of taris vs non-tari barriers, the fact that taris generate revenue (although the overall importance of these revenues has declined for some countries in recent decades) is a key dierence between them and many non-tari policies that distort trade. When considering government incentives to implement dierent kinds of market access costs, this distinction may matter. Despite these limitations, this new framework does what it aims to do. It claries when policies are similar in their eect on markets - taris, quotas, export subsidies, 35 and currency manipulations all distort on location of production - and when policies are dierent in their eects - taris and product standards create dierent cleavages - in a systematic way. 2.5.5 Limitations of this Typology In doing so, it puts the distributive politics at the center of the framework, and highlights where changes in industries over time may lead to dierences in preferences over various kinds of trade-distorting policies. 26. Production discrimination is the goal for many location-specic costs, but some costs are as- sessed based on the foreign location from which the product is shipped. Firms or importers use this as a means of jumping the market access barrier by shipping the product through an interme- diary market, but governments often use more sophisticated rules of origin to prevent this kind of chicanery. 2.6 Considering Winners and Losers from Market Access Bar- riers - Single-Country/Unilateral Policy Change Each of these kinds of market access barriers has distributive consequences: some actors win while others lose. As it is the gains and losses from policy changes that motivate political action (lobbying, supporting candidates, etc.) by market actors, understanding the changes in distributive outcomes caused by each kind of market entry cost is essential to motivating the explanation of the politics of this new pro- tectionism. Before moving to an explanation of the eects of market access barriers when raised or lowered reciprocally, we consider the eect of each kind of barrier on a domestic market when used unilaterally. These outcomes assume that, at least in the short run, rms cannot change location, variety of production, or ownership/rm structure. When these assumptions are relaxed, the complexity of tradeos increases quickly. Further, the eects discussed below are partial equilibrium outcomes. The eect of barriers on factor cost and quantity is not considered here. When applied unilaterally, each kind of barrier has the eect of dividing industries into groups of losers (who see market access barriers negatively aect their sales) and winners (who gain some of the losers sales through substitution by consumers). These winners and losers include rms headquartered within the boundaries of the protected market, rms producing goods within the borders of the market, and rms that are 36 located completely outside the protected market. y 27. This substitution depends on cross-elasticities of varieties. When no locally-produced alterna- tives exist, consumers will simply consume less overall. 2.6.1 Location-discriminating Costs When the market entry costs are applied according to the location of production, winners and losers divide based on where a rm's products are made.26 This reects the classical understanding of the eect of taris and the location-discriminating costs associated with import duties. When location-specic barriers raise the market price of foreign-produced goods, consumers will shift consumption away from the costlier varieties (the foreign-produced ones aected by location-discriminating costs) towards less expensive alternatives (those not subject to the location-specic costs) produced within the market or in other foreign markets.27 Producers in the domestic market will, at worst, see no change in their sales/prots, and may see an increase in sales due to cross-variety substitution. Even if other foreign-produced alternatives exist that are unaected by location-discriminating costs, local rms are likely to be better o when some foreign competitors' goods are made costlier. 2.6.2 Characteristic-discriminating Costs Characteristic-discriminating costs apply costs (xed entry cost or a per-unit variable cost) to the production cost of some varieties. Recall that Characteristic- discriminating costs can be applied to any subset of varieties of a good. Thus, for any policy that imposes costs on some varieties based on quality, there are winners and losers among rms in that industry. Producers of varieties aected by the policy lose, as the increased cost of compliance or outright block on sales in the market leads to 37 lower prots and fewer sales. This occurs regardless of the rm's location (domestic or foreign), the location of production (local or foreign), or the identity of the rm. Other rms, producing varieties not aected by the policy, will see gains in sales or prots as consumers alter their consumption and substitute unaected varieties for the more expensive, policy-restricted, alternatives. As with location-discriminating costs, the cross elasticity of the varieties will determine the degree to which producers of other goods will see sales and prots rise. At worst, the exclusion of some varieties from the market will not prevent consumers from buying the non-excluded ones. Thus, rms whose products are not aected by the Characteristic-discriminating policy will, at worst, see no change in prots. 28. In some models of trade, these indiscriminate costs can raise the prots of some high productiv- ity rms as lower productivity rms exit and consumers reallocate their consumption basket. Also, for some producers of inelastically-consumed goods, higher consumption taxes may lead to increased sales, as consumers reduce consumption of other goods and reallocate. 2.6.3 Firm-discriminating Costs Policies that discriminate with respect to the rm's identity create clear winners and losers. When some rms must pay an additional cost to access the market, their goods are more expensive, and they lose sales. The rms that are excluded from those additional costs may gain additional sales or prots from consumers' substitu- tion. At worst, their sales and prots are unaected. Although they work indirectly, and through more inecient means than a direct transfer, market access costs that discriminate based on rm identity work the same way as a direct payment to the winners. Alternatively, if some rms are privileged through subsidy policies or other policies that provide special access, that ease of accessing the market or direct payment to the rm represents a cost on all other rms. The eect is the same as imposing a market access cost on all non-supported rms, creating a cost dierence between the supported/privileged goods and those that are not. The resulting dif- ference in prots or sales (as costs are passed on through prices) yields the same rm-versus-rm division of winners and losers. 38 38 2.6.4 Indiscriminate Costs When a policy raises the price of all goods in a market, all producers will see sales and/or prots decline. However, the degree to which these indiscriminate costs aect rms varies. For some rms, the additional cost pushes the cost of their product above the price where consumers will purchase it. For these rms, the indiscriminate costs lead to their exit. For other rms, the increased price of their product on the market simply reduces sales or prots.28 If the indiscriminate cost applies only to one kind of good (is applied indiscrimi- nately across an industry, such as luxury taxes on all boats sold, regardless of size or cost), then consumers may shift their consumption to other goods, with some loss of utility. Alternatively, consumers may simply choose to consume less, diverting more of their resources to leisure. In either case, as the costs of the goods increase because of the market access cost, demand will fall. The impact of this cost on producers depends on how close their market price is to the indierence point of consumers in the market. Thus, costs of market entry that apply indiscriminately mean all pro- ducers are losers, but it is possible (and in fact, likely) that the eect on each rm is dierent. 2.7 Considering Winners and Losers from Market Access Bar- riers - Two/Multi-country Policy Changes 2.7 Considering Winners and Losers from Market Access Bar- riers - Two/Multi-country Policy Changes The explanations above focused on the economic eects of a policy change in only one country in a hypothetical global economy. However, many conventional treat- ments of trade barriers (and actual trade negotiations) focus on reciprocal changes (usually decreases) in market access barriers. Consider a simplied world with two 39 countries, Country A and Country B. The eect of each kind of market access bar- rier depends not only upon the dimension along which goods entering markets face additional costs, but also whether those costs are applied on both sides of a trade dyad. To illustrate the eects of dierent market access costs within a trade dyad, we consider the distortionary eects of a policy change compared to a hypothetical free trade counterfactual with no market-distorting policies. This discussion focuses only on the eects of policy changes, not on how they might arise. The focus, again, is on the distributional eects of policy changes. 2.7.1 Location-specic Costs When the market access policies used by governments to regulate their domestic markets impose location-specic costs, the eects on domestic markets and on trade are consistent with those suggested by existing theory of taris. Because these mea- sures divide market access based on location, the winners and losers in each country will divide on the basis of location of production relative to location of sale as follows. y p g y p y 30. While the disucssion of reciprocal taris is usually in the context of taris on dierent products, those discussions also generally ignore intraindustry trade. In models of intraindustry trade with heterogenous rms and monopolistic competition, taris are a general variable cost of import. 29. In some cases, it is possible that goods produced in Country B for export to Country A will instead be diverted into the Country B market, potentially displacing sales of the domestic-only Country B producers' goods and Country A's exports to Country B. 2.7.1.1 One Country Raises Location-specic Costs If, in a particular country pair, one country (Country A) imposes market entry costs on the basis of location against goods produced in Country B, rms within the protected market and rms in the market that remains access cost-free (Country B) will experience dierent distributional outcomes. In Country A, the policy that raises costs of non-Country A varieties will aect both consumers and some producers. For consumers, the increased cost of Country B- produced varieties will lead some to switch to Country A-produced alternatives. For rms producing in Country A, that substitution brings the benets of increased sales and the potential for increased margins. This may also have the eect of bringing new entrants producing substitutes for foreign-produced alternatives into the market. For 40 rms that produce in Country A and export to Country B, those sales are relatively unaected, while domestic consumption may increase. In total, the location-specic cost has the eect of improving sales for those rms that produce in Country A. In Country B, domestic consumption is relatively unaected. As goods in Country B do not have dierent costs dependent on location of production, imported varieties and domestically-produced alternatives remain at the same relative cost. Therefore, no substitution across varieties should occur. Firms that produce only for domestic consumption in Country B should see no change in their sales.29 However, rms in Country B that produced varieties that were exported to Country A for sale in that market will bear the eects of the policies in Country A. These exporting rms will see reduced prots either because of reduced margins on their goods sold to Country A or because of consumer substitution away from their varieties in Country A. In either case, the net eect for those rms will be negative. For some rms, it is possible that the new costs for reaching Country A's market will lead them to exit, if they can no longer sell protably in that foreign market. 2.7.1.2 Both Countries Raise Location-specic Costs The case of two countries raising location-specic costs reects the familiar re- ciprocal taris case discussed in existing theories of trade barriers.30 The winners and losers within Countries A and B will divide on familiar factoral, sectoral, or rm- specic lines, depending on factor mobility and product dierentiation assumptions. The specic form of the market access costs can vary. One country may use taris or quotas, while the other uses exchange rate policy or import-only restrictions. If both countries use policy to raise the cost of some varieties based on location of production, 41 the eects are symmetric to the degree that the countries are similar. The winners and losers among rms in both countries divide on the degree to which the policies reduce their export sales (if they export) versus increase their sales in the market of production, where access costs do not aect total cost of their goods, but do aect the cost of imported alternatives. 2.7.2 Characteristic-specic Costs When market access is restricted through increased costs for products with certain characteristics, the logic becomes a bit more complex. In a given trade ow dyad, it is possible that costs are applied to the same (Countries A and B both raise barriers to widgets made with material containing lead compounds), somewhat overlapping (Country A raises costs on goods made with lead in any form, while Country B only raises costs on varieties with more than a certain concentration of lead), or completely dierent sets of goods (Country A raises costs on lead-containing varieties, Country B raises costs on varieties that contain arsenic). It is also possible that one country may impose costs on certain varieties of goods, while the other does not (Country A raises costs on lead-containing varieties, Country B imposes no additional costs). In each of the markets, the additional costs will aect the at-market cost of only certain varieties, a subset of the potential varieties of the good. Consider two types of a certain good: Type J and Type K. When governments raise market access costs on one variety (for instance, restricting the sale of certain kinds of antihistamines to prescription-only, or requiring that all tires meet a content standard), it has the eect of dividing winners and losers along lines that cut across industries, not across geography. 42 2.7.2.1 One Country Raises Costs on Some Characteristics 2.7.2.1 One Country Raises Costs on Some Characteristics If Country A implements policy that raises costs on some varieties of a good based on its characteristics or how it is manufactured, it will have eects in both countries. Consider a policy that raises the cost of Type J varieties. In Country A, consumers, faced with higher prices on Type J goods (those aected by the policy), will reduce their consumption of Type J varieties. Some may substitute consumption of Type K varieties, depending on the degree to which substitution is possible. In Country B, consumer's choices will be unaected by the new policies in Country A. Producers within both countries will see benets or losses depending on whether their goods face additional costs based on the characteristics that are targeted by the policy in Country A. For producers of Type J varieties, higher costs mean lower prots or lower sales (as prices rise). If there are rms producing Type J varieties in both countries, then the losers are not conned to just those located outside Country A. As consumers in Country A move away from Type J products, rms in Country A selling only within Country A will be negatively aected. Depending on the degree to which the additional costs distort consumption, these rms may be forced to exit the market. Firms in Country A that produce Type J products both for local sale and for export will see their domestic sales fall, while export sales to Country B will be unaected. This can, in some cases, lead to rms producing only for export. Firms in Country B that produce Type J products will see the opposite, where domestic sales are unaected, but export sales fall as costs distort Country A's market. For producers of Type K products, the benets reect the other side of the cross-Type substitution. Firms in Country A that produce Type K goods and sell within Country A will benet from the increased sales of their variety. Firms in Country B that produce Type K goods will see some gains as well. For those rms that already export to Country A, this means increased sales or prots. For rms in Country B that produce only for domestic consumption, there will be little eect on sales, unless the new market 43 conditions in Country A make exporting a viable option. 2.7.2.1 One Country Raises Costs on Some Characteristics In the aggregate, Type K producers in Country B benet from the policy change in Country A, just as similar producers in Country A do. 31. This reects a market where two countries have two dierent standards for a given kind of good. 2.7.2.2 Both Countries Raise Costs on Dierent Characteristics If each of the countries implement policies that impose additional costs on dier- ent varieties, the dierence between characteristic-specic and location-specic costs becomes clearer. Consider a scenario where Country A raises costs on Type J goods, while Country B raises costs on Type K goods.31 Under these conditions, the markets in each country will have dierent varieties of products in the same category of goods. In Country A, the increased cost of Type J goods drives consumers to substitute to alternatives, including Type K varieties. In Country B, the increased cost of Type K goods drives consumers to do the opposite, substituting to Type J varieties. The eect on rms will reect this dierential substitution. Producers of Type J varieties will see lost sales in Country A, while Type K producers will see an increase in sales. For Type J producers in Country A, those lost domestic sales are the cost of the policy change. If those producers do not export to Country B, the reduced sales may lead them to exit. If the producers do export Type J goods to Country B, the substitution behavior there will oset some of the losses. For those producers in Country B that export goods of Type J to Country A, the new market entry costs will reduce prots or sales, and the reduced exports may lead them to exit the Country A market as well. Producers of Type K varieties will lose sales in Country B. For producers of Type K goods in Country B, access to the domestic market is restricted by policy changes. If a rm only produces for domestic consumption, the lost sales in Country B may mean exit, unless increases in sales in Country A (where consumers substitute to Type 44 K varieties) are sucient to lead them to export. For those producers in Country B that produce Type K products for both local and export sales, the losses will come from a reduction in local-market sales, with some relief from increased export sales. For those producers in Country A that export Type K products to Country B, the increased market access cost will have the eect of reducing those export sales, as expected. 2.7.2.2 Both Countries Raise Costs on Dierent Characteristics Because of the way that these policies restrict market access in each country, the division of winners and losers from this policy environment do not fall along neat geographic divides. Within segments of a given industry (Type J and Type K producers), rms have both domestic opponents and foreign allies. 2.7.2.3 Both Countries Raise Costs on Same Characteristic If policies in both countries raise costs on the same Type of goods, the trade dynamics look somewhat dierent from the examples above, and become more de- pendent on the distribution of producers in each country. An example of this is if both countries adopt the same product standards, thus preventing sale of (or at least rais- ing costs on) goods of Type K. In Country A, the increased costs on Type K variety goods will drive consumers to substitute Type J variety goods and other alternatives. Consumers in Country B will make similar substitutions, moving away from Type K goods as prices rise due to the new policy-related costs. In each country, consumers' behavior depends on their cross-price elasticity of demand, which may dier in each country. Given this simultaneous change in both markets, the winners and losers in each country will be similar, although not necessarily identical. Producers of Type K varieties in both countries will see lost sales or prots as consumers move away from their varieties to other, less costly varieties. These losses will occur in both domestic-only sales and trade sales. For producers of Type K goods in Country A, lost sales in Country A, combined with lost sales in Country B (for 45 those producers who were exporters to Country B) may lead them to exit. Unlike in previous examples, export sales will not provide an opportunity to mitigate some of the lost sales in the home market. Similarly, producers in Country B will face the same pattern of lost sales both foreign and domestic. Producers of Type J varieties in both countries will see benets from the new policy. These producers see gains both because of substitution towards their goods in their home markets and because of potential gains from sale to the export market, where consumers are making the same substitution. For Producers of Type J goods in Country A, when consumers in Country A increase consumption of Type J varieties, sales will increase. For those Type J producers in Country A that export to Country B, the change in consumption patterns in Country B will bring additional benets. The increased demand may also induce some rms to begin exporting, as well. A similar pattern will occur for Type J producers in Country B. 2.7.2.3 Both Countries Raise Costs on Same Characteristic However, the degree to which sales increase for producers in each market depends on how consumers divide their increased consumption of Type J varieties between domestically-produced goods and foreign-produced varieties. At the least, the substitution from Type K consumption to Type J consumption will not decrease sales of Type J varieties for individual producers. Since both countries impose market access costs on the same type of goods (Type K), consumption of Type J-variety goods in both markets increases while consumption of Type K-variety goods decrease. This leads to a division of winners and losers cleanly along lines dened by which varieties a producer makes. Within each country, the winners are those whose varieties are unaected by policy-induced market access costs, and they have allies among producers of similar varieties in the foreign market. The same division holds true for the losers among producers. 46 2.7.3 Firm-specic Costs Governments may also implement policies that privilege certain producers over others. In this case, it is useful to think of a highly-simplied competitive market- place where each country has only two producers.32 In Country A, Firms F and G produce goods within a given industry, while Country B hosts Firms Y and Z in that same industry. For these examples, we assume that all four rms, in a free-trade counterfactual, produce both for the domestic market and for export to the other country. o ow 33. This could also be considered a domestic subsidy or preferences for Firm F. 32. In some cases, the same rm may produce goods in both markets, but we set aside this featu for now. 33. This could also be considered a domestic subsidy or preferences for Firm F. 2.7.3.1 One Country Raises Costs of Some Firms If Country A implements a policy that raises market access costs for some rms, the distributive eects of the policy will split on which rms' goods enter the market with additional policy-imposed costs. These costs can be imposed through prefer- ential government purchasing rules, targeted production subsidies for some rms, restrictions on business activities, or requirements on nancing for import or sale that are tied to the rm/producer, among others. Consider a policy where Country A raises costs on all goods produced by rms G, Y, and Z.33 In this case, the market in Country B will be largely unaected, while the changes in market access in Country A will be signicant. In Country A, the policy that imposes costs on good from Firms G, Y, and Z will pass through to prices or reduced margins. Meanwhile, Firm F will have privileged access to the market. The consequences are as expected. Some consumers will shift consumption from Firm G, Y, or Z's products to Firm F's goods, with the commen- surate eects on the rms' overall welfare. This will lead both to a reduction in trade between Country A and Country B, as exports from Country B (host of Firms Y and 47 Z) decline due to higher costs and prices, and an intra-industry redistribution within Country A, as Firm F gains sales at the expense of Firm G. In this case, it is notable that the losers from Country A's policies cross national boundaries and divide within the industry. Also, had Firms Y or Z relocated production to Country A, the eect would have been the same. Consumers in Country A will also experience some utility loss through substitution or reduced consumption of their preferred varieties. Z) decline due to higher costs and prices, and an intra-industry redistribution within Country A, as Firm F gains sales at the expense of Firm G. In this case, it is notable that the losers from Country A's policies cross national boundaries and divide within the industry. Also, had Firms Y or Z relocated production to Country A, the eect would have been the same. Consumers in Country A will also experience some utility loss through substitution or reduced consumption of their preferred varieties. In Country B, where there are no additional costs levied on rms, the eects are relatively benign. 2.7.3.1 One Country Raises Costs of Some Firms Firms Y and Z, facing higher costs to export, do not sell as much abroad. However, their access to their home market is unaected, and thus prices in the home market should be unaected. Imports of goods produced in Country A by Firms F and G are similarly unaected, and so sales should remain constant there, as well. For consumers in Country B, there is no meaningful distortion that would move them from their preferred consumption pattern. 2.7.3.2 Both Countries Raise Costs of the Same Firms If, for some reason, both countries raise costs on the same rms, the single-country eect discussed in the previous paragraphs is intensied. Assume now that both Country A and Country B raise costs on Firm F, rather than lower them through subsidy or the like. The eect on Firm F will be signicant. There will also be eects on consumers in both markets, as expected. In Country A, as costs rise for Firm F, they will either incur loss through lower prot or pass on those costs to consumers as higher prices. If prices are raised, some consumers will change their consumption to varieties produced by other rms (Firms G, Y, and Z), which will have a similar deleterious eect on Firm F. Also, consumers in Country A will experience some disutility from the substitution away from their preferred variety or the reallocation of their budget to continue consuming goods from Firm F. In Country B, the same also happens. Firm F, if they are to sell in 48 that market, must bear the costs of the rm-discriminating policy or pass them on to consumers as higher prices. In the former case, the lost prots hurt the rms. In the latter, the familiar substitution occurs across varieties produced by dierent rms. For Firm F, there is a loss in both the domestic and export markets. For all other rms, the opposite is true. For consumers in both countries, there is some loss of utility from cross-Firm or cross-product substitution. The overall eect on trade is to reduce exports of Firm F's goods from Country A to Country B. While it is likely that Firm G will export more from Country A to Country B as a result of the substitution of consumers in Country B, it is unlikely that this substitution will be of sucient magnitude to fully oset the loss of trade in Firm F's goods. This increases asymmetries in trade within the industry, as well as altering the composition of goods traded within the dyad. 35. This is analogous to Firm F being given some subsidy in Country A, while Firm Y is subsidized in Country B. 34. Subsidizing national winners is analagous to raising costs on all non-subsidized rms, from the perspective of intrarm competition. 2.7.3.3 Each Country Raises Costs of Dierent Firms If each country raises costs on dierent rms, perhaps by selecting national win- ners34, this will have a dierent eect on the market. In this case, rms may nd they can sell in one market without bearing additional market access costs, while in the other market, entry requires fullling requirements or overcoming policy-induced access barriers that add additional cost to goods sold in that market. Consider the scenario where Country A raises costs on Firms G, Y, and Z, while Country B raises costs on Firms F, G, and Z.35 Since the additional costs apply to some rms in both markets and other rms in only one market, there will be market distortions in both domestic and international terms. In Country A, the additional costs imposed on goods produced by Firms G, Y, and Z will lead consumers to change their consumption towards goods produced by 49 Firm F. If the rms can absorb some of those additional costs as reduced prots, the eect on rm prots is largely the same. This substitution leads to the expected outcome: Firm F sells more goods in Country A, while Firm G sees losses, creating a redistribution among Country A rms. For Firms Y and Z, the additional costs of market access mean lower sales, fewer exports to Country A, and may lead those rms to exit the Country A market. Firm F. If the rms can absorb some of those additional costs as reduced prots, the eect on rm prots is largely the same. This substitution leads to the expected outcome: Firm F sells more goods in Country A, while Firm G sees losses, creating a redistribution among Country A rms. For Firms Y and Z, the additional costs of market access mean lower sales, fewer exports to Country A, and may lead those rms to exit the Country A market. In Country B, it is goods produced by Firms F, G, and Z that are more costly in the market. Again, if those rms can absorb the additional costs as reduced prots, they experience disutility. If they pass along costs to consumers in the form of increased prices, there will be substitution away from those varieties towards those of Firm Y (the privileged rm), with the related lost sales for the rms and loss of utility for consumers. 2.7.3.4 Costs that Apply to Firms as New Entrants Another group of policies that distort market access applies to any rm wishing to enter a market. These often take the form of a registration process or a licensing rule that ties market access to the identity of the rm selling the product. In these cases, a rm wishing to enter a market, regardless of location, may be forced to pay an additional xed cost in order to sell goods there. These kinds of costs have the eect of suppressing market entry among rms that may only sell a few units, or that make little prot o each unit sold, as the xed entry cost makes the total cost of production prohibitively high. In the context of rm-specic costs, these work somewhat dierently than those discussed above. The intraindustry division is between legacy rms and new entrants as winners versus losers in this case, assuming the policy is implemented in a way that exempts existing rms from paying the cost. If applied as a new cost to all rms in a given market, these kinds of policies can have the additional eect of distributing benets towards rms that are better able to absorb those costs for market access, much in the same way as indiscriminate costs do.36 2.7.3.3 Each Country Raises Costs of Dierent Firms In this situation, where a subset of rms have preferential access in certain mar- kets, there are cross-cutting eects for each of the rms. While Firm F may gain from its privileged market position in Country A, it loses in the export market of Country B. The same holds true for Firm Y, which gains sales in Country B, but loses them in Country A. Firms G and Z, which face additional costs both home and abroad, lose twice. Consumers in both countries also lose out, as the rm-discriminating market access costs will push them to consume less-preferred alternatives as a function of increasing costs on their more-preferred varieties. Overall trade ows should decrease, as the shipment of goods to Country B pro- duced by Firms F and G in Country A should decrease, as should the shipments of Firm Y and Z goods to Country A from Country B. This will occur across dierences in characteristics, and would even occur if those rms relocated to a third country (assuming similar underlying production costs). However, the balance of trade ows is likely less distorted than in the cases where the same rm was targeted by both countries or where only one country raised rm-discriminating costs. 50 36. This occurs as consumers substitute existing varieties in lieu of potential new varieties or those produced by more ecient rms in lieu of those produced by less ecient rms. 2.7.4.1 One Country Raises Indiscriminate Costs If one government implements a new policy that raises costs on all goods sold within a market, it will have the eect of raising all costs at once. For the most inecient producers, these additional costs will likely be too great to bear, leading to market exit. This aects both imported and locally-produced varieties. Thus, some importers, faced with total costs that exceed the price that the market will bear for their variety, will exit. Similarly, some rms engaged only in domestic production for the domestic market will exit for the same reasons. Consumers will move away from these varieties towards near-substitutes produced by rms that remain in the market, incurring some loss of utility from the substitution. For these more ecient rms, the increased sales will partially oset the decreased margins on each sale that are the result of the additional indiscriminate costs. These indiscriminate costs will also aect trade, if some imports to the market are produced by rms that are ecient enough to export when market access is cost- less, but that cannot economically sell those goods in the now higher-cost market.37 Exports from the market where indiscriminate costs are raised should be relatively unaected. 2.7.4 Indiscriminate Costs When governments implement policies that raise the cost of all goods in a market, regardless of origin, characteristics, or producer, through consumption taxes or pro- cedural costs like labeling, for example, the eect of these new costs will cut across all those divisions. These indiscriminately-applied costs will aect rms dierently, but those dierential outcomes are a matter of rm heterogeneity, not overtly discrimina- tory policies. The salient dierence among rms is the degree to which the costs can be absorbed before changing prices to market or, in the extreme, before market exit. 51 37. There may exist rms that can continue to access the market with foreign-produced goods produced eciently enough that the additional costs do not lead to market exit. 2.7.4.2 Both Countries Raise Indiscriminate Costs It is also possible that both governments implement policy that raises indiscrim- inate costs on goods sold within the respective markets. Perhaps both governments raise excise taxes or labeling costs that apply to all goods. What is consistent is that, thanks to increased costs and potential substitution of consumption, consumers in each market will have lower utility from consumption than in the alternative where such costs were not imposed. The eect on rms is from both domestic and inter- 52 national distortions. In this case, the winners and losers still divide along eciency lines as expected, but across more complex groupings of rms. The most ecient rms, whose cost structure can absorb the additional policy- induced costs and who were likely both domestic producers and exporters, will see the least negative eects of those new policies. For these rms, the eect of indiscriminate costs in both reducing the number of foreign entrants (who nd total costs of market access now too high to be economical) and some domestic competitors (who are too inecient to absorb the additional costs) will oset, in part, their own losses in margins or sales from the additional costs. For these rms, additional indiscriminate costs in both markets will have a modest eect on their prots. For rms who are moderately ecient, such that they would export to markets without the indiscriminate costs, but not to markets with the additional costs, the loss of access to an export market is clearly a loss. However, there are likely rms exporting to their home market in the same situation. Thus, while these rms lose on the international market, the eect on the domestic market is more modest than for less-ecient rms. It is even possible that, with enough exits by foreign and domestic competitors in their home market, those rms may see a small increase in sales volume (albeit with more modest prots) at home. These rms, despite reverting to domestic-only production, fare better than some. For the least ecient rms, it is the domestic eect, not the international, that matters. These rms were never going to export to the foreign market where the new costs apply. Thus, there is no direct gain or loss from the foreign market costs. 2.7.4.2 Both Countries Raise Indiscriminate Costs The eect on competitors in their own market is likely indeterminate, as some foreign rms exit and some domestic rms who exported return to the domestic-only market. However, the direct eect of new indiscriminate costs in their own market will be negative. If these rms are unable to absorb the cost in some way, either by reducing prots or raising prices, it is likely they will exit the market. In any of those cases, 53 ast ecient rms suer to a signicant degree. Overall trade ows here will be reduced as a consequence of rms in both countries reducing their exports to the other. While the exact degree of this reduction will vary according to rm-specic characteristics, net trade ows will decrease as ows in each direction decrease. 2.8 Summarizing Winners and Losers The policies that distort markets can have complex distributional consequences. For those actors that are most directly aected by these policies (rms), ending up on the winning side or the losing side of a policy change depends on a number of factors. If the policy is one that discriminates on the basis of location of a goods production, then the lines are drawn along familiar geographic lines. If the policy discriminates based on the characteristics of a good, then the benets divide within industries, along dierences in technologies or techniques. If the policy divides on the identity of the rm producing a good, the winners and losers divide according to which rm or rms are targeted by the policy. For indiscriminate costs, it is producer eciency, and the ability to absorb cost increases, that determines who loses most. These divisions become more complex when the policies are changed in reaction to the policies of another government, or when policies are harmonized or made re- ciprocal. In each of the markets where market access costs change, both the domestic eects and changes in trade ows and composition apply. Firms may nd that, with reduced location-discriminating costs in both their home market and a foreign market where they sell their goods, they face increased international competition at home but also have easier access to foreign markets. A rm whose goods previously faced characteristic-discriminating costs both home and abroad may be a clear winner from changes in policies, but if it also opens the door for other competitors with similar varieties of goods, those gains may be moderated, or another market access cost may 54 become a binding constraint on production and sales. Policies can be quite targeted or quite general, depending on the features of an industry. Location-discriminating policies may be crafted in such a way as to only aect some varieties of a good (perhaps by targeting a narrowly-dened subcategory of goods), or can take the form of broadly-dened quotas or taris that cover many varieties of a good. In the same way, policies that impose restrictions on certain varieties can allow a wide variety of goods to access the market unaected or can impose costs on all but a few varieties.38 38. In some cases, this highly-targeted policy may have the eect of privileging a single rm 2.9 Conclusions and Moving Forward This paper introduces a new framework for looking at domestic and international policies and their eects on trade, both in volume and composition. It is an attempt to resolve some points of dierence in the evaluation of non-tari barriers to trade, which we argue arise because not all non-tari barriers are alike, and some are more like taris than other non-tari measures. In moving to considering policies according to how they divide a goods market through the raising of costs, we hope to clarify the discussion not only of the eects of these policies, but also the politics that support their raising. Each of these types of policies will create dierent cleavages of winners and losers, and actual rms will have expectations of these policies eects that motivate political action. Sometimes this will occur in ways quite similar to our understanding of tari policy creation, as with most location-discriminating policies, but often will occur in quite dierent ways. Further, the types of policies that rms should demand to protect their market position will reect the manner in which each type of policy will aect them and their competitors. This will often mean intra- industry cleavages that span foreign and domestic producers, producers of competing varieties, and producers of dierent size and identity. 55 This paper does not examine the politics of how these policies come about, nor does it examine the presence or absence of these policies in dierent markets. A theory of market access barrier politics, which involves both trade-focused policy and simple domestic regulation, should follow from combining this typology and existing theories of lobbying or pressuring for distributive politics. It is also possible that such arguments would lend new insights into how markets are regulated in light of international markets. Similarly, if one were to apply this typology to existing classications of NTMs (such as UNCTAD's TRAINS data), it may be possible to measure the dierences in the types of market access costs across industries and countries, to examine when these policies are applied and how they distort markets. It may also be possible to determine how these policies distort markets (in ad valorem tari-like terms) by examining price or sales dierences across salient dimensions (for each type of market access cost) across goods in a given market. 2.9 Conclusions and Moving Forward Despite the limitations of this paper, the framework introduced herein does pro- vide some advantages for understanding trade-distorting policy. Compared to di- viding on core vs quality or at-the-border vs behind-the-border, or any of the simplifying assumptions about non-tari policies as permutations of taris, a focus on how costs apply claries the similarities among taris and some non-tari policies and the dierences between those policies and the wide variety of market-distorting policies currently considered under the broad umbrella of non-tari barriers to trade. Further, this framework puts the distributive eects (and thus the distributive poli- tics) at the core of the discussion and subsequent theories. To understand the politics of trade in an era of global rms, global supply chains, and a global goods market under mobile capital, we must revise our conception of how dierent policies aect markets, within and across national borders. 56 Market Access Barriers and Formation of Protection-Seeking Coalitions In recent years, bilateral and multilateral negotiations on trade have shifted from focusing on taris and tari-like measures, such as quotas, towards negotiations over other trade-distorting measures, such as product standards, licensing rules, intellec- tual property rights, and government support of local rms. This shift in negotiation reects the new reality of protectionism in the global goods market: taris are no longer the primary means by which governments regulate access to their domestic markets. The New Protectionism that Baldwin discussed in 1986 has become the new normal, with non-tari measures and domestic market interventions now more prevalent than ever(Baldwin 1986). The essential boundaries in the global market for goods are no longer strictly at the border. The politics surrounding modern free trade agreements, especially multilateral ones, reect the complex distributive consequences of the kinds of market integration that these agreements entail. As negotiations move from policies that apply at the border, like taris, to policies that are part of the domestic economy, like product standards, testing and labeling regulations, government support of individual rms, or licensing and intellectual property rules, the groups of political actors who rise in support (or opposition) to those agreements also changes. 57 Some industries in some countries remain protected behind taris or other mea- sures that look similar to taris. Other industries are liberalized with respect to taris, but are otherwise protected by regulations or policies that restrict market access, thus reducing trade by deterring market entry. Despite relatively low taris, trade ows do not always reach expected levels, or the goods traded are not of the varieties expected by neoclassical models. Taken together, these features of policy and trade ows raise questions: Why are some industries protected with policies like taris, while others are not? What explains not just the level, but the types of pro- tectionism dierent industries are able to obtain? When will agreement on lowering barriers between markets, of the tari or non-tari variety, be feasible? Answering these questions requires one tackle the similarities and dierences be- tween taris and non-tari policies, then explain the logic of protection-seeking in light of those similarities and dierences. This paper advances a theory of politics of industrial protection that builds o a new typology of market access barriers. Rather than taris vs. Market Access Barriers and Formation of NTBs, this typology compares policies that increase or reduce costs of goods based on the good's location of origin, process or content of production, or producing rm. That typology helps clarify when dierent policies are complements or substitutes, and thus when the politics of demand for those policies should be similar or dierent. This theory argues that features of an industry - how much FDI is present, the degree to which products are homogeneous or dierentiated, rm concentration, or the relative eciency of producers - determine what kinds of protection are politi- cally feasible. The logic of industrial protection reects tradeos among these kinds of policies. Protectionism is the result of a political process where rms demand pro- tection that benets them and oppose protectionist policies that harm their interests. In this process of lobbying and counterlobbying, rms' success at obtaining protection of dierent types depends on how other rms in the industry will lobby, and how the 58 policies they demand will aect consumer welfare. It is not feasible to implement poli- cies which raise all kinds of costs at once; the degree of market distortion would lead to political backlash. How policies aect consumer welfare depends on how signicantly the policy distorts markets, or how much protectionism there is. How other rms in an industry lobby depends on their characteristics, and thus the characteristics of the industry overall. The form of protection location-discriminating, characteristic- discriminating, rm-discriminating, or policies that impose indiscriminate costs will reect a compromise among rms and between rms and government. The logic of protection-seeking industry coalitions suggests that the presence (or absence) of foreign investment or multinational rms should lead to less (more) location-discriminating barriers to market access. In heterogeneous goods industries, characteristic-discriminating policies are politically feasible, and thus provide an (im- perfect) alternative to location-discriminating costs. When an industry is concen- trated, with one or a few large rms, those rms may successfully lobby in favor of policies that create costs for other rms, thus dividing the market with rm- discriminating costs. When enough rms in an industry are relatively ecient, or otherwise able to bear costs while still remaining protable, there may be sucient political support for policies that levy indiscriminate costs on goods within that in- dustry, harming inecient rms more than ecient ones. 1. Indeed, Jagdish Bhagwati once referred to the substitution of non-tari measures for taris as indication of a Law of Constant Protection" (Bhagwati 1988, pg. 53). Market Access Barriers and Formation of As features of an industry change due to investment, technological change, or nat- ural rm growth, death, or merging, the demands for protection from rms within that industry will change. As foreign multinationals enter a market or domestic multi- nationals invest abroad, incentives for location-discriminating barriers fade. Where possible, rms will lobby for other kinds of policies - characteristic-discriminating, rm-discriminating, or indiscriminate costs - that protect their economic interests and those of a suciently-large coalition of other rms. As technological change cre- ates new dierentiation possibilities within an industry, characteristic-discriminating 59 costs may become the most feasible option, and rms will lobby for those policies over other types. As rm concentration increases, the potential for rm-discriminating costs to be politically feasible will change. Finally, as taris, a form of location- discriminating costs, become politically infeasible, either because of agreements or because of changes in the nature of industry, the non-tari alternatives that indus- try will seek as substitutes will depend on dierences among those policies that the tari/non-tari dichotomy cannot capture, in theory or in measurement. 3.1 Considering NTMs and Existing Explanations for Trade Barriers The existing literature on trade barriers and the political incentives that motivate them in economics and political science is extensive. Literature to date has generally treated non-tari trade distorting policies in one of two ways. In one view, these measures reect the same kinds of distributive (and thus political) dynamics as taris do. Non-tari policies, then, reect the same broad logic as do taris. The politics of NTMs are treated as largely similar to the politics of taris, or are treated as a minor perturbation of that logic. In the other, NTMs are dierent in important ways from taris, and the logic underlying the politics around NTMs are distinct from that of taris. When considering the spectrum of policies that may distort trade, but that are not taris, it is not immediately clear why the politics surrounding those policies should be the dierent.1 Indeed, some research indicates that, following a decline in taris, NTBs are implemented as substitutes. In the same vein, when taris are o the table because of international agreements, NTMs appear to serve as replacement protectionism during macroeconomic downturns (Manseld and Busch 1995). 60 More recent work on the subject has made eorts to highlight the dierences be- tween NTBs and taris, even drawing distinctions within the broad category of NTBs (core vs quality, where the former captures quantitative restrictions and other "tari-like" measures while the latter focuses on safety and technical standards, for instance) (Kono 2006). These arguments have usually been focused on one specic feature of non-tari measures, distinct from taris. The result has been a body of theory on NTB that raises many plausible arguments for why the politics of protec- tionism through non-tari means is dierent from taris. Some arguments over the politics of taris vs non-tari measures focuses on the degree to which governments can get away with distributive policy via each mechanism. Taris are relatively transparent, while many non-tari measures are more dicult to understand. This lack of transparency incentivizes a switch from taris to non-tari measures, because voters observe non-tari policy only imperfectly and don't punish governments for redistributing using NTMs, especially quality NTMs (Kono 2006; Magee, Brock, and Young 1989). Research that leverages insights from new new trade theory (NNTT) of het- erogenous rms has identied patterns in trade politics that previous work focusing factors and sectors could not. 3.1 Considering NTMs and Existing Explanations for Trade Barriers An early insight from political economic analysis of lobbying for trade protection in heterogenous goods industries (new trade theory) suggests that such lobbying follows the logic of private goods. As each rm is a mo- nopolist, any lobbying eort represents a benet solely to the lobbying rm. Thus, in dierentated goods industries, where IIT occurs, rms are more likely to lobby for protection (Gilligan 1997b). However, more recent research suggests that when rms are heterogenous, the preferences over protection within a given industry will vary. When rms are heterogenous, this comes from dierences in competitiveness and the potential for reciprocal liberalizaton (Osgood 2017). An alternative view posits that industry-level demand for protection moderates as IIT causes importing 61 interests within industry lobbies to grow, and thus lobbying on trade becomes rm- specic (Madeira 2016). In some cases, these intra-industry cleavages can manifest as highly-variable tari schedules across tari lines within single industries, where individual rms lobby for protection of just their varieties (I. S. Kim 2017). Recent discussion of non-tari measures, specically technical barriers to trade, in light of NNTT, highlights intraindustry divisions. Here, when non-tari measures create xed costs for all rms then highly productive rms, including multinationals, should seek and obtain NTBs specically technical regulations, as these entry costs disproportionately benet them (Abel-Koch 2013; Gulotty 2014; Osgood 2016). The demanded level of these NTM policies again varies with respect to a rm's productivity, and may arise under certain conditions. However, there is still some disagreement on the degree to which NTMs, as xed costs, serve as substitutes for tari measures. While the existing literature has yielded many insights, an area for further theory remains. While broad factoral and sectoral theories of trade preferences explain lib- eralizing and protectionist pressures in general, they have less to say about change in policy across varieties of trade-distorting policies. Existing work on NTMs that fo- cuses on transparency and the relative opaqueness of NTMs explains only why taris are replaced by NTMs, not why the substitution varies, or why some industries retain taris or tari-like policies. 3.1 Considering NTMs and Existing Explanations for Trade Barriers New theory based on theories of heterogeneous rms has moved our understanding forward a great deal, but these tend to consider only one type of policy (tari or NTM) at a time, focus on variation within a kind of policy, or consider all NTMs as all similar usually as universal xed costs for market entry to ease inclusion in the Melitz (2003) model and its extensions. To explain the variety of policy changes across industries over time, new theory that examines the politics of protection, in light of alternative policies, is necessary. 62 2. This rm-centric approach is also consistent with approaches from Gilligan (1997b), I. S. Kim (2017), and others. 3.2 Motivations for Demanding and Supplying Protection The politics of trade barriers and the politics of industrial protection are, at their core, interactions among rms and between rms and government. The actors that drive the demand-side politics of trade protection are rms. To understand the logic of lobbying and the provision of protection, wepresent a theory of the interaction that engages in some necessary simplications. However, from these relatively simple assumptions (actors are self-interested, preferences are shaped by characteristics of the rm, etc.), it is possible to describe the politics of protectionism with some detail. This theory is a partial equilibrium one, focusing only on a single industry, and the discussion here focuses on politics within one country. Further, while each rm is a producer of it's own unique variety, no rm is assumed to be large enough on a global scale to directly aect world prices of other varieties to a signicant degree, so this may be considered a small economy example. Individual rms are assumed to be monopolistic producers of their particular variety of good. Although other rms may produce goods with similar characteristics, those alternative are distinct by virtue of producer identity, at minimum. Within each broad market category (cars, shoes, sweatshirts, wheat), one can assume an innite number of varieties. Firms are endowed with rm-specic technology to produce their good, using a single factor that, for simplicity, is assumed to be perfectly elastic in supply at xed relative prices. Thus, the determinant of a good's cost of production is that rm-specic technology, which may dier across rms. These assumptions are generally consistent with the logic of new-new trade theories of heterogenous rms and intra-industry trade (Melitz 2003). It is within this modern trade theory framework that this discussion of trade poltics resides.2 Building o the Grossman-Helpman model and the logic of menu auctions, these 2. This rm-centric approach is also consistent with approaches from Gilligan (1997b), I. S. Kim (2017), and others. Building o the Grossman-Helpman model and the logic of menu auctions, these g p g , 2. This rm-centric approach is also consistent with approaches from Gilligan (1997b), I. S. Kim (2017), and others. 63 rms propose bids across the varieties of market access restrictions (Grossman and Helpman 1994). In the original model, Grossman and Helpman assume lobbies arise exogenously, and their bids merely represent the interests of that lobby's membership. j 4. Grossman and Helpman restrict governments' policy instruments in their original model to taris and subsidies. 3. This endogenous lobby formation is discussed in other work, including Abel-Koch (2013) on the subject of NTMs. 3.2 Motivations for Demanding and Supplying Protection Here, weassume rms have the opportunity to lobby at all times, and lobbies groups of like-minded actors working in concert arise naturally.3 These bids represent promises of political support if a certain policy is implemented. The support can take many forms, and the details of that support are largely unimportant for this discussion. As in the original Grossman and Helpman model, rms provide complete bids for each level of protection that Government may provide. However, each bid now covers all levels of each type of market access restriction. The complexity of the rm's bidding is greater than in Grossman and Helpman, as the policy space is more complex. In the original model, lobbies bid on levels of protection that act as ad-valorem increases in the price of imported varieties.4 In this theory, some policies work in that fashion, while other policies create costs (xed or variable) that divide the market in other ways. This theory of protection-seeking considers lobbying when government has access to policies which discriminate on location, product characteristics, or producing rm, or that apply indiscriminately. It is analogous to the original Grossman-Helpman model in one way: location-discriminating barriers are costs that apply to imported varieties, similar to the instruments in the original model. Other variations of the G-H model, applying additional xed costs to foreign rms or xed costs to all pro- ducers, reect other kinds of location-discriminating costs and indiscriminate costs, respectively (Gulotty 2014; Abel-Koch 2013). This necessarily complicates the bid- ding, but the core logic remains the same. Governments make policy decisions in light of promised support from industrial coalitions seeking protection. In addition, 64 the insights provided by other variations of the original Protection for Sale model apply here. The interaction discussed here diverges from Grossman and Helpman's model in two other ways. First, lobbies may contain rms with interests in importing. These rms could be importers conducting business at arms-length with foreign partners or could be multinational rms with supply chains that cross borders.5 This also includes rms that might be considered foreign rms (because of home location), but that have inuence or political power within the country because of production location or other investments. This change reects the realities of trade and industrial politics in an era of global capital: domestic rms may have strong interests in importing if their supply chain crosses borders. 3.2 Motivations for Demanding and Supplying Protection Second, the policy eventually adopted may have a combination of costs from each of the categories discussed in Chapter II. The government must still set a single policy for the market, but it now has 4 dimensions, and the policy is a complete set of costs for each category. 5. The preferences of consumers of imports remain part of the social welfare function, as in Grossman and Helpman. 3.2.1 Who Can Lobby for Protection? In discussion of the logic of lobbying for protection, it is necessary to clarify which rms have the ability to lobby government for policy in their favor and which ones do not. A rm's domestic salience is the degree to which that rm is a politically- relevant actor in domestic politics. In some classic models of trade politics, rms are clearly divided into domestic and foreign rms. The former have political voice, while the latter do not. In an era of global capital ows, the division of rms into domestic and foreign is no longer as clear. Firms founded in the US play a signif- icant role in the domestic economies of countries all over the world. Firms founded in Europe, Asia, and the Americas play a signicant role in the domestic economy of the United States. These multinationals have a degree of political inuence within 65 markets that rms who do business at arms-length do not (Lee 2018; Lee and Os- good 2018; Stoyanov 2009; Gawande, Krishna, and Robbins 2006; Drope and Hansen 2004). Political salience within a country can come from two sources: capital invest- ment/presence and employment. The dependence of the state on capital motivates the rst, and the political voice of workers in a given rm motivates the second. For multinationals, political inuence in another country can come from either source. As rms invest in new markets, they gain some degree of political access, which gives them the power to lobby government for policy.6 The welfare of the rm aects the welfare of workers, and thus governments that are responsive to the public will respond more to rms as they expand and employ more workers. In general, rms that are national rms have more salience. Domestic-only producers (rms with no foreign investment) and domestic multinationals (MNCs in their home country) have the most political voice with their own government. Firms that are located and produce domestically have the most political salience for their size, as all of their investment and all of their employment is domestic. Multinationals with global supply chains generally have less inuence, given rm size, as some investment and employment is domestic, while some is foreign. 6. This is often examined in the context of foreign rm access in developing economies, but both anecdotal and empirical evidence from industrialized economies, specically the US, suggests that foreign multinationals lobby and aect policy in the countries where their investments are made.(Desbordes and Vauday 2007; Hansen and Mitchell 2000) 3.2.1 Who Can Lobby for Protection? Multinationals that produce mostly outside the borders of their home markets will still have some political voice in their home market, but it will come via the inuence that comes from capital, not from labor. Foreign multinationals have some domestic salience in their host markets, and that inuence increases in the degree to which they invest in their host market. Foreign multinationals with large production facilities (or other facilities relevant to their business) will have more political inuence than those with little investment in 66 a given market. Whether they import for further production or export, these rms are an integral part of the domestic macroeconomy. As a foreign multinational's employment and investment footprint grows or shrinks, so too will its political power. Foreign rms who only sell in the market through arms-length exchange, and thus have no direct investment or employment interests within the domestic economy, have no (or very little) political inuence.7 In discussions of rms with domestic political salience to follow, weconsider a rough ordering across types of rms that follows the logic above. Domestic rms that produce domestically are the most salient, followed by home multinationals, then foreign multinationals with signicant investment. Foreign MNCs with little investment or employment within a market and rms that do business at arms-length can be considered to have little domestic political salience, and therefore do not play a major role in lobbying for industrial protection within an economy. Other characteristics of rms, like size and employment, work in the familiar ways. This expansion of domestically politically salient is necessary for discussing the politics of industrial protection in the presence of global capital ows. Without considering the interests of rms that do not fall cleanly into the domestic versus foreign binary, any theory of trade-distorting policy would be incomplete, at best. 7. Whatever inuence they have may take the form of domestic groups with interest in continued import of that rm's goods. 8. An important omission from this framework is whether these policies raise xed or variable costs. The use of xed versus variable costs will have important implications for the eect of these policies on rms of dierent size, or with dierent levels of sales. However, that scale eect should be roughly consistent across the four types of policies, where xed costs of a given type aect small rms more than large rms and variable costs have similar eects across rms size, so are relatively better for small rms. 3.2.2 Revisiting A New Typology of Market Access Barriers Rather than dividing trade-distorting policies into taris and not-taris, the market access restriction framework situates all trade-distorting policies within a broader discussion of policies that, by adding costs to certain goods and shaping consumer choices, redistribute to certain groups within and across industries. Trade politics is redistributive politics, just as domestic industrial politics is redistributive politics. This market access-focused approach integrates the two, which is essential 7. Whatever inuence they have may take the form of domestic groups with interest in continued import of that rm's goods. 67 for understanding not only the politics surrounding modern trade negotiations and agreements but also for understanding why the nature of protectionism has changed as global capital ows have created rms with interests that span borders. This new framework considers industrial policy as imposing costs in four dierent ways. The rst kind of policies, those that are location-discriminating, impose additional market access costs on goods that are produced outside the market's borders. One might consider these policies that apply (or not) based on where it's made. The second kind, characteristic-discriminating policies, impose additional costs on goods of a certain variety, or that are produced in certain ways. These policies apply based on how it's made. The third kind of policies create additional costs for some producers, but not others, even if their goods are produced in the same manner and in the same location. These rm-discriminating costs can take a variety of forms, but all apply in a manner that divide goods market access based on who made it. The nal kind of policies impose additional costs on all goods in a particular industry. These indiscriminate policies add costs to all varieties, from all places, made by all rms. With these sorts of policies, everybody pays. All of these policies add cost to varieties of goods, thus changing the quantities that are consumed. Together, these four types comprise a typology of policies that spans all kinds of potentially-trade distorting policies, but that also separates them into groups where policies within the same category are comparable and policies in dierent categories are not.8 Each of the types of policy adds costs to some or all varieties of a good in the market. These costs are additive, as well. 9. While other arguments regarding non-tari barriers and the politics surrounding their provision may focus on potential welfare-improving features of these policies, that discussion is set aside here. It is dicult to conceive of a way of categorizing policies as welfare improving that is both consistent across markets and static over time. These sorts of welfare improving market restrictions generally reect changes in tastes within communities over time, often over matters like the environment or some normative concerns. This makes the eects of such a change dicult to measure and dicult to theorize. However, it should be clear that, if a market-distorting policy has some kind of welfare- improving eect, it will be more appealing to consumers. This means that government will be more likely to implement those policies, all else equal. 3.2.2 Revisiting A New Typology of Market Access Barriers For instance, if Government chooses a policy prole for an industry that includes a low level of indiscriminate costs, a low level of location-discriminating costs (for instance, a small ad-valorem tari), and a 68 policy that imposes moderate costs on some varieties, then dierent rms will face dierent costs for entering the market. Foreign rms manufacturing policy-targeted varieties will face the highest costs of market entry, while local rms producing va- rieties unaected by the characteristic-discriminating costs will face the lowest, only the indiscriminate costs. For some producers, the higher costs, and thus prices, will drive consumer demand low enough to force them to exit the market. 10. This can manifest in a number of ways. Democratically-elected governments may face higher 3.2.3 Consumers, Government, and Firms For consumers, the additional costs of market access-restricting policies manifest as higher nal prices for dierent varieties in the market. The exact composition of Government-provided protection with respect to type of discrimination is not impor- tant for consumers. It is merely the eect that said protection has on prices of each variety in the market that aects consumer choice. Faced with higher costs for some varieties, consumers reallocate their consumption to other varieties of goods. It is this reallocation that is critical. Consuming less or consuming less-preferred varieties due to increased cost of more-preferred varieties means a loss of welfare.9 For government, the key decision is what, if any, additional costs of market access to impose as part of a policy. The benet of imposing costs is the political sup- port that rms provide to government in exchange for policy. However, the cost of enacting policies that distort markets is borne as consumers (who also support the government politically) experience a reduction in welfare. Their support for govern- ment decreases, which makes government worse o.10 As in other theories of lobbying 69 for protection, it is the government's desire for political support from lobby groups versus its consideration of consumer welfare that determines the level of protection and the price lobby groups must pay to obtain it. For rms, whether one views a particular policy as benecial or detrimental de- pends on whether consumers divert consumption towards or away from the varieties of goods they produce.11 Firms would prefer to have policies that increase consumption of their goods. For protection-seeking rms, the critical decision they face involves lobbying in an eort to obtain protable protectionist policies or to prevent costly protectionist policies from being implemented. This decision requires the rm to de- cide how much to commit to lobbying/political support and towards which policies to commit those resources. They make this decision in light of their own preferences, and with expectations of what other rms in the industry may demand. The process by which market access policy is made or reformed can be simplied to a number of steps, which reect the logic of Grossman and Helpman's Protection for Sale model. First, rms submit bids, promises of political support that map to each combination of policies that government may adopt. Firms truthfully reveal their preferences over market access-restricting policies in these bids. risk of removal, or must expend more resources to maintain voter support. In autocracies, widespread unrest can lead to instability, but it is much less likely to aect leader/government survival. As in Grossman and Helpman's original model, one can consider the relative weights government places on benets from lobbying groups versus public/consumer support to vary. More specically, one can expect these weights to vary with the form of political institutions. p g y p 11. This is discussed in greater detail in the previous chapter. 3.2.3 Consumers, Government, and Firms Government then considers the oers of support in return for setting protectionist policies at a given level, considers its own loss in welfare from the political backlash from consumers, and sets policy at some combination of market access-restricting policies. After policy is set, consumers make purchasing decisions in the newly regulated market to maximize their utility. The government's utility is a weighted function of the political contri- butions from rms, consistent with the oers related to the implemented policy, and consumers' utility. 70 In this interaction, information is common. All rms know how others will oer support for each combination of policies. The government's weighting function on political contributions versus public/consumer welfare is also commonly known. The eect of dierent policies on consumer welfare is well understood by all parties. It is characteristics of industry and political institutions that motivate the interaction, not asymmetries of information. Also, when rms submit contribution schedules, they credibly commit to providing support once policy is set. It is these contributions that sustain government once market-distorting policies aect consumer welfare, and withholding them (or even the possibility of withholding them) would negate the incentives that cause government to implement consumer welfare-reducing policy in the rst place. 12. Broad, industry-wide subsidies are another location-discriminating policy which have been used more frequently as trade agreements have restricted the use of taris and quotas (Rickard 2012). Subsidies can be targeted, even down to the rm-level, and the dierence in use of general versus specic taris, which might be considered location- versus rm-discriminating costs, is examined in Park (2012). CVD and antidumping duties are policies which have grown out of these agreements, and have received attention in the political economy literature (Wruuck 2015; Naoi 2009). Also, it is theoretically possible for a government to implement location-discriminating policies that make locally-produced varieties more expensive, but those policies are not considered here. Deardor (1987) considers quotas against other location-discriminating policies, like taris, but argues that the quantitative limits on imports oers political benets that the others do not. The price versus quantity measure discussion is set aside here, but bears future study. 3.3 Lobbying for Protection With the basic framework of the political-economic interaction surrounding mar- ket access policy established, we can turn our attention to the way in which contribu- tion schedules the support bids are generated. At the core of the political-economic interaction of rms, consumers, and government is the process of industrial lobby- ing. Individual rms or groups of rms demand policy from government and provide support and resources necessary to make policy change feasible and desirable for gov- ernment. In the absence of industrial lobbying, one can assume government would adopt polices that privileged consumer welfare to a greater extent. It is not, however, the case that industries lobby as a homogeneous group. As discussed above, dierent sorts of policies have dierent eects, even on rms within the same industry. There is an intra-industry tension of lobbying and the potential for counter-lobbying for certain kinds of protection that comes from the distributive consequences of dierent kinds of policies. As organized, politically-salient actors, rms with a domestic presence have the ability to inuence industrial policy through 71 lobbying behavior. In comparison, consumers face signicantly higher coordination problems and experience the costs of protectionism as more diuse costs. Foreign rms may be more organized interests, but without legal or institutional access to government or political salience, do not have a seat at the table. Because of this, both are less signicant in the creation of trade and industrial policy. For clarity, werst consider lobbying over each type of market access barrier sep- arately. Simplied examples of each type of policy are used to explain how divisions within industries arise, and how lobbying for protection within industries will man- ifest. Then, weintroduce the logic of lobbying over a basket of policies (of multiple types) at once and summarize the hypotheses from this theory. 3.3.1 On location-discriminating policies Taris, quotas, countervailing duties (CVD) and the like are examples policies that add costs to goods in a market produced outside that market.12 While taris are the most obvious form of location-discriminating cost, other policies, like temporary geographic restrictions on import of certain agricultural products, also impose costs based on where a product is made. For consumers of French cheese, it is irrelevant whether the additional cost on a wedge of authentic Camembert comes from a duty schedule or from the expense of the customs procedure at the border. What is relevant is that the cheese costs more because it was produced outside the boundaries of the country. When weighing a purchasing decision, French cheese entering without duty 72 and low or no customs cost and a subsidy on locally-produced variety is similar to duty on the French cheese. What matters is that the dierence in price is due to where the cheese was produced. Positions on these policies broadly fall along expected lines domestic-only rms will prefer higher location-discriminating costs. Firms that import, either for nal sale or as part of a supply chain, will prefer lower location-discriminating costs. Each rm within the lobby has an ideal level of protection: for importers and MNCs it is low - at or near zero13 while for domestic-only rms it is higher, to drive consumers to substitute locally-produced varieties (which don't face the location-discriminating cost) for imported varieties. The benets from location-discriminating policies accrue to all domestic rms, although not necessarily equally. As consumers reduce consumption of higher-cost imported varieties, they substitute post-policy lower-cost alternatives produced do- mestically, with some loss of utility. Firms who produce their goods domestically, thus avoiding the additional costs, will earn prots as good as or better than in the market without the location-discriminating policy. Firms that produce abroad will earn less prot as a result of those policies. The lobbying around location-discriminating policies takes two possible forms that reect those benets and costs. On the pro-policy side, rms that produce their goods domestically will be willing to provide support to government if the prots from increased sales exceed the costs of that political support. 14. The free trade interests (where free trade means lower taris, in particular) of rms with global supply chains are well understood. As an example, Milner (1988) establishes the power of these interests in case studies focusing on comparable industries in the 1920s and 1970s. In the latter case, industries were divided, conditional on some degree of global intrarm trade. 13. Or, if possible, negative, such that imports are subsidized. 13. Or, if possible, negative, such that imports are subsidized. 15. A third group of rms, apart from domestic-only producers and producers with importing interests within their own industry, are rms in other industries that use imported goods as inputs. Higher (lower) location-discriminating costs mean higher (lower) production costs for these rms if they produce in the protected market. However, these rms are excluded from discussion here because of the focus on intra-industry dierences in lobbying behavior. Those input-importing rms play an important, but secondary, role in debates over protection in a given industry. This relationship is examined in greater detail elsewhere, including in Schattschneider (1935). 16. Note that characteristics is a distinct concept from quality, which is used elsewhere in the literature and generally connotes greater or lesser value. Characteristics is used here to describe an ordering which does not necessarily correspond to quality. 3.3.1 On location-discriminating policies On the other side, rms with importing interest will provide political support if the foregone prots from higher market access costs outweigh the costs of the political support they must provide to outbid those who seek location-discriminating costs.14 73 There can be only one set of government policies at a time, and so the political support that government obtains is the combination of committed support of all rms for that particular level of location-discriminating costs. For each level of location- discriminating costs, the government benets both from the support from rms and from aggregate welfare of consumers, which passes through as mass support for gov- ernment. Government sets policy to maximize the combination of the two, given the relative importance of each in the government's utility function. For location-discriminating barriers, the essential feature of the domestically- salient rms in an industry is the degree to which those rms are domestic producers. While domestic producers will increase political support of government as the level of location-discriminating costs increase, aggregate consumer welfare, and thus that por- tion of the government's utility, will fall. In an industry with only domestic producers, the level of location-discriminating costs will be set at some positive level, where the combination of political support gains and consumer welfare (which is declining) is at its maximum. This is the canonical protection for sale logic: Domestic rms who produce for the domestic market will seek protection that keeps out imported varieties of goods and will transfer some of the prots to government as political support. However, if there are rms within the industry with interests in importing in their own industry, the portion of government's utility function that comes from industrial support will be higher at lower levels of location-discriminating costs.15 Those im- porting rms will (credibly) support the government more at lower levels of location- discriminating costs. The portion of government's utility function related to political support conditional on policy is no longer as simple. Now, government can expect 74 some positive support from industry, even if it removes all location-discriminating costs. In industries where importers are an organized interest, location-discriminating barriers will be lower, as the government's maximum utility is obtained at a lower level of location-discriminating costs. Some combination of political support from importing interests, support from domestic producers and higher consumer welfare will yield maximum benets for government. g y p q y 17. In the auto industry, gas guzzler taxes in the US and CO2 emissions-indexed road taxes are two examples of this. 3.3.2 On characteristic-discriminating policies Although often not framed explicitly as trade policy, other market-access restrict- ing policies that discriminate by adding costs to some varieties of goods on the basis of product characteristics or production process can have trade distorting eects. Among rms with political voice, each would prefer the variety it produces (or is best at producing) to enter the market with no additional costs. They would prefer other varieties, with dierent characteristics, to face additional costs, thus making them less attractive to consumers. For simplication, consider a characteristic-discriminating policy that works in one of three ways. For an arbitrary ordering of varieties according to characteristics16, a policy may add costs to all varieties above some point in the ordering, to all varieties below some point in the ordering, or to all varieties between (or outside of) two points in the ordering. This policy may be a set of rules dictating a product standard, or one that limits the use of certain products in a goods production. The policy may be a luxury tax or a use tax that applies to some varieties but not others.17 Regardless of the specics of the policy, characteristic-discriminating policies have the eect of adding 75 cost to certain varieties of goods, regardless of location of production or identity of the producer. Policy that generates characteristic-discriminating costs determines both the subset of varieties that face increased costs and the magnitude of those additional costs. The benets from characteristic-discriminating policies accrue to producers of goods unaected by those policies which raise costs on other varieties.18 As con- sumers reduce their consumption of higher-cost varieties, producers of goods with characteristics unaected by the policy see increased sales and higher prot. The costs of these fall on producers who are best at producing varieties of goods with characteristics that are targeted by policy. Note that these kinds of policies create a group of winners and losers that spans the geographic boundaries that dene na- tional markets. Foreign producers of varieties targeted by these policies are aected in the same manner as domestic producers of similar varieties. Foreign produced varieties that are within the subset of varieties that access the market without addi- tional cost are winners just as domestic producers of varieties within the same subset are. These costs apply regardless of where the product is produced, or by whom. 18. For policies which lower costs of some varieties, like tax credits or refunds, being targeted by the policy is benecial. 3.3.2 On characteristic-discriminating policies Thus, characteristic-discriminating policies create coalitions of foreign and domestic producers of protected varieties against foreign and domestic producers of aected varieties, and industrial politics reect those coalitions. Domestically-salient rms that produce goods varieties aected by characteristic- discriminating costs lose out from those policies. Consumers, too, experience losses as their domain of product choices is limited or their ability to purchase certain varieties reduced due to increased costs. Being forced to choose a second-best variety (based on the consumer's preferences) or pay a higher cost to obtain their most-preferred variety is what causes the loss in aggregate welfare. Firms promise to provide the government with support in return for policies that 18. For policies which lower costs of some varieties, like tax credits or refunds, being targeted by the policy is benecial. Firms promise to provide the government with support in return for policies that 76 add costs to goods with characteristics unlike those in which they have a production advantage. The degree of support they are willing to oer is proportional to the increase in prots from consumers reallocating their consumption. If a policy aects many varieties or induces a large change in consumption habits by adding signicant cost to a few targeted varieties, rms who benet will provide more support to govern- ment than they would for a policy that has a smaller eect on their prots. For any policies that add cost to products with the characteristics of their own products, rms will oer no support to government. However, some rms may also oer some support to government that implements policies that have few characteristic-discriminating costs, as a counter-lobby against competitors who oer political support in favor of costly policy.19 When setting policy that creates characteristic-discriminating costs, government again weighs the benets in terms of political support from the winners the rms who specialize in the varieties that enter the market without additional cost against the political costs of reducing consumer welfare. As government wishes to maximize the combination of political support and aggregate welfare, setting characteristic- discriminating costs at a very high level or over a broad subset of varieties is costly. Unless the group of rms who stand to benet from reallocation of consumption be- havior provide signicant political support, government will implement more modest policies. y g y g g 20. This homogeneity can arise from natural dierences in the nature of product categories: It is relatively dicult to produce a Cavendish banana that has signicantly dierent characteristics 19. The logic here is similar to the location-discriminating case: counterlobbying in a bid occurs as a response to the pro-cost lobbying in which other rms may engage. 19. The logic here is similar to the location discriminating case: counterlobbying in a as a response to the pro-cost lobbying in which other rms may engage. 3.3.2 On characteristic-discriminating policies For the demand for and supply of policy that adds characteristic-discriminating costs, one essential feature of the industry is the degree to which goods in that indus- try are homogeneous or dierentiated. If all producers have expertise in producing goods with similar characteristics, within a tight subset of the universe of all pos- sible varieties, that is a homogeneous product industry.20 This, in part, determines 77 whether policies can be targeted or will aect many (or all) rms in a given i In industries where goods are largely homogeneous (in terms of these technical characteristics), where characteristic-discriminating costs will have broad incidence, one should expect counterlobbying to deter government from implementing signicant characteristic-discriminating costs. Put simply, the net reduction in aggregate welfare will be great, as few varieties will be unaected by the policy, forcing broad reductions in consumption, rather than substitution. In addition, there will be few rms willing to lobby in favor of these sorts of policies, as they are likely to lead to reductions in their own sales/prots. In homogeneous good industries, characteristic-discriminating policies should be less frequent and their costs less onerous. It is only if a concentrated and relatively large group of producers, outliers in terms of the variety they produce, promise to provide signicant political support that characteristic-discriminating policies should arise. Those policies would signicantly reduce consumer welfare while also leading to large increases in prots for those rms who can remain in the market producing unaected varieties. Thus, for government to implement such policies, the political support transfer must be very large, and this is unlikely to be optimal for both rms and government. In industries where goods are naturally dierentiated, where there are sucient characteristics to discriminate among varieties produced in a targeted way, a more complex set of promises of political support is oered to government. Firms will still submit oers where they provide no support for policies that add cost to their own varieties of goods, but they may provide some support for policies that impose costs on other varieties with dierent characteristics. Government can now set policies with than other Cavendish bananas. It is relatively easier to produce children's toys that have unique, or uncommon, characteristics. than other Cavendish bananas. It is relatively easier to produce children's toys that have unique, or uncommon, characteristics. An empirically-driven concept of homogeneity comes from Rauch (1999), which looks to the presence of organized commodity exchanges for a particular good as evidence of product homogeneity, and the presence of reference pricing as an intermediate between homogenous and dierentiated goods. 3.3.2 On characteristic-discriminating policies An empirically-driven concept of homogeneity comes from Rauch (1999), which looks to the presence of organized commodity exchanges for a particular good as evidence of product homogeneity, and the presence of reference pricing as an intermediate between homogenous and dierentiated goods. 78 more modest market distorting eects, excluding some varieties without reducing overall consumption as much as in the homogeneous goods case. In dierentiated goods industries, characteristic-discriminating costs will arise if politically-salient rms all produce varieties with similar characteristics. If this is the case, there are policies that redistribute sales and prots from other rms who have no ability to lobby government (either because they are foreign rms or because the costs of political support are too high) to those rms that have political access. Consumers will experience some loss of welfare from the reduction in goods market choice, which then reduces the government's welfare. It is the political support from the winners, those rms who experience growth in sales and prot and transfer some back to government, which makes this market-distorting policy outcome optimal for government. Alternatively, if domestically-salient rms produce goods with a broad variety of dierent characteristics, the combination of scarce political support for targeting any subset of varieties and potential counterlobbying against policies that raise costs of this nature will prevent government from implementing policy with signicant characteristic-discriminating costs. 3.3.3 On rm-discriminating policies Firm-discriminating policies, like policies that restrict right of sale in a market to a limited number of companies, or restrictions that raise market entry costs by requir- ing producers to obtain licenses, create starker intra-industry cleavages. Some forms of these policies, like rm-specic subsidies, bailouts, and oligopolistic/monopolistic market policies are less common. Other policies, like government procurement poli- cies that privilege domestic rms, even if they produce goods outside the domestic market, have similar eect. Some rm-discriminating policies take the form of a rm- specic cost of market entry, such as registration or license requirements that impose signicant costs on new rms. 79 79 79 These kinds of policies provide signicant benets to the winning rms, and are likely to drive some rms targeted by the policy out of the market. It is this intra-rm redistribution that drives the lobbying behavior around rm-discriminating policies. However, dierent sorts of rm-discriminating policies will have dierent lobbying dynamics. weconsider two broad kinds of policies. The rst is a policy that consis- tently and perpetually imposes costs on some producers and not others. This could be a discriminatory government procurement policy or a form of subsidy. The sec- ond is a rm-discriminating xed cost of market entry, such that a rm pays once and is then in-market with respect to additional costs. These sorts of policies are analogous to rm-discriminating policies that impose some cost only on rms that are not incumbents. If one supposes an arbitrary ordering of rms in a market, in- cluding those rms that may potentially enter a market, an entry barrier is a cost imposed on the subset of rms that are not already in a market.21 In this way, rm- discriminating policies follow a consistent logic, but the incentives for each kind of rm-discriminating cost are somewhat dierent. weexpect that in industries with one or a few large rms, those rms will lobby for and obtain targeted protection in the form of consistent rm-discriminating costs, applied to all other competitors (both domestic and foreign). Conversely, in industries with lower rm concentration, the type of rm-discriminating policy sought will bar new entrants by raising rm-specic entry costs. The logic behind each is discussed below. weconsider the consistent costs policy rst. Each rm would clearly wish to have policies in place that gave it unique advantage in the market. 21. The form the costs take, xed or variable, is a distinction set aside here for tractability. One may consider xed costs as being amortized across a rm's potential sales, and thus equivalent to a variable cost of the same level. 3.3.3 On rm-discriminating policies For any rm, the ideal policy is one that raises prohibitively high costs on all varieties of substitute goods produced by any other rm. A policy-induced monopoly is very protable for the monopolist, but not nearly as good for consumers and potential competitors. So, 80 while any rm would likely be willing to pay a high price (in terms of political support) for this sort of policy, it is unlikely that it has the resources necessary to suciently compensate government and oset whatever counter-lobbying may occur. Creating a monopoly through policy would signicantly reduce the welfare of consumers, as they are forced to change their consumption behavior to reect the new market conditions. Government pays a political price for signicantly distorting markets in this way, in accordance with institutional features. For even moderately responsive governments, creating monopolies must either have signicant benets in the form of industry contributions or modest eects on consumer welfare in order to be a utility-improving choice. In any industry with more than one rm, counterlobbying will be an important part of the politics around these rm-discriminating costs. Firms may oer some positive political contribution for governments that implement policies that do not impose costs on them. In bidding in this fashion, these rms counteract the pressures that may come from other rms' bids in favor of rm-discriminating policies. The rms most likely to bid in favor of lower rm-discriminating costs are those that ben- et most from an undistorted market: those rms who are most competitive/ecient. These rms are protable in an open market, and so may provide some benets to governments that keep costs low. Thus, governments compare the utility they derive from higher consumer utility, combined with some targeted benets from rms who prefer broadly low levels of rm-discriminating costs against the reduction in govern- ment utility from lower consumer welfare and some targeted benets from rms who benet from rm-discriminating policies. The other sort of policy, one that imposes costs solely on new entrant rms, will have somewhat dierent support. It is worth noting that these kinds of policies divide winners and losers along status quo lines: it is as if existing rms have already paid the cost, but new rms must pay costs to enter the market. Or, at the very least, policies that impose costs on new rms do not harm existing rms. One which is excludable but non-rivalrous. O at the e least olicies that i ose costs o e s do ot ha e isti g s 22. One which is excludable but non-rivalrous. One which is excludable but non-rivalrous. 23. Or, at the very least, policies that impose costs on new rms do not harm existing rms. 3.3.3 On rm-discriminating policies This will have the eect 81 of deterring entry, thus preventing newer, potentially marginally more competitive rms, from entering the market. In this sense, rm-entry cost policies create a kind of club good22 among existing rms, and thus the incentives for demanding such policies are somewhat dierent among domestic rms than in the previous type of rm-discriminating costs. Here, all existing rms benet (albeit to dierent degrees) from rm entry costs. It is new entrants and consumers who lose. Among existing rms, those that face the greatest threat from new entrants are smaller or less ecient rms. Consumers' change in consumption to new varieties produced by new entrants reduce the prots of other rms, as reallocation of the consumption basket moves away from existing varieties. For small or inecient rms, this loss in sales may be enough to induce them to exit. Preventing new market entry matters more for smaller and inecient rms than it does for large or ecient ones. However, barring new entry benets all existing rms.23 This prevents the sort of counterlobbying that existed when rm-discriminating policies divided winners and losers among existing rms. For these sorts of policies, smaller and more inecient rms will be willing to contribute more (relative to their resources) to obtain rm- discriminating policies, but larger and more ecient rms may contribute as well to obtain the benets of deterred market entry, as well. For government, these greater contributions are appealing, relative to lost utility from lost consumer welfare. Also, there is no positive support incentive in the form of rm counterlobbying for low costs against which government must weight their choices. It is only the prospect of signicant consumer welfare loss that deters government from creating policy that imposes cost on new entrants. Firm-discriminating cost policies will arise under dierent conditions for each kind of rm-discriminating cost. Policies that always impose costs on some rms are less likely to arise in industries with inter-rm competition. When there are many rms 82 of similar size, the creation of a suciently-large coalition of rms lobbying in favor of such policies is dicult. Counterlobbying against any such policy and the utility loss from market distortions will reduce incentives for government to provide such a policy. 3.3.3 On rm-discriminating policies However, as an industry becomes less competitive (domestically), a coalition of large rms may be able to overcome the problems of providing suciently large incentives for government to implement rm-discriminating policies. As the rm concentration, for instance, within an industry rises, the likelihood that rm-discriminating policies (in favor of the large rms) will arise also increases. The few large rms have resources to lobby for targeted protection, and the smaller rms with commensurately less inuence cannot counterlobby suciently to make provision of that protection undesirable for government. For policies that add costs to new entrants, the existence of domestically-salient rms with lobbying power signicantly increases the likelihood that such policies will be implemented. (Perhaps unsurprisingly, this is common.) However, some variation in the likelihood of such policies being implemented does exist. When all rms in an industry are relatively ecient (compared to potential new entrants), there is less incentive to demand rm-discriminating policy, and it is somewhat less likely to be implemented. Existing rms are unlikely to face signicant competition from new entrants, and so devote few resources to obtaining policy that would deter them. When all rms in an industry are relatively inecient, thus making new entrants highly competitive bad for existing rms, but good for consumers it is possible that existing rms will be unable to contribute suciently to make these policies attractive to the government. Consumers would benet from access to a broader market with more ecient producers. It is when the existing rms are relatively similar (on average) to potential entrants that rm-entry-discriminating policies may arise. 83 83 24. This follows the same logic as the concept of nontari barriers in Abel-Koch (2013), Gulotty (2014), and Osgood (2016). 3.3.4 On policies that add costs indiscriminately Other kinds of policies eectively add cost to all goods sold within a market. Consumption taxes, labeling requirements, and testing or certication requirements are all examples of indiscriminate costs. These additional costs shift the price of all goods sold upward, although this price change may dier across products if the cost is a xed one. By increasing the cost-to-market of all goods within an industry, these kinds of policies redistribute among rms by inducing rm (or variety) exit. As these indiscriminate costs raise the price of goods, consumers will consume less of each variety, all else equal. For some producers, reduced demand for their goods induces them to exit. The reduced demand makes continued production unprotable at their particular level of eciency total costs exceed the price consumers are willing to pay for their variety. The most ecient ones may be able to absorb some of the costs by taking lower prots, but costs will still be passed on to nal prices. The least-ecient producers exit rst, and increasing costs leads to more producers exiting the market as demand falls. Faced with higher costs for all varieties, consumers will consume less, or will consume less-preferred varieties, thus reducing their welfare as well. In some circumstances, where consumer demand for a certain class of goods is fairly inelastic overall (and therefore consumers will substitute across varieties more than they reduce aggregate consumption) and some ecient rms can therefore trade o reduced prots per unit for increased volume (from consumer substitution), it may be possible for a segment of the industry to gain from indiscriminate costs. If these conditions exist, it is the most ecient rms that will lobby for indis- criminate costs, while the least ecient rms will oppose them.24 Among relatively ecient rms, each will have an optimum level of indiscriminate costs, where su- ciently many inecient rms exit the market to increase sales, but where the costs 84 are not so high as to lead to a net reduction in their own sales. In general, a rm's preferred level of indiscriminate costs is increasing in its eciency. 25. This might, alternatively, be considered as small/inecient rms lobbying in favor of less regulation. 3.3.4 On policies that add costs indiscriminately To obtain this higher level of indiscriminate costs, ecient rms will be willing to support the gov- ernment politically (as long as the required contribution does not exceed the benets from increased, post-inecient rm exit sales), and submit oers to government that include positive support for policy with some positive level of indiscriminate costs. On the other hand, inecient rms may oer some positive support to government for setting indiscriminate costs at or near zero.25 For government, increasing indiscriminate costs can negatively aect its utility by reducing consumer welfare. As consumers either consume less or are forced to choose among fewer remaining varieties (after rms exit), their overall level of welfare de- clines. Consistent with the eects of other kinds of market-distorting policies, this has a negative eect on government, depending on how much weight government places on consumer welfare. For rms seeking indiscriminate costs, then, it is necessary to provide contributions sucient to overcome the eects of lost consumer welfare. Additionally, low-eciency rms may counterlobby, oering government with posi- tive support for implementing low-cost policy. Thus, when government decides what policy to set, it is deciding between the higher political support and lower consumer welfare at higher levels of cost versus some (lower) level of political support from inecient rms, but a higher level of consumer welfare. It is the distribution of rm eciency among producers that determines which will be greater and at what level indiscriminate cost policy will be set. This suggests the conditions under which indiscriminate costs should arise. If all rms within an industry are similarly-ecient, the intra-industry redistribution that motivates lobbying for indiscriminate costs is unlikely to exist. If consumers demand for an industry's goods is relatively elastic, indiscriminate costs are likely to reduce 25. This might, alternatively, be considered as small/inecient rms lobbying in favor of less regulation. y g y , y 25. This might, alternatively, be considered as small/inecient rms lobbying in favor of less regulation. 85 demand more than it redistributes it, which eliminates the motivation for rms to demand these kinds of policies. 3.3.4 On policies that add costs indiscriminately However, if there is some rm heterogeneity and demand overall is inelastic, then the level of indiscriminate costs should be higher, and should increase as the number of ecient rms or size of the most ecient rm increases.26 Industries with rm heterogeneity with respect to ineciency and some degree of inelastic demand should be where indiscriminate costs play a role in intra- industry redistribution. 26. Again, this relationship will be non-monotonic. If the number of relatively ecient rms increases too much, the eect on consumer welfare will be greater and the number of less-ecient rms forced to exit will decline. 3.3.5 Considering all policies at once, and government incentives As discussed above, these policies are not mutually exclusive. It is possible to implement some combination of location-, characteristic-, and rm-discrmininating costs, as well as indiscriminate costs, at once. However, the additional costs of these policies are additive. If imported orange juice (from concentrate) from Juiceco (an imaginatively-named subsidiary of ConglomCo) faces import taris based on its im- ported status and additional regulatory costs related to content of concentrate juices, buyers of that juice will pay more than if only one (or neither) of the policies applied. The process of lobbying over multiple policies at once, then, is one of rms opti- mizing their oers, and thus their promises of political support, to reect the status of the industry. Firms would like to be monopolists, bidding for a combination of policies that just eliminate all competitors. However, such policy would be incredibly distortionary and would reduce consumer welfare to a signicant degree, thus making it very costly. Also, it is clear that every other rm within an industry would bid for a monopoly-creating policies, but ones that met their needs within the market. Thus, oering support for modest levels of policy, which reduce some competition, but not all, is the politically-feasible solution. As one form of policy becomes politically in- 86 feasible, rms will lobby instead for the next-best option, in order to maintain some degree of protection. This infeasibility arises when the politically-salient rms in an industry have certain characteristics. When FDI means many rms have interests in cross-border trade, location-discriminating costs become infeasible. When goods produced by rms in the industry are homogenous, providing few opportunities for divisions within that industry, characteristic-discriminating costs become infeasible. If an industry is composed of many rms of similar size, targeted rm-discriminating costs will either be counterlobbied or will raise costs on consumers too much for government to support them. Indiscriminate costs are generally unpalatable to con- sumers (and thus government pays a political cost for imposing them), and when there are inecient rms among the domestically-salient within industry, these costs will be opposed and thus less feasible. Faced with increased opposition to one's most-preferred policy, a rm may instead substitute a second-best policy in their oer of political support in order to maintain a suciently-large protection-seeking coalition. 3.3.5 Considering all policies at once, and government incentives In doing so, a coalitions of rms within an industry will demand protection that reects the best feasible compromise among domestically-salient rms. Firms that cannot obtain policy which suits them will simply keep their lobby resources in their pockets. To clarify, an industry with signicant FDI, which produces homogeneous goods (or heterogeneous goods where a wide variety are produced by domestically-salient rms), which is composed of many similarly-sized rms, and where domestically-salient rms are not signicantly more productive than their competitors is an industry where market access costs due to policy should be low overall. Demands for protection, and thus the structure of market access barriers, can change over time. Consider an industry that is composed solely of domestic rms (no signicant FDI inows or outows), where varieties are relatively homogenous, and rms are generally modestly productive and have modest market share. In this hy- 87 87 pothetical baseline economy, location-discriminating costs are the most likely. The winners and losers from these policies cleave along the same lines as political salience does. Domestic rms will demand protection along the most feasible dimension for creation of a suciently-large coalition, where most or all domestic rms are willing to provide government with support in exchange for policy that redistributes to them. This is location-discriminating policy. This lobbying dynamic changes as the features of an industry change. As new rms enter or existing rms leave, or existing rms change their industrial behavior, the incentives of industry actors also change. The arrival of more rms with supply chains that span national borders will aect the lobbying for location-discriminating barriers. A sudden technological shift that leads to new market entrants with new varieties of goods will change demands for certain kinds of characteristic-discriminating policies. When rms merge or a previously-small rm grows rapidly, the lobbying and counter- lobbying for rm-discriminating policies will change. Changes in productivity within a suciently-large segment of a market will alter demands for indiscriminate costs. In addition to the implications of intra-industry demands for protection, there are general implications of the relative weight that government places on social welfare versus political support from industry. As discussed above, governments that are more beholden to public support will be more responsive to changes in consumer welfare. t a ess ep ese tat ve o es 28. One may consider this a kind of distortionary policy production frontier. 27. This also implies that more representative democracies will have lower market access barriers than less representative ones. 3.3.5 Considering all policies at once, and government incentives In general, market distorting costs will be lower overall or will have lower net eect on consumers in countries where governments are chosen democratically.27 While rms may wish to demand more protection, in democracies the political costs that government faces for distorting markets too much places a limit on the degree of total distortionary policy that is politically feasible.28 All of the action of lobbying, counterlobbying, and determination of type and level of policy occurs within that 88 limit, shaped in part by political institutions. The substitution of one policy for another, therefore, is not merely a demand-side matter, but also a practical reality of politics under constraint: governments cannot maximize policies across all varieties, lest they distort markets to such a degree that they face political costs. If there are no domestically-salient rms within an industry29, there will be no lobbying to support a government that imposes costs on products within an industry. In the absence of this political support, governments of all types should, in order to maximize consumer welfare, keep market access costs low. If there are no domestic producers, any location-discriminating costs would increase costs on imports the only available varieties. Characteristic-discriminating costs would limit the varieties of good that consumers can access, with no benet to government from rms produc- ing the protected varieties. The same logic holds true for rm-discriminating costs: the beneciaries are not politically salient, and government bears the costs of lost consumer welfare. For indiscriminate costs, in the absence of producers who benet from intra-industry redistribution, there are no benets to lowering consumer welfare. Thus, in the absence of such rms, there is little political incentive for government to create policies that raise market access costs. One notable exception, raised here merely for completeness, is if these policies have revenue-seeking opportunities for gov- ernment.30 The discussion of incentives for creating costly policy has been discussed with the implicit assumption that any revenues from policy were small, especially when compared to the political costs to government of reducing consumer welfare. In systems where government is relatively unresponsive to mass/consumer interests, revenue-seeking may be optimal government policy. 30. This, of course, reects the incentives for imposing taris in many countries before adoption of broad scale income and wealth taxation. Taris and other duties were essential revenues for government. Other policies that raise revenue for government may have one or more of the cost implications discussed in the theory. 29. For example, if there are no domestic producers and all varieties of a good are produced by foreign rms without domestic investment. 3.3.5 Considering all policies at once, and government incentives The majority of this discussion, however, has assumed a government that is at least minimally responsive to consumer 29 Fo e a le if the e a e o do estic od ce s a d all a ieties of a good a e od ced b Thus, in the absence of such rms, there is little political incentive for government to create policies that raise market access costs. One notable exception, raised here merely for completeness, is if these policies have revenue-seeking opportunities for gov- ernment.30 The discussion of incentives for creating costly policy has been discussed with the implicit assumption that any revenues from policy were small, especially when compared to the political costs to government of reducing consumer welfare. In systems where government is relatively unresponsive to mass/consumer interests, revenue-seeking may be optimal government policy. The majority of this discussion, however, has assumed a government that is at least minimally responsive to consumer 89 welfare. 3.3.6 Hypotheses for unilateral policy creation The dynamics of lobbying for protection within a single country suggest a number of testable hypotheses that can be taken to the data to test the theory. These are generated by considering all of the motivations for rms to seek one or another form of protection (as best ts their own preferences and the preferences of others in their industry), and the incentives for government to use dierent forms of costs to regulate market access. Each of the types of policies requires an industrial coalition to demand protection and the absence or relative weakness of a strong politically-salient counterlobbying group to oppose it. The degree of market distortion possible, and the price rms must pay in political support to obtain it, is determined by political institutions. In considering the manner in which industry features aect lobbying, and thus the provision of policy that imposes location-discriminating costs, weadvance these hypotheses: H1: Industries with greater degrees of foreign investment, both inward and outward, will face lower location-discriminating costs of market access. H2a: In dierentiated goods industries, characteristic-discriminating costs will be imposed on varieties of goods with characteristics unlike those of politically- salient producers. H2b: When an industry's goods are homogeneous, or politically-salient rms produce a wide variety of dierentiated goods, characteristic discriminating costs will be low. H2b: When an industry's goods are homogeneous, or politically-salient rms produce a wide variety of dierentiated goods, characteristic discriminating costs will be low. H3a: When rm concentration is high, politically-salient large rms will obtain rm- discriminating policy that raises costs on other incumbent producers. H3a: When rm concentration is high, politically-salient large rms will obtain rm- discriminating policy that raises costs on other incumbent producers. 90 H3b: When rm concentration is low and politically-salient rms are, on average, inecient, rms will successfully lobby for policies that raises costs on new entrants. H4a: In industries where demand is inelastic and rm eciency is heterogeneous, the most-ecient rms will lobby for indiscriminate costs policies, which will be provided if ecient rms are politically salient. H4b: When all rms within an industry are of similar eciency, and the government will not raise indiscriminate costs. 3.3.6 Hypotheses for unilateral policy creation In recognizing that market access distorting costs are additive, and thus the costs to consumers, to government, and to rms (in terms of osetting contributions) are the same, weargue: H5: Higher levels of one kind of market-distorting policy will be associated with lower levels of other policies, all else equal. H5: Higher levels of one kind of market-distorting policy will be associated with lower levels of other policies, all else equal. Finally, in considering the general incentives that government faces under dierent economic and institutional conditions, weargue: Finally, in considering the general incentives that government faces under dierent economic and institutional conditions, weargue: H6a: In the absence of a domestic industry to lobby for protection, government will erect few barriers to market access, consistent with maximizing consumer welfare. H6a: In the absence of a domestic industry to lobby for protection, government will erect few barriers to market access, consistent with maximizing consumer welfare. H6b: More representative governments, those selected by a larger portion of the population, will set policies that are less distortionary overall. H6c: Governments that are not responsive to consumer welfare demands may set policies that extract revenues. 91 91 These hypotheses link the characteristics of an industry and the features of politi- cal institutions to the type of policy that should arise from the lobbying process over industrial policy. Governments have a wide variety of policies from which to choose, as well as the option to reduce intervention in markets. The types of policy chosen, and the way in which policy changes over time, should reect the hypotheses above. 31. There are other options, but these are unique to the context of each country's trade agreement obligations. This also means that some location-discriminating policies can be used with some trade partners, but not others. Provisions of GATT/WTO also allow specic location-discriminating 3.4 Discussion and Conclusions Understanding and explaining the politics of trade openness and domestic indus- trial protection in a global economy requires examining trade-focused policies and potential imperfect substitutes for those policies. In practice, no two policies are exactly alike. However, many of the same protections aorded by a tari can be provided to rms with a quota or a voluntary export restraint. In comparison, it is unlikely that a product quality standard, even if it aects many imported varieties and thus reduces trade to some extent, will provide similar eects. If both quotas and standards are considered under one broad umbrella, it is unclear how we should expect the politics of taris to compare to the politics of non-taris. Existing liter- ature has generally treated all NTMs as like taris, but dierent in one critical way. By integrating a new typology of market access barriers with core insights about protection-seeking politics, we can improve our understanding of not just why trade appears to have opened as it has, but also from where challenges to other forms of agreement and cooperation may arise. This logic explains why, in industries where trade agreements limit a government's ability to implement a most-preferred policy (like taris), rms will attempt to obtain other market access barriers of similar type that are not prohibited, like buy national campaigns instead of taris.31 Even if the industry does not change in foreign 92 investment, product heterogenity, rm concentration, or relative industry eciency other policies within the same class of costs are an option. In this way, changes in policy from taris to other non-tari measures can be better understood as a substitution of one prohibited policy for another similar, but permitted, policy. But in some cases, the decline in taris and rise in non-tari measures represents a change in the contours of industrial protection, reecting a change in the demands for protection within an industry. The theory advanced here is one of unilateral policy change. It does not incorpo- rate incentives that arise from reciprocal policy change, or the logic of cooperation central to some theories of trade liberalization. Indeed, for a comprehensive discus- sion of trade liberalization, the dynamics of protection-seeking discussed here should be integrated with insights from that research. 1. Trade in services is another critical part of the global economy, and non-tari barriers of various type are a critical distortion to this trade. Much of the logic of market access barriers which motivates this empirical study applies to services trade, as well, but the focus in this work is on goods trade and data related to goods trade. 3.4 Discussion and Conclusions However, by focusing on the logic of changing incentives for or against dierent types of policy, this theory contributes to our understanding of changes in the use of various trade policy instruments, and by extension trade ows. Much existing work has focused primarily, or solely, on changes in tari rates and agreements, or has treated non-tari measures in a manner similar to taris. A look to the ways in which these policies may be demanded dierently from one another, and demanded dierently on each side of a negotiation, may im- prove understanding when returning to the study of agreement (or disagreement) in trade and market liberalization in bilateral or multilateral contexts. Critically, the implications of this argument are important for understanding a long-run trend in the global goods market. Taris have, through unilateral reduc- tions and bilateral and multilateral agreements, generally fallen. However, other forms of protection have risen both in importance and level. The other major trend of the post-WWII global economy, the rise in multinational rms and growth of international investment, is a plausible cause of this new pattern in trade and market barriers. As policies countervailing duties and anti-dumping duties specically to disincentivize raising of general taris. 93 more rms with political salience within an industry have global ties, the demand for location-discriminating policies will fall. With less demand (or more counterlobby- ing) for location-discriminating costs, rms will demand other kinds of policies, like characteristic-discriminating or rm-discriminating, instead. As the characteristics of demand for protection shift, responsive government changes policies to suit. more rms with political salience within an industry have global ties, the demand for location-discriminating policies will fall. With less demand (or more counterlobby- ing) for location-discriminating costs, rms will demand other kinds of policies, like characteristic-discriminating or rm-discriminating, instead. As the characteristics of demand for protection shift, responsive government changes policies to suit. 94 CHAPTER IV 4.1 Introduction While taris have generally declined and their signicance as barriers to trade fallen in the period of globalization following the Second World War, the importance and scale of other barriers to trade have risen. This is due in part to the success of multilateral agreements on taris, which have structured a freer-trade system. How- ever, as challenges to deepening cooperation beyond taris and oft-repeated claims of trade discrimination behind borders suggest, taris are not the sole means of re- stricting or distorting international goods trade.1 Global trade today is increasingly intra-industry trade and intra-rm trade, and varieties of similar goods tailored to tastes and regulations in dierent markets pass each other on the oceans. Adoption of common product standards and common certication measures are two ways trade partners may seek to lower trade costs. Meanwhile, exporters attempting to enter new markets often report regulatory barriers as an important factor in preventing entry. However, larger multinational competitors producing in the same markets ship 95 goods back to their home markets with ease. This pattern, where trade appears free and yet not, where intra-industry and intra- rm ows are prevalent, and where access to markets appears unequal both within and across industries, raises questions of how and why market access restrictions vary in this way. Chapter II introduces a framework for considering taris, non-tari barriers, and other policies as market access barriers, moving beyond the tari/non- tari barrier distinction which is the basis of much of the existing discussion. This framework considers that barriers impose additional costs on goods based on where they are made, how they are made, who makes them, or indiscriminately. This new typology motivates a new theory of industrial coalition politics around market access barriers, as discussed in Chapter III. In this theory, individual rms change the type of protection demanded2 in response to features of the rm and of the industry as a whole. This framework provides conceptual and theoretical answers to the questions of which industries receive protection, how they are protected, and why they receive protection, inclusive of a broad variety of policies that may be either substitutes or complements. To improve the answer to these questions and test the claims of the model of market access politics, we turn to data on extant trade barriers. 2. For example, a shift from location-discriminating barriers, like taris or quotas, to characteristic-discriminating ones, like safety requirements that reect technologies that local rms have already developed. 3. Baldwin (1970) does suggest that a broader denition including both domestic and international distortions would be possible, but in practice such an approach is unmanagable, likely a warning to the future. 4.1 Introduction In this paper, we examine an existing data source on trade barriers, UNCTAD's Trade Analysis Information System (TRAINS) Non-Tari Measures database, and adapt those data to meet the market access barriers classication. After a description of the state of aairs in market access restriction, we then turn to analyze some potential causes of dierences in both the level and features of market access restrictions. Using industry- level measures of foreign direct investment, rm concentration, industry size, and product heterogeneity, we examine the relationship between some potential demand 96 side causes of market access barriers and the contours of market access restriction across a wide variety of products in the United States. The results suggest that the presence or absence of FDI is associated with dierent market access barriers and dierentiated goods face dierent barriers than more homogeneous ones. Discussions of dierences in eciency loss, compared to taris, were common, but the distri consequences were often considered to be similar, if they were considered at all. One of their claims is that successive GATT rounds constrained tari policy suciently tive consequences were often considered to be similar, if they were considered at all. 5 O f th i l i i th t i GATT d t i d t i li i tl t y of their claims is that successive GATT rounds constrained tari policy suciently to tive consequences were often considered to be similar, if they were considered at all. 5 One of their claims is that successive GATT rounds constrained tari policy suciently to of Protection The discussion of taris and non-tari barriers to trade is by no means new. In the wake of the Kennedy Round of GATT, early work on nontari trade-distorting policies focused on those that appeared quite similar to taris or that were applied at a border. Measures of any type that caused goods and services to be allocated in such a way as to reduce potential real world income were considered non-tari barriers, and with import duties, comprised all types of trade-distorting policies (Baldwin 1970).3 However, as expressed by Bhagwati (1988), much of the thinking of the time considered NTMs as a substitute for tari policy Bhagwati's Law of Constant Protection. As trade economists looked to NTMs and NTBs, the discussion of the politics around these policies largely paralleled that of taris, with slightly dierent economic eects.4 However, over time, it has become clear that the politics surrounding non-tari policies is more complex than that. Subsequent research focused directly on the politics of NTMs. In an attempt to adjudicate whether pressure politics or a broader national interest and institutions drive protectionist policy in a cross-national context, Manseld and Busch (1995) look to NTMs as a means to better examine distributive politics.5 While the empirical 97 models they estimated were designed as ambivalent to whether NTMs were substi- tutes or complements to taris, their argument generally treats NTMs as substitutes, created by the same political pressures as taris might be. In contrast, Ray (1987) considers the US case alone, but focuses on the change in protectionist policy from taris to NTMs. In part, the motiviation for this change, Ray argues, is changes in political pressures and the qualitative dierences between taris and NTMs. Ray specically focuses on dierences in revenue generation, ability to target protection towards groups where tari distortions would be too distortionary, the public's rela- tive diculty of assessing their eect, and the ability of NTMs to be more specically targeted, thus reducing free rider problems and increasing pressure for NTMs among declining industries. The idea of NTMs as less observable, and thus preferable for democracies where public backlash is a concern, appears in some cases to have broader cross-national support (Kono 2006). Other work has focused on specic types of non-tari policies that have trade implications. limit governments' responsiveness to pressure. So NTMs were considered a better measure because they were less constrained by international agreements. This approach, especially focusing on core NTMs, like quotas, is adopted elsewhere (Busch and Reinhardt 1999). of Protection Research on subsidies has shown some similarities to taris in political and institutional causes, but suggests that subsidies may be more targetable, which can change the political dynamics behind their provision (McGillivray 2004; Rickard 2012). The discussion of how targeting of export subsidies aects success of an in- dustrial support program also suggests that targetability matters, and it aects both distributive and aggregate economic outcomes (Rodrik 1993). Another body of research focuses on the sources of domestic market regulation, including by private actors, and global trade. The move to harmonization of product and regulatory standards in the 1980s, 1990s, and 2000s, while the body of regulations expanded, was seen by some as an eort to reduce nontari barriers to trade. Where standards are amended to meet an international standard, reducing barriers between 98 markets, this may occur due to domestic regulatory institutions that themselves are consequences of historical events. Here, it is the standards setters and the degree to which they share information and adapt rules at the international level that shape regulation, harmonization and the degree to which standards are barriers to trade (Mattli and Büthe 2003). Alternatively, there is some evidence that trade networks, and the pressures of export demand, can shape adoption of new standards to harmo- nize exporting and importing markets, even if that means more standards/regulation (Prakash and Potoski 2006). Building o economic models of trade Heckscher-Ohlin, Ricardo-Viner, Lan- caster's, Dixit and Stiglitz's, Krugman's and Melitz's new trade theories to posit the preferences of market actors and then subsequently which factors, sectors, or in- dustries obtain protection in a microfoundational way, has a long tradition in political economy (Brock and Magee 1978; Rogowski 1987; Gilligan 1997b; Bombardini 2008; Osgood 2016; I. S. Kim 2017).6 Most of this work focuses on tari barriers to trade, as those are the foundation of the economic work on which the political economy work is built. More recently, researchers are adapting these arguments to t the logic of NTMs by adapting the underlying economic models with cost structures that reect an interpretaton of NTMs' eects (Abel-Koch 2013; Gulotty 2014). The discussion of non-tari trade barriers does leave some avenues for further re- search. First, whether NTMs are substitutes or complements for taris remains an open question. 6. Scheve and Slaughter (2001) summarizes the sectors vs factors discussion. of Protection Research comparing use of dierent trade instruments across indus- tries and time in Japan point one way forward (Naoi 2009). However, the broader discussion contains work based on each assumption, without much sign of resolution. Second, there is some disagreement in the literature over which types of non-tari measures are indicative of NTM politics generally, and which kinds of policies are unique in a way that makes them less comparable to other NTMs. These two ten- 99 sions suggest that, both empirically and theoretically, the tari/non-tari measure divide has led to some compromises that make inquiry and debate more complicated. The central contribution of this paper, and the larger research agenda in which it resides, is a shift to a types of costs framework that makes comparisons between and across dierent types of trade- (and domestic market-) distorting measures more consistent. 4.3 Hypothesized Patterns of Market Access Barriers There is no shortage of good scholarship on the matter of trade protection, es- pecially in light of the growth of global supply chains and global capital ows. The shift to a discussion of market-access barriers is meant to reconcile the similarity in ndings from the study of taris and NTMs that look like taris while also clarifying why some studies of NTMs suggest other forces at work. A theory of unilateral policy change in response to lobbying from industry-specic interests is presented in Chapter III. In that paper, individual rms tailor their demands for protection to reect both their own interests and those of their domestically-salient competitors. As the set of other rms changes in terms of interest in international supply chains, relative size or rm concentration, and natural product heterogeneity and the similarity of varieties the nature of individual rm demands and the dominant demands of the industry as a whole will change. This theory, which parallels the core logic of Grossman and Helpman (1994), generates a set of testable hypotheses that link those industry characteristics to the varieties of protection sought and obtained. For each of the four types of market access barriers location-discriminating, characteristic-discriminating, rm-discriminating, and indiscriminate dierent features of an industry's composition are expected to lead to more or less demand for a particular form of market access costs. In addition, the level of each type of market access cost for a particular variety of good will 100 aect the incentives for governments to raise additional market access costs of the same or other types on that variety of good. As dierent industries have dierent characteristics more foreign investment or less, greater rm heterogeneity or industry concentration, perhaps a naturally-homogeneous product the types of barriers each may request (and obtain, in many cases) should vary according to underlying industry features. A main claim of the theory is that the growth in global capital ows drove demands for protection (in the aggregate) away from location- discriminating barriers in some industries (where foreign investment, both inward and outward) is greater, to other kinds of market-access-restricting policies. This paper will attempt to test some of the claims of Chapter III, using the typology of market access barriers introduced in Chapter II. 8. It is the implication that these measures may increase trade ows, or distort them in terms of composition, which makes consideration of the net trade eect of NTMs so complex. Unlike taris, which generally result in net trade ow declines, some non-tari measures may block some trade ows, but also domestic alternatives, resulting in more imports, or merely reallocate import demand among available alternatives. Discussion of these possible eects, in the context of distributive consequences of dierent kinds of market access barriers, is discussed in Chapter II. 4.3 Hypothesized Patterns of Market Access Barriers First, this paper focuses on testing the hypothesis that the level of location-discriminating costs on a given product should be inversely related to the level of foreign direct investment in the industry that produces that good. This is claimed to occur because politically-salient rms with global supply chains disprefer protectionist policies that may raise costs on their varieties of goods, even if they aect other foreign competitors as well. As a result, the policies that a protection-seeking industry coalition will demand from government should include fewer location-discriminating costs, and instead substitute other kinds of costs to protect those politically-salient rms. This paper also tests a hypothesis about the presence or absence of characteristic- discriminating costs. The theory suggests that, in homogeneous goods industries, attempts to raise characteristic-discriminating costs will either lead to ineective pol- icy (that doesn't serve the rent-seeking interests of protection-demanding rms) or would raise costs on many or all producers. In either case, the theory suggests that in homogeneous-goods industries, characteristic-discriminating costs should be raised infrequently, if at all, on that industry's varieties of goods. The theory also generates additional hypotheses about when rm-discriminating 101 and indiscriminate-cost policies should arise, and those are tested against the data here, as well. The number of rms and the degree of concentration within an industry should aect how many rm-discriminating costs the products of that industry face. In particular, rm-discriminating costs are more appealing when a domestic industry is more concentrated. However, it is the eciency of politically-salient rms that determines whether policies that raise indiscriminate costs on producers are raised.7 When those rms with political access are relatively inecient, they should lobby less for these kinds of policies, which hurt them more than competitors. 7. This is somewhat similar to the logic in Gulotty (2014), where NTMs are all generally considered as xed costs of market entry, paid by all producers. ers Collection of data on non-tari barriers to trade is complex. The variety of policies that may serve as a barrier to international trade, but that are not taris, is exten- sive. Further, some of the policies are dicult to observe. Some non-tari measures (NTMs) apply at national borders, while others are applied behind the border. It is also sometimes unclear which policies cause negative distortions in trade ows and which do not. It is therefore common for these policies to be referred to as non- tari measures, rather than non-tari barriers, in UNCTAD reporting.8 Unlike taris, which as a single kind of policy have the benet of being relatively easy to measure and compare ad valorem and specic duties can be transformed fairly easily non-tari measures must be categorized and coded across a number of di- mensions. For this reason, UNCTAD's own coding of NTMs has changed a number 102 of times in the past 20+ years. The most recent classication system was nalized and released in 2012 (Group 2015). The 2012-coded UNCTAD TRAINS database categorizes trade barriers according to a coding scheme that splits NTMs into sixteen chapters, split among technical measures, non-technical measures, and export-related measures.9 These categories are descriptive, and focus primarily on how the policies are implemented or the proximate goals of the policy (anti-dumping versus counter- vailing duties and technical barriers to trade (TBT) versus sanitary and phytosanitary standards (SPS), for instance). The current TRAINS database is constructed by collecting data on legal measures from ocial sources in each country in the sample. Industry and voluntary standards, which are standards and policies enforced explicitly or through mutual agreement by rms in a given industry, but that are not explicit legal requirements enforced by government, are excluded from this collection eort (Knebel and Rial 2016). In- ternational standards are also not considered in the UNCTAD database, unless a government adopts the standard through legislation. These data come from cen- tralized government sources, such as ocial registers or periodical announcements of regulatory changes, or from decentralized data collection by UNCTAD or UNCTAD- supporting organizations and data collectors. At times, UNCTAD uses data pur- chased from private companies that consolidate regulations, if ocial sources and data collected directly by UNCTAD-aliated bodies are insucient. After iden- tifying sources, UNCTAD collects and organizes all documents obtained from those sources. 9. A complete summary of the codebook can be found in Appendix B. ers Then each document is examined to identify all regulations contained in each document, with an eye to languge that identies products and countries as targets, when possible. Once individual regulations are identied, each is classied according to which measure type, tari line, partner, and objective applies for each regulation. Particular attention is paid to whether a policy applies in a way that is meant to 9 A l t f th d b k b f d i A di B 103 support some other policy objective protection of the environment distinguishes an SPS measure from a TBT, for instance and whether a policy contains multiple measures that must be coded separately.10 The TRAINS database has grown in scale in a number of ways over its his- tory. In more recent years, more countries are covered by the data collection ef- forts. Within each country, subsequent years often contain more varieties of policies, as well. Whereas much of the publicly-available early versions of TRAINS contains only tari-like measures quantitative restrictions and the like later years in the database expand into more categories of non-technical measures, some categories of technical measures, and export measures.11 Whereas early country-years in the database Japan in 1989, for example are relatively sparse in terms of product and measure coverage, later country years are much more complete. Because of the nature of the data collection, even the best TRAINS country-year databases are likely to be incomplete. Specically, measures that are more opaque, measures that have older legal origins, or those that are enforced in an informal way will be missed by the data collection eort. However, compared to the alternatives, the TRAINS database remains the best option for comprehensive study of a variety of NTMs. 11. This expansion in data coverage follows increased eorts of UNCTAD, a more developed frame- work for collecting and classifying measures, and also increased participation in data collection eorts by governments, especially when data collection is explicitly dened as a responsibility in trade and other international agreements. 10. The specics of these classications, as well as discussion of more detailed coding principles can be found in Knebel and Rial (2016), which is ocial guidance for coders of NTMs. 12. The TRAINS database identies 182 dierent types of non tari measures, including categories for measures not elsewhere specied. Some of these are quite general, others much more specic. If time and resources permit, it would be possible to code each specic measure contained in the database according to the location/characteristics/rm/indiscriminate costs typology. For the US in 2012, this is 5752 dierent measures. Each of these is a particular type of NTM that covers one or more product lines with one or more trade partners. This measure-level coding is not done here. 4.4.1 Translating NTMs to Market Access Barriers However, TRAINS measures NTMs according to a classication that raises a number of problems for understanding the distributive politics behind trade policy. To use this existing database, it is necessary to transform the TRAINS typology, which categorizes measure by specic type phytosanitary standards that are temporary versus import licensing requirements, for instance into something that reects 104 the typology described in Chapter II. From the typology of market access barriers discussed in Section 4.3, it is possible to map each specic type of policy12 to its type of market access restriction. We do this by examining the description and coding instructions for each type of TRAINS NTM code, then considering how each type of measure divides a hypothetical global market with many producers, manufacturing goods of dierent characteristics in dierent markets. If the policy adds costs to some producers and not others, it is not an indiscriminate cost. If it discriminates in some way, we then consider which dimension of the goods dening elements determines whether the producer must bear additional costs to enter a market. The measures recorded in the database sometimes clearly identify a particular type of market access barrier. Quotas code E2 are location-discriminating costs, for instance while other measures overlap multiple types of costs. Importer approval or registration requirements for agricultural products for phytosanitary reasons codes A13, A14, or A15 captures both location-discriminating costs only imports are restricted by this policy and rm-discriminating costs each rm must bear the costs to obtain a license or complete the registration process if they wish to import. Policies that require testing on certain varieties of goods A82 or B82 can be considered to impose both indiscriminate and characteristic-discriminating costs. This is common among policies coded in the TRAINS data. Conceptually, one can imagine this as a policy imposing costs on multiple orthogonal dimensions at once, or a vector of costs through a multidimensional cost space. There are two options for addressing this issue. In the rst, a policy can be categorized as imposing multiple varieties of costs at once. 4.4.1 Translating NTMs to Market Access Barriers In the second, one can select only one cost the most expansive in coverage of rms, or the one that benets 105 the smallest number of rms, or the cost that best matches the general eects of the policy and identify a policy as representing only one kind of market access barrier.13 In this paper, we choose to use the former, inclusive coding. This has the consequence of making individual policies count multiple times, albeit across dierent categories of market access barriers. As many products face a variety of non-tari measures in the TRAINS data, this choice means that comparing the number of costs per product across products and across types of barriers is roughly equivalent. A product with more NTM lines will appear to have more Market Access Barriers in each category and overall. The specic coding of every NTM code in the TRAINS data can be found in Appendix B. In general, dierent sections of the codebook represent similar kinds of market access barriers, although important dierences do arise. For instance, in both the TBT and SPS sections of the UNCTAD NTM coding, some measures generally dene location- or characteristic-discriminating costs, while other measures in the same section of the codebook those requiring testing or certication apply indiscriminate costs that all producers must bear. In the same vein, similar sorts of measures exist in dierent parts of the codebook, as well. With the TRAINS data translated to a market access barriers framework, it is possible to use the database as a set of outcomes against which the hypothesized causes of dierent forms of protectionism discussed above can be tested. 13. In this case, one may consider the import licenses for phytosanitary reasons discussed above as only a rm-discriminating or only a location-discriminating cost, for instance. 4.5 Data and Analysis The data used in this analysis come from a variety of sources. Testing the presence of hypothesized relationships between industry characteristics and varieties of market access barriers requires a variety of measures at dierent intra-industry levels of gran- 106 ularity. For many countries and times, the necessary data are either not distributed outside of government statistics agencies or not collected at all. In order to maintain focus on the dierences of interest dierences in the kinds of protections provided for dierent industries and rms the analysis in this manuscript is restricted to a case where adequate and reliable data are available for many industries. Testing the potential sources of demand for dierent forms of protection requires systematic data on industry-level foreign investment positions, characteristics of in- dustry size and concentration, and a measure of the degree of underlying product dierentiation within a particular industry. These data are all available for the United States in a form that allows for intra- industry comparisons of market access barriers to dierent varieties of goods. The dataset compiled for this paper covers US market access barriers in 2012, and a variety of industry measures in that year and preceding years. we discuss the data in more detail below. 4.5.1 Market Access Barriers The outcomes of interest are the varieties of protection imposed on dierent prod- ucts (and thus granted to dierent industries and rms). As discussed above, the source for these measures is UNCTAD's TRAINS database, which catalogs reported and measured incidences of non-tari barriers to trade at the 6-digit Harmonized System product code level. The 6-digit level is a fairly granular level of product dif- ferentiation. For example, HS code 020741 the meat of whole ducks, either fresh or chilled is distinct from 020711 the meat of whole chickens, fresh or chilled and 020743 just the fatty livers of duck, again fresh or chilled. Many policies in the TRAINS data cover a variety of product lines, but some cover only one or a handful of lines. In the TRAINS data, this appears as a single measure that applies to a number of product lines. 107 107 As data in some years in the UNCTAD database are more comprehensive than others, case selection is driven in part by the availability of data across many varieties of NTMs and industries/products simultaneously.14 The TRAINS data collection eort requires the presence of organized ocial reporting on NTMs, so the most complete country-years are those for countries with a robust national statistics and legal archiving framework, or those countries that have been given specic assistance for creation of these data. Although the TRAINS data cover over 150 countries with observations since 1988, there is signicant missingness, both at the country-year level and within the country-year level. For many of the 150 countries, only one or two reports are available over the panel's time coverage. Some country-year panels may cover only some products or some varieties of measures. This fact, combined with the need for consistent and broad coverage of the covariates of interest discussed below, limits the set of cases that provide sucient data for a test of the market access demands hypotheses. Given the variety of data availability issues, we restrict the analysis in this paper to one country in one year: The United States in 2012. In some ways, the United States is a straightforward choice. As a large economy with signicant trade ows, there should be suciently large industries with both political power and economic motive to engage in pressure politics. However, in other ways, the United States is an outlier case. , 15. For example, a particular agricultural import licensing policy is the same whether Chile or 14. This is discussed above, as well. 17. Here, a distinction is drawn between sectors and industries. Sectors are larger groups that may include a variety of similar industries, like mining, which contains several distinct industries of ferrous and nonferrous metal mining as well as coal mining and the like. In some interpretations sectors span broad categories of goods or services produced, like export oriented rms. By comparison, individual industries are more cohesive and represent the producers of a narrower class of products just iron mining, for instance that still often comprise a number of rms. 4.5.1 Market Access Barriers Despite the size of trade volumes, the US is nowhere near the global economy's most trade-intensive economy. Future work should examine these patterns in other countries and across time, but for a variety of reasons discussed above, and in consideration of using the most complete case feasible, analysis is restricted to this single country-year. The format of the TRAINS data lists observations at the product-policy-trade partner-year level, which signicantly increases the number of observations per coun- try year. However, many of these are functionally duplicates15, which requires some 108 initial summarization of the data. To reduce overcounting of the same policies, only a single instance of a policy16 for a particular product is considered. This essen- tially collapses the data on the trade partner dimension of the database, making all observations equivalent to one where the world is the counterparty. Were this analysis cross-sectional or k-adic in nature, this would be a greater concern (Poast 2010). Because this is a case study of one country-year, it is possible to keep this home v foreign comparison consistent across other measures in the dataset and mit- igate some of the concerns that would be present if focusing on specic bilateral or multilateral trade and trade barrier relationships. p y The TRAINS database has a specic observation code for each reported policy. China is listed as the counter-party. 4.5.2 Industry Characteristics The hypothesized causes of varying forms of market access barriers vary not just at the country, factor, or sector levels, but at the industry and rm levels.17 As the demands for protection will vary according to features of a given industry, it is nec- essary to measure those features in a systematic way. In the US case, comprehensive data are available for a broad swath of industries that covers the features in question. The US Census Bureau collects data on industry size and concentration in the Eco- nomic Census, and the Bureau of Economic Analysis collects data on the net foreign direct investment positions both inward and outward of US industries. The degree of product heterogeneity or homogeneity in a given industry, and therefore for each product, is measured using a 2007 revision of Rauch's commodity classication scheme(Rauch 1999). The concentration of an industry, or subsector of an industry, can be measured 109 a variety of ways. The asset value or market capitalization of a rm may represent its size in a way that is meaningful for the ability to invest in political pressure. A rm's employment base may serve as a dierent measure of political inuence. Sales or revenues data reveal the share of a market that the rm commands, and indicates the degree to which a few rms' (or many rms') products dominate the market. Looking to revenues also has the advantage of being roughly comparable across industries, whereas measures of market capitalization or employment may vary according to the capital or labor intensity of a given industry. Every ve years, the US Census Bureau surveys rms across the country to obtain measures of business activity in the United States. Their sampling methodology includes a complete sample of all large- and medium-sized rms, as well as all multi-establishment rms.18 For small rms with only one establishment, summary data from other federal agencies are used in the sample. While this does raise questions about accuracy for the smallest rms, for the rms that comprise the majority of economic activity, the complete coverage gives a good picture of sectors, down to a detailed level. The Economic Census database provides information on both market value sales (for retail and service industries) and value added sales (for manufacturing indus- tries) for each rm or company in utilities, wholesale and retail trade, services, and manufacturing sectors. 18. According to Census denitions, an establishment is a single physical location where business activities are performed. Firms or companies can be composed of more than one establishment, or only one. Also, for establishments performing more than one economic activity, the establishment is coded according to whatever activity comprises the majority of the economic activity at the location. 19. This is a consequence of matching products only to their producing industries, and sections 31- 33 cover industrial manufacturing. For many agricultural products, the matching section in NAICS Section 11 is not present in the Economic Census data. 21. This authority comes from the International Investment and Trade in Services Survey Act, the present iteration of the International Investment Survey Act of 1976. More information on the history of BEA statistics of this nature can be found in Mataloni (1995). 22. These are often from specically-censored reporting to protect information about individual rms in industries where few rms are engaged in FDI. g g 23. This dierence reects dierences data availability for the two series. 20. HHI = PN 1 s2 i , where N is number of rms and s ∈[0, 100] is a rm's market share, and HHI ∈[0, 10000]. 4.5.2 Industry Characteristics From this, estimates of total industry size and the size of the top 4, 8, 20, and 50 rms or companies in each industry are computed. For the analysis in this paper, only rms and industries coded in NAICS Chapters 31 to 33 the manufacturing section of the industrial classication are used.19 For these sections, only the number of establishments and total value added are summarized. 110 The Census Bureau computes a Herndahl-Hirschman Index for the top 50 rms in the industry20 and the share of industry value added produced by the top 4, 8, 20, and 50 rms, from these data. With these three measures, we know the size and concentration of each industry. The aggregate industry value added sales and num- ber of establishments (roughly the number of locations of business) are included in models as measures of domestic industry size. We use the Herndal Index of the top 50 companies in each subsector as a measure of industry concentration. Metrics of foreign investment, both inward and outward, are necessary for estimat- ing the relationship between foreign rms with domestic political salience, domestic rms with international interests, and demands for kinds of market access restrictions. In the United States, the BEA collects data on the US business activity of foreign multinationals (inward FDI) via mandatory surveys completed by all US aliates of foreign multinationals.21 This same mandate also dictates that US rms must report on their economic activity with foreign aliates, giving estimates of outward FDI ac- tivity. From these annual reports, the BEA summarizes the net investment position of a number of industries separately in terms of inward and outward ows. These data are released annually, but have omissions22 and occasionally very sudden shifts from year-to-year. As the analysis is cross-sectional in nature, we sacrice the time series coverage for completeness of observations and some degree of smoothing by taking the average of years leading up to 2012. In addition, this averaging moderates some of the time trend present in this measure following the 2008 global nancial crisis. For FDI inows, this is 2008 to 2012, while for outows years 2009 to 2012 are averaged.23 These two variables, average FDI inow stock and average FDI outow 111 stock, are then used to capture the degree of multinationalization in the industry that produces a given product. 4.5.2 Industry Characteristics The coding of products as dierentiated or homogeneous comes from Rauch's commonly-used classication of global commodity markets. This measure classies goods as dierentiated, reference-priced, or traded on an organized exchange. From the rst to the last category, goods are increasingly homogeneous, even at the same product code level (4-digit SITC v2 code). While many products' heterogeneity reects that of their codebook neighbors, variation within broad product groups does exist. The Rauch coding is cross-sectional and time invariant24, but for this inter- industry cross-sectional analysis the time-invariance is not a concern. For use in these models, the Rauch coding is transformed to a zero-to-one scale, where homogeneous goods are coded zero, reference-priced goods are coded at 1 2, and dierentiated goods are coded as one. 24. The initial classication was devised for a 1999 paper, and the codebook was revised in 2007 to match the revised SITC coding scheme. 4.5.3 Product/Industry Coding and Concordance One challenge of integrating data from these varying sources, collected and coded for dierent purposes, is that economic activity is divided dierently and coded with dierent goals in mind. The TRAINS data, primarily meant for comparison to tari data, codes policies according to how they apply to particular products using the UN Harmonized System (HS) coding. These 6-digit codes classify goods from broad categories down to variants on similar products. Consider the HS code for Vegemite 210690. It falls in Chapter 21, Miscellaneous Edible Preparations, Heading 2106, Food preparations not elsewhere specied or included, and subheading 210690, "Other". Vegemite's place in a tari schedule is dened almost by exclusion, but the grouping narrows as other, dierent, potentially substitute products are partitioned 112 o into other product codes.25 These are the codes used by many customs agencies to levy duties, and are the basis of duty schedules for many trade agreements. The focus of this coding scheme, therefore is on grouping products in a way that makes the process of customs and duty as consistent as possible. This is not the case for other coding schemes in the data used here, and those codebooks reect dierent goals. Measures of industry size and concentration are generated according to the North American Industry Classication System (NAICS) codebook. NAICS is primarily designed for collection of economic data at the rm and industry level. These di- visions do not always align with product codings like those in the HS system. The measures of US inward and outward FDI are coded according to the International Surveys Industry (ISI) codebook, an adaptation of the NAICS codebook for the task of surveying both US rms and foreign multinationals.26 The Rauch product homo- geneity/heterogeneity measure is developed for the SITC Version 2 codebook. Each of the measures in question market access barriers, industry size and concentra- tion, foreign direct investment positions, and measures of product heterogeneity are collected for slightly dierent partitions of the US economy, as dened by dif- ferent codebooks for industry classication. This prevents easy comparison of these measures in an empirical model. Given these dierent coding schemes, it is necessary to link the data according to a set of concordance rules. Data merging is done in a number of steps. First, the 6-digit HS code for each product serves as the base for all observations. g g 27. It should be noted that every code in the HS codebook is present in the 2012 United States NTM data; there are no products at the 6-digit level with no market access barriers of any kind. This also means that there is no missingness at the product-code level, although missingness within product codes policies missed, for instance may exist. 26. This adaptation is largely straightforward, but sometimes splits individual NAICS codes across multiple ISI codes, or vice versa. This requires one rst translate data from ISI coding to NAICS coding before taking steps discussed below. 25. We thank Marta Bengoa for bringing this particular example to our attention. 29. There are a number of ways this matter could have been resolved, but we believe a parsimonious method like this maintains clarity while also acknowledging that, for some products, industrial interests have characteristics which reect multiple groups' preferences. 4.5.3 Product/Industry Coding and Concordance Market access barriers are summarized for each of the HS codes in the TRAINS data.27 All other data are then linked to the market access measures by matching them to the 113 HS codes. As most of the additional data used here come from US Census sources, we use concordance tables from the Census to make these connections. With every major release of the Economic Census, an updated concordance table is created to use the most recent versions of other codebooks for linking. We use the concordance tables prepared for the 2012 Census data release for linking across the four coding schemes in the data. To bridge industry and product data and create single observations, we rst nd the highest level of granularity for each of the independent variables for which data is consistently available.28 For the FDI measures, this is the 4-digit NAICS coding level. For measures of industry size and concentration, this is the 5-digit NAICS level. For Rauch's product heterogenity measure, we match at the 4-digit SITC classication. The merging of these data require overcoming two more matching problems. There are instances in which multiple codes in one of the source classications match a given product code for instance, if two or three SITC codes match a single HS product code. In this case, a simple average of the values for each of the matches is used.29 In other cases, a single industry in the FDI or concentration data maps on to a number of dierent products within a certain range. In these cases, those observations apply to all of those products, a one-to-many merge with repeating values across multiple products. For each of the variables in the data, the level of aggregation in the data is consistent across all observations, which requires these transformations. 28. The highest level of granularity represents the measurement level with the least aggregation from individual observations to sub-industry or industry totals. 4.5.4 Data Summary With these concordance issues addressed, we have a dataset ready for analysis. We are unable to match some products to complete industry-level data due to missing 114 observations in FDI or industry concentration data. There are 5207 product-line level observations, of which 3080 are complete. These data present both the number of policy barriers identied as additional costs imposed on some varieties of goods and the underlying characteristics of the industries that produce those goods. Those 3080 products represent a sample of the total US goods market, but are the most complete sample possible with these resources. A summary of the variables can be found in Table 4.1. The outcome variables, the number of market access barriers, dier somewhat in the full sample versus the sample used in the analysis. For the measures of industry characteristics, the full sample and the complete-case sample used in analysis dier somewhat, as well. The measures of market access barriers are strongly correlated.30 Statistic N Mean St. Dev. Min Max # Loc-Disc Barriers 5,207 18.061 13.909 3 344 # Char-Disc Barriers 5,207 24.297 21.629 4 413 # Firm-Disc Barriers 5,207 9.887 8.776 1 182 # Indiscrim Barriers 5,207 9.702 8.418 0 139 Avg. FDI Out 3,660 10.849 11.517 0.020 53.532 Avg. FDI In 3,601 12.922 17.733 0.009 106.962 Rauch Di Good 4,674 0.741 0.326 0.000 1.000 Herf. Index, top 50 rms 4,552 535.971 515.844 18.000 3,755.100 Establishments 4,552 1,642.822 2,480.778 24.000 24,707.000 Value Added 4,552 21.006 25.385 0.246 129.479 Table 4.1: Summary Statistics, Full Sample # Loc-Disc # Char-Disc # Firm-Disc # Indiscrim # Loc-Disc Barriers 1 # Char-Disc Barriers 0.889 1 # Firm-Disc Barriers 0.911 0.769 1 # Indiscrim Barriers 0.616 0.642 0.735 1 Table 4.2: Correlation of Barrier Count Across Measures 30. The correlation table is presented in Table 4.2. Statistic N Mean St. Dev. Min Max # Loc-Disc Barriers 5,207 18.061 13.909 3 344 # Char-Disc Barriers 5,207 24.297 21.629 4 413 # Firm-Disc Barriers 5,207 9.887 8.776 1 182 # Indiscrim Barriers 5,207 9.702 8.418 0 139 Avg. FDI Out 3,660 10.849 11.517 0.020 53.532 Avg. FDI In 3,601 12.922 17.733 0.009 106.962 Rauch Di Good 4,674 0.741 0.326 0.000 1.000 Herf. 30. The correlation table is presented in Table 4.2. 30. The correlation table is presented in Table 4.2. 4.5.4 Data Summary Index, top 50 rms 4,552 535.971 515.844 18.000 3,755.100 Establishments 4,552 1,642.822 2,480.778 24.000 24,707.000 Value Added 4,552 21.006 25.385 0.246 129.479 Table 4.1: Summary Statistics, Full Sample Table 4.1: Summary Statistics, Full Sample # Loc-Disc # Char-Disc # Firm-Disc # Indiscrim # Loc-Disc Barriers 1 # Char-Disc Barriers 0.889 1 # Firm-Disc Barriers 0.911 0.769 1 # Indiscrim Barriers 0.616 0.642 0.735 1 Table 4.2: Correlation of Barrier Count Across Measures 30 The co elatio table is ese ted i Table 4 2 Table 4.2: Correlation of Barrier Count Across Measures 115 31. Negative binomial regression is chosen over Poisson regression because of concerns about overdispersion, where the conditional variance is signicantly greater than the conditional mean. A test of the Poisson model's assumption of equal conditional mean and conditional variance is presented in Appendix A. p pp 32. Specically, the variable is ln(Barrier + 1), to address occasional zeroes in the data. 32. Specically, the variable is ln(Barrier + 1), to address occasional zeroes in the data. 4.5.5 Model Design The design of the measures of market access restrictions counts of number of barriers per product code motivates the empirical model design. Models are es- timated using both ordinary least squares and negative binomial regression.31 The former is chosen for parsimony and ease of interpretation, but is not strictly appro- priate for these data, where negative values are not possible. In the OLS framework, we estimate two sets of models. In the rst, the count variables of market access bar- riers are included as the count measures explained above. Then, the market access barriers count is log-transformed32 to address the long right tail in the distribution of the measure count variable. The negative binomial model is presented as it is a more appropriate model for count data, but brings additional restrictions on model specication and a more complex interpretation. By presenting all three, we aim to demonstrate when the results are consistent and ease interpretation of the direction and magnitude of the results. All models are estimated with and without the count of other kinds of market access restriction as covariates, to capture the hypothesis that an increase in one variety of costs ought to lead to a decrease in other varieties of market access costs. Aside from substitution of dierent varieties of market access barriers as out- come variables and control variables, the models are structured in the same way. The models are estimated with clustered standard errors to address concerns of het- eroskedasticity across larger product groups due to legal or industry frameworks that lead to dierences that divide on larger product group lines. The clusters are dened on the 2-digit level product code to address concerns about correlated errors within 116 similar groups of products.33 4.6 Results Fitting these models to the data provides some insights on why markets for dier- ent varieties of goods are restricted in dierent ways. Table 4.3 outlines the general results from the models. They are described in more detail below. The model es- timates for each of the four types of market access barriers are presented in Tables 4.4 to 4.15 in their respective subsections. A number of patterns arise that indicate that dierent kinds of market access restricting policies are associated with dierent industry features. Some of the hypotheses of the theory are supported, while oth- ers are statistically or substantively not strongly supported by the data. In some cases, evidence from the US case suggests a more complex relationship between industry features and the nature of protectionism, where it exists. As models are estimated for each of the four types of market access-restricting costs separately, the results from those models are considered individually rst. Then, the models estimates are considered together to draw summary conclusions. 33. As the product coding scheme necessarily puts similar goods in adjacent or nested product codes, using a higher level of coding, the 2- or 4-digit code, for instance, provides an eective method for grouping goods that are similar. 4.6.1 Location-discriminating Costs In models where the outcome of interest is the number of location-discriminating measures aecting a particular product line, results generally suggest that FDI and product dierentiation are related to the contours of protection, while other industry characteristics are not as consequential. The industry's average FDI inows and outows in the years preceding 2012 ap- pear to have dierent associations with the number of location-discriminating mea- sures. In industries where there have been signicant outows of direct investment 117 Industry Feature Location- disc Char-disc Firm-disc Indisc FDI Outow +, mixed signicance +, mixed signicance +, mixed signicance +, mixed signicance FDI Inow -, mixed sig- nicance ∅ ∅ -, weak/mixed signicance Prod. Di. -, signicant - (no con- trols), ∅(w controls) - (no con- trols), ∅(w controls) ∅ (no con- trols), + (w controls) Concentration ∅ ∅ -, weak ef- fect inconsistent # of Estab. -, weak/small eect ∅ -, weak ef- fect -, weak/small eect Value Add ∅ ∅ ∅ ∅ Control Loc-disc NA + + - Char-disc + NA - + Firm-disc + - NA + Indisc - + + NA Table 4.3: Summary of model results Table 4.3: Summary of model results Table 4.3: Summary of model results capital, where rms have built supply chains or subsidiaries in foreign markets, prod- ucts of those industries tend to face more location-discriminating barriers on average. However, for products produced in industries where there have been signicant FDI inows, fewer location-discriminating costs restrict market access. In this case, prod- ucts in the US that are produced by industries where the United States has seen signicant FDI inows are covered by fewer location-discriminating policies. This pattern is somewhat inconsistent with the expectations from the model of protection-seeking coalitions discussed above. Whereas the theory suggested that pressures from domestic rms investing abroad (FDI outows) and foreign rms in- vesting in the US (FDI inows) would both lead to fewer location-discriminating costs, this does not appear to be the case. In these data, only inward ows have that eect. While this relationship does not appear in every model, it is roughly consistent across specications and estimators. 118 # Loc-Disc Barriers (1) (2) Avg. FDI Out (USD bil) 0.456∗ 0.002 (0.267) (0.036) Avg. 4.6.1 Location-discriminating Costs FDI In (USD bil) −0.131 −0.063∗∗ (0.178) (0.029) Rauch Di Good −12.074∗∗∗ −1.058∗∗∗ (0.604) (0.187) Herf. Index, top 50 rms −0.001 0.0004∗ (0.001) (0.0002) Establishments −0.001∗∗ 0.00003 (0.0003) (0.0001) Value Added 0.153 0.032 (0.096) (0.022) # Char-Disc Barriers 0.304∗∗∗ (0.060) # Firm-Disc Barriers 1.219∗∗∗ (0.098) # Indiscrim Barriers −0.425∗∗∗ (0.129) Constant 21.895∗∗∗ 3.203∗∗∗ (4.396) (0.923) N 3,080 3,080 R2 0.369 0.949 Adjusted R2 0.368 0.949 Residual Std. Error 9.998 (df = 3073) 2.844 (df = 3070) ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.4: OLS with clustered standard errors Table 4.4: OLS with clustered standard errors Table 4.4: OLS with clustered standard errors With other industry characteristics, such as product dierentiation, industry size, and industry concentration, the relationships vary. Products face somewhat fewer location-discriminating market access barriers when they are dierentiated goods. As the range of this variable is zero-to-one, the overall eect on the market is modest, but the relationship is statistically signicant in most specications. However, the number of location-discriminating barriers to market access do not appear to have a statistically or substantively signicant relationship to industry concentration or the measures of industry size in these models. 119 log(# Loc-Disc Barriers + 1) (1) (2) Avg. FDI Out (USD bil) 0.030∗∗ 0.003∗ (0.013) (0.001) Avg. FDI In (USD bil) −0.009 −0.001 (0.008) (0.001) Rauch Di Good −0.480∗∗ −0.057∗ (0.187) (0.031) Herf. Index, top 50 rms −0.00003 0.00001 (0.0001) (0.00001) Establishments −0.00004∗∗∗ −0.00000∗ (0.00001) (0.00000) Value Added 0.006 0.001 (0.004) (0.001) log(# Char-Disc Barriers + 1) 0.484∗∗∗ (0.042) log(# Firm-Disc Barriers + 1) 0.590∗∗∗ (0.041) log(# Indiscrim Barriers + 1) −0.279∗∗∗ (0.035) Constant 2.864∗∗∗ 0.675∗∗∗ (0.199) (0.069) N 3,080 3,080 R2 0.365 0.963 Adjusted R2 0.364 0.963 Residual Std. Error 0.478 (df = 3073) 0.115 (df = 3070) ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.5: Log-linear model with clustered standard errors Table 4.5: Log-linear model with clustered standard errors Table 4.5: Log-linear model with clustered standard errors In a model where the counts of other policies are included as controls, it is possi- ble to examine the relationships among the various types of market access barriers, given the industry characteristics considered above. In these models, the relation- ship between the number of location-discriminating barriers and quality- and rm- discriminating barriers is positive. 4.6.1 Location-discriminating Costs Also, given the changes in magnitude of other co- variates in the model, when added as controls the other barriers capture a signicant portion of the variance in location-discriminating costs in the model. Products for which there are more characteristic- and rm-discriminating barriers in the TRAINS 120 # Loc-Disc Barriers (1) (2) Avg. FDI Out (USD bil) 0.027∗∗ 0.009∗∗∗ (0.012) (0.003) Avg. FDI In (USD bil) −0.011 −0.007∗∗∗ (0.008) (0.003) Rauch Di Good −0.583∗∗∗ −0.129∗ (0.188) (0.071) Herf. Index, top 50 rms −0.00000 0.00002 (0.0001) (0.00002) Establishments −0.00005∗∗∗ −0.00000 (0.00002) (0.00000) Value Added 0.009∗∗ 0.002 (0.004) (0.001) # Char-Disc Barriers 0.011∗∗∗ (0.002) # Firm-Disc Barriers 0.043∗∗∗ (0.006) # Indiscrim Barriers −0.004 (0.004) Constant 2.976∗∗∗ 2.116∗∗∗ (0.197) (0.086) N 3,080 3,080 ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.6: Negative Binomial Model with clustered standard err ble 4.6: Negative Binomial Model with clustered standard errors Table 4.6: Negative Binomial Model with clustered standard errors data also have more location-discriminating barriers. data also have more location-discriminating barriers. data also have more location-discriminating barriers. data also have more location-discriminating barriers. This relationship does not hold between location-discriminating and indiscrimi- nate costs. The greater the number of indiscriminate cost barriers, the fewer the number of location-discriminating costs a particular product faces. This suggests that, when accounting for industry-specic factors, the measures that divide markets along production location, product characteristic, or producer identity lines are com- plements, but indiscriminate cost policies and location-discriminating cost policies are substitutes. Given the choices made in coding the TRAINS measures as market access barriers, this is expected. While the theory of protection-seeking might suggest 121 that all of the policies should be substitutes34, this analysis does not support that hypothesis as clearly. These model estimates are consistent with some of the hypothesized relationships between industry characteristics (and the other kinds of market access barriers) that arise from theories of protection-seeking coalitions. Products of industries where rms with global links (as measured by inward FDI) are more politically salient face fewer location-discriminating barriers. 34. This is due to the aggregate eect on prices and utility as all policies distort goods markets. 4.6.2 Characteristic-discriminating Costs Results from models testing an association between industry characteristics and the presence or absence of characteristic-discriminating costs imposed on a particular product are somewhat less clear. The relationships between industry characteristics and the number of characteristic-discriminating measures dier signicantly when other market access costs are included as controls versus when they are not. In mod- els where characteristic-discriminating costs are regressed on industry characteristics without those controls, many of the industry characteristics in the model have statis- tically signicant relationships with the number of barriers. However, when including other measures as controls, many of the industry characteristics measures no longer covary with the outcome of interest in a meaningful way. The primary hypothesis related to characteristic-discriminating barriers is that they should be less frequent in homogeneous-good industries. However, evidence from the models estimated here does not support that hypothesis. In some cases, there is no statistically-distinguishable relationship between product dierentiation and the presence or absence of characteristic-discriminating costs. In other mod- els, a relationship that is opposite to the theory appears to hold. Products that the Rauch classication categorizes as dierentiated goods face fewer characteristic- 122 # Char-Disc Barriers (1) (2) Avg. FDI Out (USD bil) 0.599 0.014 (0.365) (0.060) Avg. FDI In (USD bil) −0.315 0.023 (0.246) (0.040) Rauch Di Good −17.771∗∗∗ −1.579 (6.510) (1.857) Herf. Index, top 50 rms −0.001 −0.001 (0.002) (0.001) Establishments −0.001∗∗∗ −0.0001 (0.0004) (0.0001) Value Added 0.253∗ −0.011 (0.144) (0.019) # Loc-Disc Barriers 2.133∗∗∗ (0.194) # Firm-Disc Barriers −2.360∗∗∗ (0.332) # Indiscrim Barriers 1.287∗∗∗ (0.240) Constant 31.176∗∗∗ −1.869 (6.659) (1.845) N 3,080 3,080 N 3,080 3,080 R2 0.242 0.860 Adjusted R2 0.241 0.859 Residual Std. Error 17.492 (df = 3073) 7.535 (df = 3070) ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.7: OLS with clustered standard errors Table 4.7: OLS with clustered standard errors Other industry characteristics do not appear to be strongly related to these mea- sures of characteristic-discriminating market access costs. In some specications, es- pecially those without the additional measures as controls, some parameter estimates appear statistically signicant at canonical thresholds, but the eect sizes appear modest. When additional market access barriers are included in the models, these estimates generally do not hold, and in some cases an even weaker eect of oppo- 123 log(# Char-Disc Barriers + 1) (1) (2) Avg. FDI Out (USD bil) 0.037∗∗∗ −0.001 (0.002) (0.001) Avg. 35. This may be in part because characteristic-discriminating barriers are sometimes administered 4.6.2 Characteristic-discriminating Costs FDI In (USD bil) −0.015∗∗∗ −0.001 (0.001) (0.0005) Rauch Di Good −0.508∗∗∗ 0.007 (0.038) (0.012) Herf. Index, top 50 rms −0.00003 −0.00002∗∗ (0.00002) (0.00001) Establishments −0.00005∗∗∗ 0.00001∗∗∗ (0.00001) (0.00000) Value Added 0.007∗∗∗ −0.001∗∗ (0.001) (0.0002) log(# Loc-Disc Barriers + 1) 1.365∗∗∗ (0.018) log(# Firm-Disc Barriers + 1) −0.713∗∗∗ (0.019) log(# Indiscrim Barriers + 1) 0.559∗∗∗ (0.012) Constant 3.056∗∗∗ −0.456∗∗∗ (0.038) (0.029) N 3,080 3,080 R2 0.282 0.934 Adjusted R2 0.281 0.934 Residual Std. Error 0.635 (df = 3073) 0.193 (df = 3070) ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.8: Log-linear model with clustered standard errors Table 4.8: Log-linear model with clustered standard errors Table 4.8: Log-linear model with clustered standard errors Table 4.8: Log-linear model with clustered standard errors site direction appears. The instability of these estimates, combined with their small magnitude, suggests no strong relationship in these models. site direction appears. The instability of these estimates, combined with their small magnitude, suggests no strong relationship in these models. When they are included as controls, the relationship between other forms of market access barriers and characteristic-discriminating costs is both strong and signicant. In general, across the models, the number of location-discriminating barriers is pos- itively associated with the number of characteristic-discriminating ones. The same positive relationship holds true for indiscriminate costs, as well.35 However, these 124 # Char-Disc Barriers (1) (2) Avg. FDI Out (USD bil) 0.027∗∗ 0.015∗∗∗ (0.013) (0.005) Avg. FDI In (USD bil) −0.015∗ −0.006 (0.009) (0.004) Rauch Di Good −0.605∗∗∗ −0.041 (0.205) (0.102) Herf. Index, top 50 rms 0.00001 −0.00003 (0.0001) (0.00004) Establishments −0.0001∗∗∗ −0.00002 (0.00002) (0.00001) Value Added 0.010∗∗ −0.001 (0.005) (0.002) # Loc-Disc Barriers 0.058∗∗∗ (0.006) # Firm-Disc Barriers −0.063∗∗∗ (0.012) # Indiscrim Barriers 0.053∗∗∗ (0.007) Constant 3.309∗∗∗ 2.033∗∗∗ (0.226) (0.123) ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.9: Negative Binomial Model with clustered standard erro Table 4.9: Negative Binomial Model with clustered standard errors data suggest that when there are more rm-discriminating costs, fewer characteristic- discriminating costs are present, on average and with all other covariates considered. As discussed in relation to location-discriminating costs, the positive relationships may be a consequence of the coding rules chosen, specically the decision to count single measures as imposing multiple types of costs. 4.6.2 Characteristic-discriminating Costs From these results, one may conclude that if there exists a relationship between industry characteristics and these characteristic-discriminating costs is not well iden- tied using these measures. This may be in part because of the way UNCTAD codes the NTMs in the database. Many dierences in technical standards are not consid- using policies that raise indiscriminate costs on products. 125 ered, or are only considered in some cases. These standards are likely to be the most prevalent type of characteristic-discriminating cost, and their omission from the data may go some way towards explaining the inconsistent ndings here. These results also suggest that, in commodities markets, or markets where goods are considered commodities or index-priced goods, characteristic-discriminating policies are a means of creating dierentiated markets, conferring dierent benets than the theory of protection-seeking coalitions would suggest. In either case, this evidence does not strongly support the theory as it is. 4.6.3 Firm-discriminating Costs Results from the models where rm-discriminating costs are the outcome of inter- est are mixed. For some industry characteristics, like FDI ows, ndings are unstable. For others, like the number of establishments in a given sector, results are stable, but modest in magnitude. As with the previous models, links between varieties of market access costs remain strong in this coding. Theory suggests that rm-discriminating costs should be more prevalent in in- dustries where a small cadre of rms can collude to block new entrants, or where a suciently-concentrated segment of rms can raise costs on others. However re- lationships between the number of rm-discriminating costs and both the number and concentration of rms from these models do not match those predictions. While the parameter estimates for the association between concentration and the number of rm-discriminating costs may be statistically signicant, they are substantively very modest. Also, the modest relationship is opposite to that predicted by the theory. While theory would suggest a positive relationship between the Herndal index (that is larger the greater the degree of concentration) and the number of rm-discriminating barriers, the opposite appears to be the case. The same modest relationship appears for the link between number of establish- 126 # Firm-Disc Barriers (1) (2) Avg. FDI Out (USD bil) 0.329 0.031 (0.205) (0.035) Avg. FDI In (USD bil) 0.018 0.047∗∗ (0.132) (0.023) Rauch Di Good −5.596∗∗ −0.547 (2.230) (0.467) Herf. Index, top 50 rms −0.001 −0.0005∗∗∗ (0.001) (0.0002) Establishments −0.001∗∗ −0.00004 (0.0003) (0.0001) Value Added 0.054 −0.014 (0.042) (0.011) # Loc-Disc Barriers 0.505∗∗∗ (0.067) # Char-Disc Barriers −0.139∗∗∗ (0.044) # Indiscrim Barriers 0.501∗∗∗ (0.049) Constant 10.718∗∗∗ −0.541 (2.520) (0.450) N 3,080 3,080 R2 0.415 0.955 Adjusted R2 0.414 0.955 Residual Std. Error 6.589 (df = 3073) 1.831 (df = 3070) ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.10: OLS with clustered standard errors Table 4.10: OLS with clustered standard errors ments in a given industry and the number of rm-discriminating costs that products of that industry face. However, where models reveal a substantively weak but statis- tically signicant relationship, it is in the direction that theory would predict. The greater the number of US establishments associated with a certain product line, the fewer the number of rm-discriminating policies that apply to that product line. 4.6.3 Firm-discriminating Costs How- ever, over the range of the variable in the sample, this is not consequential when all covariates are considered. Other industry characteristics, like the degree of foreign investment (inward and 127 log(# Firm-Disc Barriers + 1) (1) (2) Avg. FDI Out (USD bil) 0.033∗∗ −0.002 (0.016) (0.002) Avg. FDI In (USD bil) −0.006 0.003∗∗ (0.010) (0.001) Rauch Di Good −0.458∗∗ −0.005 (0.188) (0.039) Herf. Index, top 50 rms −0.0001 −0.00003∗∗∗ (0.0001) (0.00001) Establishments −0.0001∗∗ 0.00000 (0.00002) (0.00000) Value Added 0.006 −0.001 (0.004) (0.001) log(# Loc-Disc Barriers + 1) 1.018∗∗∗ (0.069) log(# Char-Disc Barriers + 1) −0.436∗∗∗ (0.056) log(# Indiscrim Barriers + 1) 0.560∗∗∗ (0.039) Constant 2.182∗∗∗ −0.559∗∗∗ (0.215) (0.100) N 3,080 3,080 R2 0.336 0.961 Adjusted R2 0.334 0.961 Residual Std. Error 0.626 (df = 3073) 0.151 (df = 3070) ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.11: Log-linear model with clustered standard errors Table 4.11: Log-linear model with clustered standard errors Table 4.11: Log-linear model with clustered standard errors outward) and the degree of product dierentiation, have similarly weak relationships with the number of rm-discriminating barriers. In some models, the presence of outward FDI ows appears related to more rm-discriminating market access costs, but this relationship is somewhat inconsistent across models. Similarly, inward FDI ows appear positively related to the number of rm-discriminating barriers in some models, but negatively so in others. Across the models where other market access barriers are included, the number of rm-discriminating market access barriers is positively related to the number of 128 # Firm-Disc Barriers (1) (2) Avg. FDI Out (USD bil) 0.034∗∗ 0.014∗∗∗ (0.015) (0.005) Avg. FDI In (USD bil) −0.007 −0.005∗ (0.011) (0.003) Rauch Di Good −0.464∗∗ −0.104 (0.188) (0.070) Herf. Index, top 50 rms −0.00001 −0.0001∗ (0.0001) (0.00004) Establishments −0.0001∗∗ −0.00002∗ (0.00003) (0.00001) Value Added 0.007∗ 0.0001 (0.004) (0.002) # Loc-Disc Barriers 0.033∗∗∗ (0.005) # Char-Disc Barriers −0.008∗∗ (0.004) # Indiscrim Barriers 0.041∗∗∗ (0.005) Constant 2.231∗∗∗ 1.298∗∗∗ (0.217) (0.095) N 3,080 3,080 ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.12: Negative Binomial Model with clustered standard er Table 4.12: Negative Binomial Model with clustered standard errors Table 4.12: Negative Binomial Model with clustered standard errors location-discriminating and indiscriminate cost barriers, but negatively associated with the number of characteristic-discriminating ones, when all of the industry-specic features are included in the models. 4.6.3 Firm-discriminating Costs The estimates from these models do not clearly indicate a relationship between the industry-specic characteristics discussed above and the number of rm-discriminating barriers that apply to a particular product. In cases where estimated coecients are consistent in direction and statistical signicance, their magnitude is modest. For those variables where magnitudes are more substantial, direction of relationship and statistical signicance are not consistent. While these measures are clearly associated with the presence or absence of other policies, those relationships are a consequence 129 of coding decisions when translating TRAINS data into the typology of market access barriers. of coding decisions when translating TRAINS data into the typology of market access barriers. 36. An avenue for future research is comparing eciency to these costs using measures of Total Factor Productivity, although use of these measures for inter-industry comparison can be dicult. 4.6.4 Indiscriminate Costs In looking to policies that raise costs on all products of a given type, such as xed costs of market entry, the relationships are somewhat dierent than those for other types of market-access costs. The theory of protection-seeking coalitions suggests that it is industry eciency that determines whether the products of a given industry face costs that apply indiscriminately to all varieties. Without a direct measure of domestic industry eciency, it is not possible to test this hypothesis directly.36 However, the weak link between value added (which is associated with greater output and productivity) and the number of indiscriminate barriers suggests that there isn't strong evidence for that relationship here. The link between foreign investment (both inward and outward) and indiscriminate- cost market access barriers is mixed. In the models where other measures are not included as controls, there appears to be a positive relationship between outward FDI ows and a greater number of indiscriminate-cost barriers. However, when including the other measures as controls, that relationship fades. On the other hand, where FDI inows appear to be unrelated to indisciminate-cost policies in the models without other policies as controls, when considering them in the model, a weak and negative relationship emerges. As with other measures as outcomes, this does not suggest a strong relationship. For dierent reasons, the link between product dierentiation and the number of indiscriminate costs a particular good faces is inconclusive. In the models where other barriers are not included, any relationship where one appears to be distinguishable is negative. However, when controlling for other kinds of market access barriers, 130 # Indiscrim Barriers (1) (2) Avg. FDI Out (USD bil) 0.301∗ −0.019 (0.182) (0.037) Avg. FDI In (USD bil) −0.013 −0.033 (0.126) (0.023) Rauch Di Good −2.838 2.161∗∗∗ (1.862) (0.735) Herf. Index, top 50 rms −0.00003 0.001∗∗ (0.001) (0.0004) Establishments −0.001∗∗ −0.00003 (0.0003) (0.0001) Value Added 0.052 0.004 (0.034) (0.015) # Loc-Disc Barriers −0.435∗∗∗ (0.142) # Char-Disc Barriers 0.187∗∗ (0.076) # Firm-Disc Barriers 1.236∗∗∗ (0.111) Constant 9.053∗∗∗ −0.519 (2.294) (0.961) N 3,080 3,080 R2 0.276 0.887 Adjusted R2 0.275 0.887 Residual Std. 4.6.4 Indiscriminate Costs Error 7.279 (df = 3073) 2.875 (df = 3070) ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.13: OLS with clustered standard errors Table 4.13: OLS with clustered standard errors Table 4.13: OLS with clustered standard errors dierentiated goods appear to face more indiscriminate costs in the US market. This inconsistency makes it impossible to draw strong conclusions about an association between product dierentiation and the raising of indiscriminate cost policies. For measures of industry size and concentration, there is no strong evidence that any of those metrics are strongly associated with greater or fewer indiscriminate cost policies. The Herndal index of the industry producing a particular good doesn't have a consistent positive or negative relationship across the models, and even in cases where it is statistically signicant, the magnitude of the eect is modest at best. 131 log(# Indiscrim Barriers + 1) (1) (2) Avg. FDI Out (USD bil) 0.038∗∗ 0.002 (0.016) (0.002) Avg. FDI In (USD bil) −0.010 −0.002∗ (0.010) (0.001) Rauch Di Good −0.334∗ 0.083∗ (0.174) (0.049) Herf. Index, top 50 rms −0.00005 0.00003∗ (0.0001) (0.00002) Establishments −0.0001∗∗∗ −0.00001∗∗∗ (0.00003) (0.00000) Value Added 0.007∗∗ 0.001 (0.003) (0.001) log(# Loc-Disc Barriers + 1) −1.005∗∗∗ (0.132) log(# Char-Disc Barriers + 1) 0.714∗∗∗ (0.077) log(# Firm-Disc Barriers + 1) 1.170∗∗∗ (0.069) Constant 2.068∗∗∗ 0.212 (0.207) (0.132) N 3,080 3,080 R2 0.269 0.934 Adjusted R2 0.268 0.933 Residual Std. Error 0.723 (df = 3073) 0.218 (df = 3070) ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.14: Log-linear model with clustered standard errors Table 4.14: Log-linear model with clustered standard errors Table 4.14: Log-linear model with clustered standard errors In contrast, while the coecient estimates for the variable capturing the number of establishments in a producing industry is consistently negative, the magnitude of that parameter is quite small. Only at the very maximum of the sample range for that variable would there be a relationship large enough to reach the magnitude of one additional policy. As with the other models where location-, characteristic-, and rm-discriminating costs were the outcomes of interest, when including other market access costs as con- trols, there are strong and signicant relationships. Location-discriminating costs ap- 132 # Indiscrim Barriers (1) (2) Avg. FDI Out (USD bil) 0.031∗∗ 0.010∗∗ (0.015) (0.004) Avg. FDI In (USD bil) −0.008 −0.010∗∗ (0.011) (0.004) Rauch Di Good −0.185 0.170∗ (0.176) (0.088) Herf. 4.6.4 Indiscriminate Costs Index, top 50 rms 0.00003 0.00002 (0.0001) (0.00003) Establishments −0.0001∗∗ −0.00002∗∗ (0.00003) (0.00001) Value Added 0.007∗ 0.001 (0.004) (0.002) # Loc-Disc Barriers −0.042∗∗∗ (0.009) # Char-Disc Barriers 0.022∗∗∗ (0.004) # Firm-Disc Barriers 0.099∗∗∗ (0.013) Constant 2.104∗∗∗ 1.175∗∗∗ (0.217) (0.132) N 3,080 3,080 ∗p < .1; ∗∗p < .05; ∗∗∗p < .01 Table 4.15: Negative Binomial Model with clustered standard er ble 4.15: Negative Binomial Model with clustered standard errors Table 4.15: Negative Binomial Model with clustered standard errors pear to have a negative relationship with indiscriminate costs. However, characteristic- and rm-discriminating costs appear positively associated with the number of indis- criminate cost policies. pear to have a negative relationship with indiscriminate costs. However, characteristic- and rm-discriminating costs appear positively associated with the number of indis- criminate cost policies. From these models, it appears that any relationship between industry characteris- tics and the presence or absence of policies that raise costs on goods indiscriminately is modest. While the FDI measures appear to have some eect, it is modest or in- consistent. Dierentiated goods appear to have face more indiscriminate costs, but only when controlling for other kinds of policies. Industry size and concentration do not appear to be consistently related to more or fewer of these policies, either. The strongest relationship, as in the other models, is with other kinds of policies. 133 4.6.5 Summary of Results The results from the individual models taken together provide some insights into the nature of market access across products in the US. First, foreign investment, both inward and outward, is related to the number of market access costs of dierent types. While the theory suggested that FDI should aect location-discriminating costs, there is evidence that both FDI inows and FDI outows are related to the presence or absence of multiple types of market access barriers. Where there is more outward FDI in an industry, those products are generally face more market access costs of all types. Another observation from these results is that the relationship between industry characteristics and the number of policies of any type in place is modest. In the models where other policies are included as controls, the coecients suggest that the dierence in outcome of counts of any type of policy is at most one or two additional policies of a specic type over the range of the industry characteristic variables. The areas of dierence in the relationships across the measures are in FDI in- ows, industry concentration, product dierentiation, and the size of an industry as measured by the number of establishments. The strongest relationships from these models are the relationships among the varieties of market access costs. With this coding and these data, it appears that there is complementarity among most pairs of policy types, but not all. The negative relationship between location-discriminating costs and indiscriminate costs, as well as between characteristic-discriminating and rm-discriminating costs, suggests a degree of substitution between them. 134 4.6.6 Areas for Future Research The evidence here is by no means conclusive. In some ways, it raises more ques- tions than it answers and also points towards improvements that can be made in future research. Some of those improvements concern measurement of core concepts raised by the theory of market access barriers, while others concern expanding the scope of the project across cases and time. The market access costs coding scheme used for this analysis can be improved. The high degree of correlation across the measures, created partially by construction, also makes examining dierent relationships between industry characteristics and protectionist policies more dicult. While the goal of being inclusive, or conservative, when translating the descriptions of measures from the TRAINS codebook to the 4 costs typology has some benets, it also created a situation where one measure counted multiple times, and is somewhat inconsistent with the theory's conception of those costs as independent dimensions of the barriers facing a particular good. Rather than measuring the policy as imposing greater discriminatory costs on one feature of a product and lesser costs on others, this choice makes all costs imposed by an NTM equally signicant. A weighting scheme across dimensions for NTMs where descriptions suggest multiple types of costs may be more appropriate, although more dicult to construct. More critically, this coding scheme, while a best eort to adapt existing data to a new theory, also conicts with a core argument of the market access barriers frame- work. Each of the types of policies is, in theory, orthogonal to the others. While a given policy may raise multiple types of costs, those costs are themselves independent factors. The one policy, multiple costs coding for those TRAINS measures that are not clearly one type or another is a second-best solution for measuring these costs. Another avenue for future improvement may be text analysis of policies where content dimensions are constrained to match these four types of costs more closely. Alterna- 135 tively, with more detailed market data, it might be possible to recover price wedges across these dimensions, albeit with signicant model structure and assumptions. Expanding the domain of study across cases is also necessary for one to have greater condence in the validity of the theory of protection-seeking coalitions. 4.6.6 Areas for Future Research While limiting analysis to the United States meant better access to ne-grained data, it also reduces condence in the external validity of the ndings. It is possible that the US's trade politics at the particular period of study is anomalous in some way. Institutional features of the US may make the dynamics of protection-seeking behavior signicantly dierent than in other countries. While they rarely exist for more than one year, there are NTM panels for other countries in the TRAINS dataset that may serve as comparison cases for the US. Looking to these other cases may reveal whether the nding here are artifacts of the US specically, or other fundamental patterns, including those hypothesized above. 4.7 Conclusion This evidence, provided by looking to existing data on NTMs through a slightly dierent lens, links empirical insights to a new body of thinking on how trade- distorting measures work. By looking to dierent kinds of policies based not on whether they are import taxes or not, but rather based on what features of a hypo- thetical good they aect, we see a slightly dierent picture of protectionism. While there are relationships that persist across all varieties of policies, there is also some dierence in the relationships across measures. This suggests, to some degree, that NTMs are not all alike, and ought not be treated as such. These estimates provide initial insights into what drives the provision of dier- ent kinds of protectionist policies for dierent segments of an economy. Some of the ndings are consistent with existing evidence from the study of tari and non-tari measures. In particular, the relationships between industries with greater degrees 136 of international investment and market access barriers reect existing argument and evidence. Other ndings, such as the varying covariance of product dierentiation and industry size and concentration across dierent market access barriers open the door for further inquiry. The substitution-versus-complementarity evidence from the models with controls may suggest a more complex relationship among the measures than extant theory suggests, but is more likely the product of the process that gen- erated the data. Future research, less reliant on existing databases of NTMs (which necessarily focus on the location-discriminating nature of a policy) or armed with a revised coding strategies that more clearly distinguish between policies, may yield stronger and more consistent ndings with respect to the varieties of protection and the ways in which and reasons why global goods market access has changed over time. The overarching takeaway from this analysis is that not all trade-distorting policies are driven by the same industry pressures. Moving away from theory and empirical analysis that either treat all NTMs as similar to taris, or all NTMs as fundamentally dierent from taris (but still homogeneous as a group) is a step forward in under- standing the policies that regulate and shape the global goods market, both across and within countries. 137 APPENDICES 138 Testing the Negative Binomial Model's Overdispersion Assumption One of the motivations for using the negative binomial model, which like the Poisson model estimates expected values of count variables, is a concern that the conditional mean and conditional variance of the outcome variable are not equal. In a Poisson model, both the mean and variance are dened by a single parameter. If the outcome variable (here, counts of market access barriers) are overdispersed, it may be necessary to include additional model structure to address that overdispersion. In the negative binomial model, this is captured by an additional parameter α, which is xed to unity in the Poisson model. To justify use of the negative binomial model, rather than the Poisson, it is necessary to test the additional assumption. One way of testing this assumption is by comparing the model t when estimated as a Poisson model (where α is xed at unity) against the negative binomial model. As both are estimated using maximum likelihood, a likelihood ratio test can be used to test the hypothesis that the negative binomial model (where α is a free parameter) has better t than the Poisson model (where α is constrained). If the conditional means and variance are not equal, the t of the negative binomial model will be superior to that of the Poisson model in a statistically-distinguishable way. 139 The assumption of overdispersion is tested for each of the models specied in the body text. Each of the models is reestimated as a Poisson model, then the log- likelihood ratio statistic of the negative binomial model and the Poisson model is computed.1 Then, a classical likelihood ratio test is performed. The one-tailed p- value of this statistic, which is distributed chi-squared, is the measure that indicates whether there is a signicant dierence in t between the two models. Both the statistic and the p-value are presented in Table A.1. Here, the negative binomial is the alternative model and the Poisson model the null model Testing the Negative Binomial Model's Overdispersion Assumption Model D p-value Model 1a 6, 575.022 0 Model 1b 295.301 0 Model 2a 20, 634.060 0 Model 2b 3, 590.341 0 Model 3a 4, 851.644 0 Model 3b 437.502 0 Model 4a 7, 025.719 0 Model 4b 1, 266.179 0 Table A.1: Overdispersion Assumption Test Model D p-value Model 1a 6, 575.022 0 Model 1b 295.301 0 Model 2a 20, 634.060 0 Model 2b 3, 590.341 0 Model 3a 4, 851.644 0 Model 3b 437.502 0 Model 4a 7, 025.719 0 Model 4b 1, 266.179 0 Table A.1: Overdispersion Assumption Test Table A.1: Overdispersion Assumption Test The results are fairly conclusive. For every specication of the model examined, the negative binomal model clearly ts better than a Poisson model. The overdisper- sion in the count data for market access barriers is too great to support the assump- tions of the Poisson model in this case, and thus including the extra free parameter is appropriate. 140 Coding of Market Access Barriers, UNCTAD Table B.1: Recoding of TRAINS NTM Measures NTM Code Subcode NTM Name Loc. Char. Firm Indiscr. A Sanitary and Phytosani- tary Measures A1 Prohibitions/restrictions of imports for SPS reasons A11 A110 Temporary geographic probibitions for SPS reasons 1 1 0 0 A12 A120 Geographical restrictions on eligibility 1 1 1 0 A13 A130 Systems Approach 1 1 0 0 A14 A140 Special authroization re- quirement for SPS rea- sons 1 1 1 0 A15 A150 Registration require- ments for importers 1 0 1 0 A19 A190 Probibitions n.e.s. 1 1 0 0 A2 Tolerance limits for residues and restricted use of substances 141 NTM Code Subcode NTM Name Loc. Char. Firm Indiscr. A21 A210 Tolerance limits for residues of or con- tamination by certain non-microbiological substances 0 1 0 0 A22 A220 Restricted use of certain substances in foods and feeds and their contact materials 0 1 0 0 A3 Labelling, marking, and packaging requirement A31 A310 Labelling requirements 0 0 0 1 A32 A320 Marking requirements 0 0 0 1 A33 A330 Packaging requirements 0 1 0 0 A4 Hygienic requirements A41 A410 Microbiological criteria of the nal product 0 1 0 0 A42 A420 Hygenic practices during production 0 1 0 0 A5 A500 Treatment for elimi- nation of animal pests and disease-causing organisms in the nal product 0 1 0 0 A51 A510 Cold/heat treatment 0 1 0 0 A52 A520 Irridation 0 1 0 0 A53 A530 Fumigation 0 1 0 0 A59 A590 Treatment for elimina- tion of animal pests and disease-causing organ- isms in the nal product, n.e.s. 0 1 0 0 A6 A600 Other requirements on production of post- production processes 0 1 0 0 A61 A610 Plant-growth processes 0 1 0 0 A62 A620 Animal-raising or - catching processes 0 1 0 0 A63 A630 Food and feed processing 0 1 0 0 A64 A640 Storage and transport conditions 0 1 0 0 A69 A690 Other requirements n.e.s. 0 1 0 0 A8 A800 Conformity assessment related to SPS 0 1 0 1 142 NTM Code Subcode NTM Name Loc. Char. Firm Indiscr. Coding of Market Access Barriers, UNCTAD A81 A810 Product registration re- quirement 1 1 0 0 A82 A820 Testing requirement 1 1 0 0 A83 A830 Certication requirement 1 1 0 0 A84 A840 Inspection requirement 1 1 0 0 A85 A850 Traceability requirement 1 1 1 0 A851 A851 Origin of materials and parts 1 1 0 0 A852 A852 Processing history 0 1 0 0 A853 A853 Distribution of products after delivery 0 1 0 0 A859 A859 Traceability require- ments, n.e.s. 0 1 0 0 A86 A860 Quarantine requirement 1 1 0 0 A89 A890 Conformity assessment related to SPS, n.e.s 0 0 0 1 A9 A900 SPS measures, n.e.s. 0 1 0 0 B Technical Barriers to Trade B1 Prohibitions/restrictions of imports for objectives set out in the TBT agreement B11 B110 Probibition for TBT rea- sons 1 1 0 0 B14 B140 Authorization require- ment for TBT reasons 1 1 1 0 B15 B150 Registration requirement for importers for TBT reasons 1 1 1 0 B19 B190 Prohibitions/restrictions of imports for TBT agreement reasons, n.e.s 1 1 0 0 B2 Tolerance limits for residues and restricted use of substances B21 B210 Tolerance limits for residues of or con- tamination by certain substances 0 1 0 0 B22 B220 Restricted use of certain substances 0 1 0 0 B3 Labelling, marking, and packaging requirement 143 NTM Code Subcode NTM Name Loc. Char. Firm Indiscr. B31 B310 Labelling requirements 0 0 0 1 B32 B320 Marking requirements 0 0 0 1 B33 B330 Packaging requirements 0 1 0 0 B4 Production or post- production requirement B41 B410 TBT Regulations on pro- duction processes 0 1 0 0 B42 B420 TBT regulations on transport and storage 0 1 0 0 B49 B490 Production or post- production requirements, n.e.s. Coding of Market Access Barriers, UNCTAD 0 1 0 0 B6 B600 Product identity require- ment 0 1 0 0 B7 B700 Product-quality or - performance requirement 0 1 0 0 B8 B800 Conformity assessment related to TBT 1 1 1 1 B81 B810 Product registration re- quirement 1 1 1 1 B82 B820 Testing Requirement 1 1 1 1 B83 B830 Certication requirement 1 1 1 1 B84 B840 Inspection requirement 1 1 1 1 B85 B850 Traceability information requirements 1 1 1 1 B851 B851 Origins of materials and parts 1 0 0 1 B852 B852 Processing history 1 1 0 1 B853 B853 Distribution of products after delivery 1 0 1 1 B859 B859 Traceability require- ments, n.e.s. 1 1 1 1 B89 B890 Conformity assessment related to TBT, n.e.s 0 1 1 1 B9 B900 TBT measures, n.e.s. 0 1 1 1 C1 C100 Pre-shipment inspection 1 0 0 0 C2 C200 Direct consignment re- quirement 1 1 0 0 C3 C300 Requirement to pass through specied port of customs 1 0 0 0 144 NTM Code Subcode NTM Name Loc. Char. Firm Indiscr. C4 C400 Import-monitoring and - surveillance requirements and other automatic li- censing measures 1 0 1 0 C9 C900 Other formalities, n.e.s 1 0 0 0 D1 Antidumping Measures D11 D110 Antidumping Investiga- tion 1 0 0 0 D12 D120 Antidumping Duty 1 0 0 0 D13 D130 Price undertaking 1 0 0 0 D2 Countervailing measure D21 D210 Countervailing investiga- tion 1 0 0 0 D22 D220 Countervailing Duty 1 0 0 0 D23 D230 Undertaking 1 0 0 0 D3 Safeguard measures D31 D310 General (multilateral) safeguard 1 0 0 0 D311 D311 Safeguard investigation 1 0 0 0 D312 D312 Safeguard duty 1 0 0 0 D313 D313 Safeguard quantitative restriction 1 0 0 0 D314 D314 Safeguard measures, other form 1 0 0 0 D32 Agricultural special safe- guard D321 D321 Volume-based agricul- tural special safeguard 1 0 0 0 D322 D322 Price-based agricultural special safeguard 1 0 0 0 D39 D390 Safeguard , n.e.s. Coding of Market Access Barriers, UNCTAD 1 0 0 0 E1 Non-automatic import- licensing procedures other than authorizations for SPS or TBT reasons E11 Licensing for economic reasons E111 E111 Licensing procedure with no specic ex ante criteria 1 0 1 0 E112 E112 Licensing for specied use 1 1 1 0 E113 E113 Licensing linked with lo- cal production 1 0 1 0 E119 E119 Licensing for economic reasons, n.e.s. 1 0 1 0 145 NTM Code Subcode NTM Name Loc. Char. Firm Indiscr. E12 E120 Licensing for non- economic reasons 1 0 1 0 E121 E121 Licensing for religious, moral, or cultural reasons 1 0 1 0 E122 E122 Licensing for political reasons 1 0 1 0 E129 E129 Licensing for non- economic reasons, n.e.s. 1 0 1 0 E2 Quotas E21 E210 Permanent Quotas 1 0 0 0 E211 E211 Global allocation 1 0 0 0 E212 E212 Country allocation 1 0 0 0 E22 E220 Seasonal Quotas 1 0 0 0 E221 E221 Global allocation 1 0 0 0 E222 E222 Country allocation 1 0 0 0 E23 E230 Temporary 1 0 0 0 E231 E231 Global allocation 1 0 0 0 E232 E232 Country allocation 1 0 0 0 E3 Prohibitions other than for SPS and TBT reasons E31 Prohibition for economic reasons E311 E311 Full prohibition (import ban) 1 0 0 0 E312 E312 Seasonal prohibition 1 0 0 0 E313 E313 Temporary prohibition, including suspension of issuance of licenses 1 0 0 0 E314 E314 Prohibition of importa- tion in bulk 1 1 0 0 E315 E315 Prohibition of products infriging patents or other intellectual property rights 1 1 0 0 E316 E316 Prohibition of used, repaired, or remanufac- tured goods 1 1 0 0 E319 E319 Prohibition for economic reasons, n.e.s. 1 0 0 0 E32 Prohibition for non- economic reasons E321 E321 Prohibition for religious, moral, or cultural reasons 1 1 0 0 146 NTM Code Subcode NTM Name Loc. Char. Firm Indiscr. E322 E322 Prohibition for political reasons (embargo) 1 0 0 0 E329 E329 Prohibition for non- economic reasons, n.e.s. 1 0 0 0 E5 Export-restraint arrange- ment E51 Voluntary export- restraint arrangements (VERs) E511 E511 Quota agreement (VER) 1 0 0 0 E512 E512 Consultation agreement (VER) 1 0 0 0 E513 E513 Administrative coopera- tion agreement (VER) 1 0 0 0 E59 E590 Export-restraint arrange- ments, n.e.s. Coding of Market Access Barriers, UNCTAD 1 0 0 0 E6 E600 Tari-rate quotas (TRQ) 1 0 0 0 E61 E610 WTO-bound TRQs, in- cluded in WTO schedules 1 0 0 0 E611 E611 Global allocation, WTO- bound TRQ 1 0 0 0 E612 E612 Country allocation, WTO-bound TRQ 1 0 0 0 E62 E620 Other TRQs included in other trade arrangements 1 0 0 0 E621 E621 Global allocation, other TRQs 1 0 0 0 E622 E622 Country allocation, other TRQs 1 0 0 0 F1 F100 Administrative measures aecting customs value 1 0 0 0 F11 F110 Minimum import prices 1 0 0 0 F12 F120 Reference prices 1 0 0 0 F19 F190 Other administrative measures aecting the customs value, n.e.s 1 0 0 0 F2 F200 Volutary export-price re- straints (VEPRs) 1 0 0 0 F3 F300 Variable charges F31 F310 Variable levies 1 1 0 0 F32 F320 Variable components 1 1 0 0 F39 F390 Variable charges, n.e.s. 1 0 0 0 F4 F400 Customs surcharges 1 0 0 0 F5 F500 Seasonal duties 1 1 0 0 147 NTM Code Subcode NTM Name Loc. Char. Firm Indiscr. F6 F600 Additional taxes and charges levied in con- neciton to services provided by the gov- ernenment F61 F610 Customs-inspection, - processing, and -servicing fees 1 0 0 0 F62 F620 Merchandise-handling or -storing fees 1 0 0 0 F63 F630 Tax on foreign exchange transactions 1 0 1 0 F64 F640 Stamp tax 1 0 0 0 F65 F650 Import license tax 1 0 1 0 F66 F660 Consular invoice fee 1 0 0 0 F67 F670 Statistical tax 1 0 0 0 F68 F680 Tax on transport facilities 1 0 0 0 F69 F690 Additional charges, n.e.s. 1 0 0 0 F7 Internal taxes and charges levied on imports F71 F710 Consumption taxes 0 0 0 1 F72 F720 Excise taxes 0 0 0 1 F73 F730 Taxes and charges for sensitive product cate- gories 0 0 0 1 F79 F790 Internal taxes and charges levied on im- ports, n.e.s. 0 0 0 1 F8 F800 Decreed customs valua- tions 1 0 0 0 F9 F900 Price-control measures, n.e.s. Coding of Market Access Barriers, UNCTAD G1 Advance payment re- quirement G11 G110 Advance import deposit 1 0 1 0 G12 G120 Cash margin requirement 1 0 1 0 G13 G130 Advance payment of cus- toms duties 1 0 0 0 G14 G140 Refundable deposits for sensitive product cate- gories 1 1 0 0 G19 G190 Advance payment re- quirements, n.e.s. 1 0 1 0 G2 G200 Multiple exchange rates 1 0 0 0 148 148 NTM Code Subcode NTM Name Loc. Char. Firm Indiscr. G3 Regulation on ocial for- eign exchange allocation G31 G310 Prohibition of foreign ex- change allocation 1 0 0 0 G32 G320 Bank authorization 1 0 1 0 G33 G330 Authorization linked with non-ocial foreign exchange G331 G331 External foreign ex- change 1 1 1 0 G332 G332 Importers' own foreign exchange 1 0 1 0 G339 G339 License linked with non- ocial foreign exchange, n.e.s. 1 0 1 0 G39 G390 Regulation on ocial for- eign exchange allocation, n.e.s. 1 0 1 0 G4 G400 Regulation concerning terms of payment for imports 1 0 1 0 G9 G900 Finance measures, n.e.s. H1 State-trading enterprises, for importing: other se- lective import channels H11 H110 State-trading enterprises, for importing 1 0 1 0 H19 H190 Other selective import channels, n.e.s. 1 0 1 0 H2 Compulsory use of na- tional services H21 H210 Compulsory national in- surance 1 0 1 0 H22 H220 Compulsory national transport 1 0 1 0 H29 H290 Compulsory national ser- vice, n.e.s. 1 0 1 0 H9 H900 Measures aecting com- petitions, n.e.s. 1 0 1 0 I1 I100 Local content measures 1 1 0 0 I2 I200 Trade-balancing mea- sures 1 1 1 0 I9 I900 Trade-related investment measures, n.e.s. 1 1 1 0 149 NTM Code Subcode NTM Name Loc. Char. Firm Indiscr. Coding of Market Access Barriers, UNCTAD J1 J100 Geographical restriction (on distribution) 0 0 0 1 J2 J200 Restriciton on resellers (on distribution) 1 0 1 0 K K000 Restricitons on post-sales services 1 1 1 0 L L000 Subsidies 1 0 0 0 M M000 Government procurement restrictions 0 0 1 0 N N000 Intellecutal property 0 0 1 0 O O000 Rules of origin 1 0 0 0 P Export-license, -quota, - prohibition, and other quantitiative restricitons 1 0 0 0 P11 P110 Export prohibition 1 0 0 0 P12 P120 Export quotas 1 0 0 0 P13 P130 Licensing- or permit re- quirements to export 1 0 1 0 P14 P140 Export registration re- quirements 1 0 1 0 P19 P190 Export quantitative re- strictions, N.e.s 1 0 0 0 P21 P210 State-trading enterprises, for exporting 1 0 1 0 P29 P290 Other selective export channels, n.e.s. 1 0 1 0 P3 P300 Export price-control measures 1 0 0 0 P4 P400 Measures on re-export 1 0 0 0 P5 P500 Export taxes and charges 1 0 0 0 P6 P600 Export technical mea- sures 1 1 0 0 P61 P610 Inspection requirement, for export 1 1 0 0 P62 P620 Certication required by the exporting country 1 1 0 0 P69 P690 Export technical mea- sures, n.e.s. 1 1 0 0 P7 P700 Export subsidies 1 0 1 0 P8 P800 Export credits 1 0 1 0 P9 P900 Export measures, n.e.s. 1 0 1 0 150 BIBLIOGRAPHY 151 151 BIBLIOGRAPHY Abel-Koch, Jennifer. 2013. 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https://openalex.org/W4297826504	https://ejbpc.springeropen.com/counter/pdf/10.1186/s41938-022-00585-9	English	null	Correction to: Optimizing production of a biopesticide protectant by black yeast	Egyptian Journal of Biological Pest Control	2,022	cc-by	597	The original article can be found online at https://doi.org/10.1186/s41938- 018-0078-4. The original article can be found online at https://doi.org/10.1186/s41938- 018-0078-4. Correspondence: Ahmed_bt@mans.edu.eg 1 Botany Department, Faculty of Science, Mansoura University, Mansoura 355111, Egypt 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/. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. fi The affiliation has since been updated in the published article, and the corrected affiliation may be found in this erratum. Correction to: Egyptian Journal of Biological Pest Control (2018) 28:72 Following publication of the original article (Saleh et al. 2018), the authors flagged that the article had published with an incomplete version of affiliation 2; ‘Agriculture research center (ARC)’ had been erroneously omitted from the affiliation. 1 Botany Department, Faculty of Science, Mansoura University, Man‑ soura 355111, Egypt. 2 Agriculture Research Center (ARC), Agricultural Genetic Engineering Research Institute (AGERI), Giza, Egypt. © 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/. Saleh et al. Egyptian Journal of Biological Pest Control (2022) 32:94 https://doi.org/10.1186/s41938-022-00585-9 Saleh et al. Egyptian Journal of Biological Pest Control (2022) 32:94 https://doi.org/10.1186/s41938-022-00585-9 Egyptian Journal of Biological Pest Control Saleh et al. Open Access CORRECTION Correction to: Optimizing production of a biopesticide protectant by black yeast Hany Saleh1, Ahmed Abdelrazak1 , Ashraf Elsayed1, Hisham El‑Shishtawy2 and Yehia Osman1 Reference Saleh H, Abdelrazak A, Elsayed A et al (2018) Optimizing production of a biopesticide protectant by black yeast. Egypt J Biol Pest Control 28:72. https://doi.org/10.1186/s41938-018-0078-4 Reference Saleh H, Abdelrazak A, Elsayed A et al (2018) Optimizing production of a biopesticide protectant by black yeast. Egypt J Biol Pest Control 28:72. https://doi.org/10.1186/s41938-018-0078-4 Author details 1 1 Botany Department, Faculty of Science, Mansoura University, Man‑ soura 355111, Egypt. 2 Agriculture Research Center (ARC), Agricultural Genetic Engineering Research Institute (AGERI), Giza, Egypt. The original article can be found online at https://doi.org/10.1186/s41938- 018-0078-4. *Correspondence: Ahmed_bt@mans.edu.eg 1 Botany Department, Faculty of Science, Mansoura University, Mansoura 355111, Egypt Full list of author information is available at the end of the article
https://openalex.org/W591828384	https://link.springer.com/content/pdf/10.1007/s00427-015-0504-5.pdf	English	null	Germ line transformation and in vivo labeling of nuclei in Diptera: report on Megaselia abdita (Phoridae) and Chironomus riparius (Chironomidae)	Development, genes and evolution	2,015	cc-by	6,174	Dev Genes Evol (2015) 225:179–186 DOI 10.1007/s00427-015-0504-5 Dev Genes Evol (2015) 225:179–186 DOI 10.1007/s00427-015-0504-5 SHORT COMMUNICATION * Steffen Lemke steffen.lemke@cos.uni-heidelberg.de Introduction Abstract To understand how and when developmental traits of the fruit fly Drosophila melanogaster originated during the course of insect evolution, similar traits are functionally studied in variably related satellite species. The experimental toolkit available for relevant fly models typically comprises gene expression and loss as well as gain-of-function analyses. Here, we extend the set of available molecular tools to piggyBac-based germ line transformation in two satellite fly models, Megaselia abdita and Chironomus riparius. As proof- of-concept application, we used a Gateway variant of the piggyBac transposon vector pBac{3xP3-eGFPafm} to generate a transgenic line that expresses His2Av- mCherry as fluorescent nuclear reporter ubiquitously in the gastrulating embryo of M. abdita. Our results open two phylogenetically important nodes of the insect order Diptera for advanced developmental evolutionary genetics. The insect order Diptera (true flies, Fig. 1a) aggregates several traits that have facilitated its use as framework to link the molecular evolution of genomes and genetic networks with phenotypic divergence and novelty across consecutive mac- roevolutionary timescales (Rafiqi et al. 2011). Diptera have been studied for over a century, they cover about 250 million years of insect radiation within a well-established phylogeny, and they contain Drosophila melanogaster, one of the best- studied model systems in developmental biology (Anderson 1966; Wiegmann et al. 2011). Common traits of early embry- onic development in flies are the absence of a posterior growth zone and segmentation within the blastoderm embryo prior to gastrulation (long germ insects, Davis and Patel 2002). During fly gastrulation, the lateral ectoderm elongates by convergent extension (germband extension), midgut precursors involute at the anterior and posterior pole, the mesoderm internalizes on the ventral side, the lateral ectoderm gives rise to neuronal progenitors, and extraembryonic tissue forms from cells of the dorsal blastoderm (Fig. 1b–d, Anderson 1966). The molecular basis of fly segmentation and morphogenesis is best under- stood in D. melanogaster (Fig. 1b). In D. melanogaster, satu- rated genetic screens and the molecular reconstruction of de- velopmental circuits that pre-pattern the blastoderm embryo have established a powerful genetic reference system (Jaeger et al. 2012), to which development of satellite fly species can be compared (Cicin-Sain et al. 2015; Lemke et al. 2010). Two such satellite fly models, which have been established for these purposes at informative positions within the dipteran phylogeny, are Megaselia abdita (Phoridae) and Chironomus riparius (Chironomidae) (Fig. 1a). Keywords Germ line transformation . Chironomus riparius . Megaselia abdita . Germ line transformation and in vivo labeling of nuclei in Diptera: report on Megaselia abdita (Phoridae) and Chironomus riparius (Chironomidae) Francesca Caroti1 & Silvia Urbansky1 & Maike Wosch1 & Steffen Lemke1 Received: 23 December 2014 /Accepted: 20 May 2015 /Published online: 5 June 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com 1 Centre for Organismal Studies, Universität Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany Introduction EvoDevo Communicated by Siegfried Roth Electronic supplementary material The online version of this article (doi:10.1007/s00427-015-0504-5) contains supplementary material, which is available to authorized users. The scuttle fly M. abdita represents the basal branch of cyclorrhaphan flies; it shared the last common ancestor with Dev Genes Evol (2015) 225:179–186 180 contrast to D. melanogaster, however, the amnioserosal fold extends further in M. abdita, ruptures, and gives rise to a serosa that detaches from the embryo proper, crawls over, and eventually encloses it. d Gastrulation in Chironomus shares with M. abdita and D. melanogaster the overall domains of the blastoderm embryo that give rise to endoderm, mesoderm, and ectoderm and the extraembryonic epithelia; and, like in other flies, the germband of Chironomus elongates by convergent extension. In contrast to M. abdita and D. melanogaster, Chironomus additionally shares characteristics with embryonic development of non- dipteran insects: blastoderm cellularization leads to a much less- pronounced columnar epithelium; pole cells are internalized during cellularization, and prior to the onset of germband extension, Chironomus embryos lack a cephalic furrow, mesoderm invagination is neither characterized by a deep furrow nor a ventral tube, and prominent dorsal folds cannot be observed during germband extension. The dorsal blastoderm develops by extension of the amnioserosal fold into the extraembryonic serosa. Unlike in M. abdita, the serosa does not detach from the remaining embryo and fuses, like in Tribolium castaneum, on the ventral side of the embryo and thus forms a ventral amnion. cf cephalic furrow, meso mesoderm, pmg posterior midgut, amg anterior midgut, atf anterior transverse furrow, ptf posterior transverse furrow, gbe germband extension, af amniotic fold, as amnioserosa, s serosa; gray area: yolk; dotted lines in top row: ingrowing front of cell membranes; green arrows: global cell movements; area with oblique lines: extraembryonic tissue, amnioserosa in D. melanogaster and serosa in M. abdita and Chironomus. In all schematics, anterior is to the left and dorsal up Fig. 1 The insect order Diptera as evolutionary framework for early embryonic morphogenesis. a Dipteran phylogeny, with approximate age of last common ancestor given for major taxonomic groups (Wiegmann et al. 2011). b–d Schematic drawings and descriptions of successive stages of embryonic morphogenesis during gastrulation of Drosophila melanogaster (b), Megaselia abdita (c), and Chironomus spec (d), modified from and based on (Campos-Ortega and Hartenstein 1997; Lye and Sanson 2011; Rafiqi et al. 2008; Wotton et al. Cloning procedures and have a long history of being used in developmental stud- ies (reviewed in Sander 2000). and have a long history of being used in developmental stud- ies (reviewed in Sander 2000). and have a long history of being used in developmental stud- ies (reviewed in Sander 2000). and have a long history of being used in developmental stud- ies (reviewed in Sander 2000). To generate in vitro-transcribed messenger RNA (mRNA) of the piggyBac transposase, the coding sequence was amplified by PCR and cloned into pSP35; the resulting vector pSPiggyHelp was linearized by EcoRI, and capped mRNA was synthesized using mMessage mMachine SP6 transcrip- tion kit (Life Technologies). To allow for three-way Gateway assembly of DNA fragments into piggyBac, two piggyBac destination vectors were generated, pBacDestA{3xP3- eGFP} and pBacDestB{3xP3-eGFP}: PCR-amplified ccdB cassettes with flanking attR4 and attR3 sites were inserted into the AscI and BglII sites of pBac{3xP3-eGFPafm} (Horn and Wimmer 2000) to generate pBacDestA{3xP3-eGFP} and pBacDestB{3xP3-eGFP}, respectively. The piggyBac vector pBacDest{His2Av-mCherry} was used to generate the trans- genic nuclear reporter line His2Av/sqh::His2Av-mCherry; it was assembled by LR recombination of three Gateway entry vectors into pBacDestA{3xP3-eGFP}, i.e., 5′-pENTR- His2Av, which contained the M. abdita His2Av locus from position −585 to +834 (+1 being the beginning of the ORF) followed by a fragment encoding the 20 C-terminal amino acids of D. melanogaster His2Av, middle-pENTR-mCherry, which contained the mCherry CDS, and 3′-pENTR-sqh, which contained 1077 bp immediately downstream of the M. abdita spaghetti squash CDS. For M. abdita and C. riparius, transcriptome and genome resources are either available or in the process of being established (Jimenez-Guri et al. 2013; Marinković et al. 2012; S. Lemke and U. Schmidt-Ott, unpublished). Embryos of M. abdita have been shown to be very suitable for func- tional studies (Rafiqi et al. 2012; Stauber et al. 2000); similar results have been obtained for embryos of C. riparius (Klomp et al. 2015). Both species, like many other non-drosophilid fly models used to study the evolution of development, lack pro- tocols for stable germ line transformation. In D. melanogaster, germ line transformation has been used for in vivo functional dissection of cis-regulatory DNA, for precisely targeted gene expression and gene knockdown during later stages of em- bryogenesis, or for in vivo cell tracking and lineage analyses (St Johnston 2013; Rebollo et al. 2014). Fly cultures M. abdita Schmitz (Sander strain) was obtained from Johannes Jäger (CRG, Barcelona, Spain); the culture descends from the M. abdita strain maintained in the Schmidt-Ott lab- oratory and was maintained as described (Rafiqi et al. 2011). C. riparius Meigen was obtained from Urs Schmidt-Ott (The University of Chicago, Chicago, USA), who obtained the cul- ture from Gerald K. Bergtrom (University of Wisconsin, Milwaukee, WI) in 2004 (Klomp et al. 2015). The culture was maintained at 25 °C to 28 °C and a constant 17/7-h day/ night cycle. Embryos, larvae, and pupae were reared in food safe containers (Cambro) as aqueous cultures with constant aeration. Larvae were fed with a suspension of food paste prepared from sterilized milled parsley (Tro-Kost) and active dry baker’s yeast (0.65 %, w/w); eclosed flies were collected regularly and transferred to a separate cage with a dish of water for the deposition of egg packages. Cloning procedures Germ line transfor- mation in other insects has been achieved previously by using transposon elements such as piggyBac, Hermes, Minos, and mariner (Horn et al. 2002; O’Brochta and Atkinson 1996). Here, we report successful transgenesis in M. abdita and C. riparius based on the piggyBac transposon system. As proof of concept and tool for future cell tracking and lineage analyses, we generated a His2Av-mCherry fusion construct in M. abdita, which was expressed ubiquitously in the early gastrulating embryo. Introduction 2014; Ritter 1890) and own observations of fixed and live embryos. b Just prior to gastrulation, embryonic development in D. melanogaster is characterized by cellularization of the initially syncytial blastoderm. Germ cells have formed and are characterized by their round shape and position at the posterior pole of the cellularizing blastoderm. Gastrulation starts with the involution of the presumptive mesoderm along the ventral midline of the embryo, which first forms a furrow and then an epithelial tube that eventually collapses. Concurrent with mesoderm involution, the cephalic furrow forms, the posterior pole plate tilts, and the germband elongates by convergent extension of the lateral ectoderm. During germband extension, dorsal folds form between cephalic furrow and the front of the germband, and pole cells as well as anterior and posterior midgut are internalized. Cells of the dorsal blastoderm flatten and stretch to give rise to an extraembryonic epithelium (amnioserosa), which covers the dorsal opening of the embryo until its closure after germband retraction. c Gastrulation in M. abdita is qualitatively similar to D. melanogaster. Major differences compared to D. melanogaster have been described for extraembryonic development, which, in M. abdita, originates from the dorsal blastoderm like in D. melanogaster. In extraembryonic epithelium in D. melanogaster (Rafiqi et al. 2012). D. melanogaster approximately 145 million years ago and has been introduced as comparative system to bridge major differ- ences in embryonic development of mosquito-related flies (Culicomorpha) and D. melanogaster (Rafiqi et al. 2011). While overall similar to Drosophila (Fig. 1c), embryonic de- velopment in M. abdita retained ancestral features that have helped to understand the evolution of highly diverged traits of the Drosophila model, such as the transition from an ancestral bipartite extraembryonic development (formation of a sepa- rate amnion and serosa) toward the amnioserosa as a single The midge C. riparius represents the nematoceran subor- der, which constitutes the basal branch of this insect order and shared the last common ancestor with D. melanogaster ap- proximately 250 million years ago (Wiegmann et al. 2011). Within the nematoceran suborder, Chironomidae are closely related and share common developmental traits with mosqui- toes (Culicidae) (Anderson 1966) but are less laborious to maintain, easy to manipulate, accessible for in vivo imaging, Dev Genes Evol (2015) 225:179–186 181 Material and methods M. abdita embryos were injected at the posterior pole with pre-mixed pBac{3xP3-eGFP} plasmid and in vitro-synthe- sized mRNA encoding the piggyBac transposase at DNA/ RNA concentrations of 100:300 ng/μl. C. riparius embryos were injected into the center with pre-mixed plasmid and mRNA at DNA/RNA concentrations of 500:300 ng/μl or 100:300 ng/μl. Screening for enhanced Green Fluorescent Protein (eGFP) expression either in the adult eye (M. abdita) or in the nervous system of late-stage larvae (C. riparius) was performed using fluorescent binoculars (Olympus MVX 10, light source: X-cite Series 120Q; Nikon AZ 100, light source: Nikon Intensilight C-HGFI). To maintain transgenic lines, in- dividuals were constantly inbred and screened for the fluores- cent reporter in each generation. Details on fly culture maintenance, cloning procedures, germ line transformation, and the molecular analyses of transgenic lines are provided in Supplementary Methods. Using germ line transformation to generate fluorescent nuclear in vivo reporter for M. abdita Recent analyses in D. melanogaster and the flour beetle Tribolium castaneum have demonstrated how the dynamics of early embryonic development and gastrulation can be cap- tured and quantitatively analyzed by employing ubiquitous fluorescent cell labeling in combination with in toto high- speed imaging (Krzic et al. 2012; Strobl and Stelzer 2014). In D. melanogaster, Histone2Av fused to a fluorescent protein is widely used to label nuclei as proxy for cell position with a high signal to noise ratio during all stages of the cell cycle (Krzic et al. 2012). To test whether piggyBac-based germ line transformation could be used to generate an equivalent tool in M. abdita, we first identified the M. abdita orthologue of the His2Av locus, including 0.5 kb of putative 5′ regulatory region in conserved synteny with bällchen. Poor genome assembly in the 3′ genomic region of M. abdita His2Av precluded cloning of its 3′ UTR and regulatory DNA that may account for ubiq- uitous gene expression. The missing sequence information was substituted with the 3′ UTR of M. abdita spaghetti squash, which, in D. melanogaster, encodes the ubiquitously expressed regulatory myosin light chain (Kiehart et al. 2000). The different DNA fragments were combined by three-way Gateway reaction into one of two newly generated piggyBac destination vectors (pBacDest{His2Av-mCherry}, Supplemental Fig. 1), and injection of pBacDest{His2Av- mCherry} for germ line transformation yielded 92 larvae (92/2100 injected embryos=4.3 %), of which 36 adult flies eclosed (G0, 36/92=39.1 %). Crosses among G0 flies were set up in small pools as outlined above. In two independent crosses, transgenic offspring was identified based on green fluorescence in the eyes (2/36=5.5 %) and used to establish two independent His2Av/sqh::His2Av-mCherry lines through repeated inbreeding. Following injection, embryos were kept under oil in a moist chamber at 25 °C until hatching. After around 28 h, larvae started to hatch and were transferred to M. abdita cul- ture vials; about 1 week after injection, the larvae pupated; 3 weeks after injection, the adult flies eclosed. Of 1100 injected embryos, 103 larvae hatched (103/1100=9.4 %), and 38 adult flies eclosed (G0, 38/103=36.9 %). Crosses among G0 flies were set up in small pools, and adults were screened after eclosure for eGFP expression in the eyes. Despite strong and dark pigmentation of the eyes, flies could be positively scored based on a specific green fluorescent signal in the compound eyes and ocelli (Fig. piggyBac-mediated germ line transformation in M. abdita To establish piggyBac-based germ line transgenesis in M. abdita, a standard 3xP3-eGFP piggyBac vector was used, which, in D. melanogaster, drives eGFP reporter expression in the adult eyes and the larval nervous system (Horn et al. 2000). M. abdita embryos were collected from 30-min depo- sitions (25 °C), covered with halocarbon oil, and injected through the chorion. Vector and in vitro-synthesized mRNA encoding the piggyBac transposase were pre-mixed and injected at the posterior pole of M. abdita embryos, which was determined by its rounder, less pointy tip, its larger diam- eter, and a retraction of the embryo from the vitelline mem- brane prior to the formation of pole cells. Microscopy and image analysis Expression of His2Av-mCherry in early developing M. abdita embryos was analyzed on a MuVi-SPIM. Briefly, embryos were obtained from 30- to 45-min depositions, dechorionated, washed, and mounted for imaging as described previously for D. melanogaster (Krzic et al. 2012). For the calculation of nuclei number, image stacks were segmented with iLastik (1.0) and the position of each nucleus was extracted with Dev Genes Evol (2015) 225:179–186 182 Matlab (R2013a). Nuclear density was measured manually by counting the number of nuclei within a defined area. Based on green fluorescence in the eyes, transgenic animals were obtained from two independent G0 crosses, indicating a germ line transformation rate of 5.2 % (2/38). Green fluores- cence in the eyes varied depending on the age of the flies, but throughout the lifetime of an adult it was strong enough to distinguish transgenic from wild type. To stably maintain a transgenic insertion, Drosophila genetics offer balancer chro- mosomes, which carry visible markers, suppress genetic re- combination, and are homozygous lethal. In M. abdita, bal- ancer chromosomes are not available. To maintain transgenic lines in M. abdita, flies were inbred and each generation was screened for green fluorescence in the eyes. Using germ line transformation to generate fluorescent nuclear in vivo reporter for M. abdita This corresponds to a mean nuclear density of 1.8 nuclei per 100 μm2, which is very similar to the 2.0 nuclei per 100 μm2 that have been measured in fixed and DAPI-stained material (Wotton et al. 2014). The His2Av-mCherry fusion protein continued to be ubiquitously expressed during later stages of embryonic development, but the signal to noise ratio de- creased, and it was not longer possible to segment nuclei. sufficient nuclear-associated fluorescence to allow for visual and computational segmentation of individual nuclei in the blastoderm. Compared with an analysis based on in vivo bright-field microscopy and fixed specimen (Wotton et al. 2014), we found overall development, the order of events, and the timing between individual developmental events ac- curately recapitulated (Fig. 3, Supplemental Movie 1): in late blastoderm stage, all nuclei in the periphery were elongated and could be distinguished by their shape from the more spherical shape of the pole cells (Fig. 3a, a’); onset of meso- derm internalization was characterized by basally descending nuclei along the ventral midline (Fig. 3b, b’) and was followed about 10 min later by the onset of germband extension, for- mation of the ventral furrow, and a basal nuclear shift in the cephalic furrow initiator cells (Fig. 3c, c’). During germband Fig. 3 His2Av-mCherry expression in M. abdita embryos at late blastoderm stages and during the onset of gastrulation. a–d’ Embryos are shown as mid-sagittal (left column) and transverse sections (right column) in late blastoderm stage (a, a’), at the onset of gastrulation (b, b’), briefly after the onset (c, c’), and during germband extension (d, d’). During late blastoderm stage, nuclei in the periphery were elongated and could be distinguished from the more spherical shape of the pole cells (insets in a’). Onset of cephalic furrow formation was observed after the onset of germband extension (white triangles in c’, d, d’); dorsal folds appeared along the dorsal midline during germband extension (asterisk in d). e Overlay of nuclear positions (gray spheres) extracted by automated image segmentation and raw image of embryo at late blastoderm stage. The embryos showed additional fluorescence in the yolk, which was significantly higher than in non-transgenic flies and suggested that not all of the His2Av-mCherry fusion protein was associated with chromatin. Using germ line transformation to generate fluorescent nuclear in vivo reporter for M. abdita All embryos are shown with anterior to left; scale bar (in e) is 200 μm Based on green fluorescence in the eyes, the two transgenic His2Av/sqh::His2Av-mCherry lines were maintained for over 30 generations. In contrast to expression of the eGFP reporter, however, the expression of the His2Av-mCherry reporter was not stably maintained. After approximately ten generations, we observed continuous decrease in fluorescence indepen- dently in both lines, and after approximately 20 generations, expression of His2Av-mCherry in nuclei had been eventually lost. We were able to detect a single bona fide piggyBac in- sertion in the last maintained transgenic line (Supplemental Fig. 2). His2Av-mCherry expression in this line could not be restored after repeated outcrosses against wild-type flies, sug- gesting that expression loss had not been due to homozygousing of the flies. Expression of His2Av-mCherry could have been lost due to selective silencing of the His2Av- mCherry transgene. Alternatively, our transgenic lines had initially carried multiple piggyBac insertions as previously reported for Bombyx mori (Tamura et al. 2000) and Ceratitis capitata (Handler et al. 1998), and loss of piggyBac insertion copies over time led to the decreasing levels of His2Av- mCherry transgene expression. To avoid a possible loss of piggyBac insertions after germ line transformation, future transgenic lines will need to be screened for eGFP reporter as well as marker gene expression and, from the F1 onward, repeatedly outcrossed against wild type. Inbreeding of indi- vidual lines will then start only after the separation of poten- tially multiple piggyBac insertions. To independently increase overall expression levels of the His2Av-mCherry transgene, future piggyBac insertions could be generated to contain a tandem duplication of the His2Av-mCherry fusion locus. Fig. 3 His2Av-mCherry expression in M. abdita embryos at late blastoderm stages and during the onset of gastrulation. a–d’ Embryos are shown as mid-sagittal (left column) and transverse sections (right column) in late blastoderm stage (a, a’), at the onset of gastrulation (b, b’), briefly after the onset (c, c’), and during germband extension (d, d’). During late blastoderm stage, nuclei in the periphery were elongated and could be distinguished from the more spherical shape of the pole cells (insets in a’). Onset of cephalic furrow formation was observed after the onset of germband extension (white triangles in c’, d, d’); dorsal folds appeared along the dorsal midline during germband extension (asterisk in d). Using germ line transformation to generate fluorescent nuclear in vivo reporter for M. abdita 2a–d); screening for eGFP expression in the larval nervous system was not possible due to autofluorescence of fish food in the gut. Fig. 2 Expression of 3xP3-eGFP in transgenic M. abdita. a–d Heads of M. abdita wild type (wt; a, b) and transgenic fly (tg; c, d) shown with white light (a, c) and fluorescent illumination using a GFP long-pass filter set (488+GFP LP; b, d). Transgenic animals showed fluorescence in ocelli (arrowheads) and ommatidia (brackets, asterisk in d) as reported for D. melanogaster (Horn et al. 2000). Presumably due to the dark pigmentation, fluorescence in the ommatidia is restricted to a small area of the ommatidia that are directly facing the microscope lens. Scale bar (in a) is 0.2 mm Fig. 2 Expression of 3xP3-eGFP in transgenic M. abdita. a–d Heads of M. abdita wild type (wt; a, b) and transgenic fly (tg; c, d) shown with white light (a, c) and fluorescent illumination using a GFP long-pass filter set (488+GFP LP; b, d). Transgenic animals showed fluorescence in ocelli (arrowheads) and ommatidia (brackets, asterisk in d) as reported for D. melanogaster (Horn et al. 2000). Presumably due to the dark pigmentation, fluorescence in the ommatidia is restricted to a small area of the ommatidia that are directly facing the microscope lens. Scale bar (in a) is 0.2 mm To analyze how His2Av/sqh::His2Av-mCherry was expressed in M. abdita, we imaged the transgenic line in a late blastoderm embryo and during the onset of gastrulation using a multi-view light-sheet microscope (Krzic et al. 2012). The M. abdita His2Av/sqh::His2Av-mCherry line showed Dev Genes Evol (2015) 225:179–186 183 extension, a third dorsal fold could be discerned in addition to two previously described transverse folds along the dorsal midline between cephalic furrow and the amnioproctodeal invagination (Rafiqi et al. 2008) (Fig. 3d, d’). The signal to noise ratio of His2Av-mCherry-labeled nuclei during blasto- derm stage was sufficient to apply automated image segmen- tation routines and extract nuclear positions for embryos in the late blastoderm stage. Just before the onset of gastrulation, the M. abdita embryo contained, in total, 4534 nuclei in the pe- riphery, of which 23 nuclei were classified as pole cell nuclei based on their position and spherical appearance (Fig. 3e). Using germ line transformation to generate fluorescent nuclear in vivo reporter for M. abdita e Overlay of nuclear positions (gray spheres) extracted by automated image segmentation and raw image of embryo at late blastoderm stage. The embryos showed additional fluorescence in the yolk, which was significantly higher than in non-transgenic flies and suggested that not all of the His2Av-mCherry fusion protein was associated with chromatin. All embryos are shown with anterior to left; scale bar (in e) is 200 μm piggyBac-mediated germ line transformation in C. riparius To test whether the procedure for germ line transformation in M. abdita was, in principle, applicable to other species within 184 Dev Genes Evol (2015) 225:179–186 Fig. 4 Expression of 3xP3-eGFP in C. riparius. a–d C. riparius wild type (wt; a, b) and transgenic larvae (tg; c, d) shown with white light (a, c) and fluorescent illumination using a GFP long-pass filter set (488+GFP LP; b, d). Transgenic larvae showed fluorescence in the head (bracket in d) and individual segments (arrowheads in d) as reported for 3xP3-eGFP expression in the segmented nervous system of various dipteran larvae (Horn et al. 2000; Ito et al. 2002; Kim et al. 2004; Kokoza et al. 2001). Scale bar (in a) is 0.2 mm the insect order Diptera, we focused on the midge C. riparius as representative for the nematoceran suborder. C. riparius embryos were collected from <1-h-old egg packages, which were gently bleached to disintegrate the gelatinous string enclosing the individual eggs. Embryos were aligned on a glass slide along a capillary, briefly dried, and covered with halocarbon oil. While preliminary mock injections had indi- cated that even young embryos (i.e., 15–60 min after deposi- tion of the egg package) survive physical penetration of the vitelline membrane, the survival rate decreased tremendously when injected with transposon and transposase prior to pole cell formation. All attempts to generate germ line transforma- tion were therefore carried out by injecting C. riparius embry- os at the two-pole-cell stage, or slightly later, into the center of the embryo. Fig. 4 Expression of 3xP3-eGFP in C. riparius. a–d C. riparius wild type (wt; a, b) and transgenic larvae (tg; c, d) shown with white light (a, c) and fluorescent illumination using a GFP long-pass filter set (488+GFP LP; b, d). Transgenic larvae showed fluorescence in the head (bracket in d) and individual segments (arrowheads in d) as reported for 3xP3-eGFP expression in the segmented nervous system of various dipteran larvae (Horn et al. 2000; Ito et al. 2002; Kim et al. 2004; Kokoza et al. 2001). Scale bar (in a) is 0.2 mm Following injection, embryos were kept on the slide and under oil in a moist chamber at 25 °C. piggyBac-mediated germ line transformation in C. riparius After about 3.5 days (i.e., roughly 12 h before injected larvae would hatch), the slides with the embryos were placed in a petri dish and water was slowly added until most of the oil detached from the glass and started to float on the water surface. Surviving embryos typically stayed with their vitelline membrane attached to rem- nants of oil on the glass of the capillary or the slide, and hatched larvae were found usually at the oil/water interface of these droplets. Following hatching, the larvae were trans- ferred with a small loop to a food safe container with pre- aerated water and parsley suspension (see “Material and methods” section). Of 720 injected embryos, 27 larvae hatched (27/720=3.8 %). Because wild-type single crosses set up in 50-ml Falcon tubes showed fertilization rates below 10 %, all G0 adults were instead crossed in one single pool. From this G0 cross, five egg packages were obtained, each of which was let develop in a separate tank. packages, which most likely stem from at least two indepen- dently transformed females present in the pooled G0 cross. Positive larvae were collected and transferred to separate tanks; pupation of these larvae followed after about 3 weeks; and by 4 weeks, latest, all adults had eclosed. F1 adults were intercrossed in a small plastic box, which contained the water tank for egg package deposition. To maintain the line, larvae were screened each generation for eGFP marker expression in the nervous system. Conclusions Using the TTAA piggyBac element, we established protocols for successful germ line transformation in the two dipteran species M. abdita and C. riparius. With a net transformation rate of about 2–5 % per fertile G0 for M. abdita and C. riparius, our results are in the range of piggyBac-mediated germ line transformation events previously reported for other insects (Handler et al. 1998; Lorenzen et al. 2002; Pinkerton et al. 2000), suggesting that the rate of integration for piggyBac is relatively uniform throughout insects. In both our fly species analyzed, the overall survival rate of injected embryos was noticeably low and possibly affected by a high transposase activity provided through mRNA, suggesting that our protocol will benefit from additional fine-tuning of injec- tion conditions. C. riparius adult flies proved sensitive to CO2 and died after minimal exposure. However, as reported previously for D. melanogaster, Aedes aegypti, Anopheles stephensi, and Anopheles gambiae (Horn et al. 2000; Ito et al. 2002; Kim et al. 2004; Kokoza et al. 2001), we were able to observe fluorescence of eGFP in the nervous system of C. riparius larvae during the final instar stage, approximately 2 to 4 days prior to pupation. To identify putative transgenic animals, all F1 larvae were transferred during their last instar stage in batches of two to five individuals into wells of a 24-well plate and screened for eGFP expression in the central nervous sys- tem. From four out of five tanks, we obtained individual larvae showing a strong and specific green fluorescent signal in the larval brain and a segmental pattern throughout the abdomen (Fig. 4a–d). Since all transgenic animals were descendants of a single pooled G0 cross, we conservatively estimated germ line transformation at a rate of 3.7 % (1/27). Similar rates were obtained in two additional and independent experiments. The actual rate may be higher; not all G0 larvae eclosed and we obtained transgenic animals from more than two egg With two new pBacDest vectors, we introduced Gateway variants of the widely used pBac{3xP3-eGFP} vector system, which extends piggyBac-based transgenesis in insects to ligation-free cloning, the fast and efficient assembly of multi- ple fragment constructs based on recombination, and the use of modular Gateway extensions such as Golden GATEway 185 Dev Genes Evol (2015) 225:179–186 Horn C, Schmid BGM, Pogoda FS, Wimmer EA (2002) Fluorescent transformation markers for insect transgenesis. Insect Biochem Mol Biol 32:1221–1235 (Kirchmaier et al. 2013). Conclusions As proof of concept, we have suc- cessfully tested this system in M. abdita by generating a trans- genic line that expresses His2Av-mCherry as fluorescent nu- clear reporter for in vivo time-lapse recordings. 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PLoS ONE 8, e76117 Klomp J, Athy D, Kwan CW, Bloch NI, Sandmann T, Lemke S, Schmidt- Ott U (2015) A cysteine-clamp gene drives embryo polarity in the midge Chironomus. Science 348:1040–1042 Acknowledgments Work on C. riparius was begun when SL was a member of the Schmidt-Ott lab, and we thank Urs Schmidt-Ott for shar- ing unpublished Chironomus protocols and sequence data. We thank Stefan Günther and Lars Hufnagel for help with MuVi-SPIM recordings and Paula Gonzalez for help with Matlab and iLastik, Lucas Schütz for help with IT, and Jochen Wittbrodt for sharing equipment. Conclusions For comments and suggestions on the manuscript, we thank Urs Schmidt-Ott, Annika Guse, and Liz Hambleton. Kokoza V, Ahmed A, Wimmer EA, Raikhel AS (2001) Efficient trans- formation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. 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Insect Mol Biol 11:399–407 Lye CM, Sanson B (2011) Tension and epithelial morphogenesis in Drosophila early embryos. Curr Top Dev Biol 95:145–187 Marinković M, de Leeuw WC, de Jong M, Kraak MHS, Admiraal W, Breit TM, Jonker MJ (2012) Combining next-generation sequencing and microarray technology into a transcriptomics approach for the non-model organism Chironomus riparius. PLoS ONE 7, e48096 References O’Brochta DA, Atkinson PW (1996) Transposable elements and gene transformation in non-drosophilid insects. Insect Biochem Mol Biol 26:739–753 Anderson DT (1966) The comparative embryology of the Diptera. Annu Rev Entomol 11:23–46 Campos-Ortega JA, Hartenstein V (1997) The embryonic development of Drosophila melanogaster. Springer, Berlin Pinkerton AC, Michel K, O’Brochta DA, Atkinson PW (2000) Green fluorescent protein as a genetic marker in transgenic Aedes aegypti. Insect Mol Biol 9:1–10 Cicin-Sain D, Hermoso Pulido A, Crombach A, Wotton KR, Jimenez- Guri E, Taly J-F, Roma G, Jaeger J (2015) SuperFly: a comparative database for quantified spatio-temporal gene expression patterns in early dipteran embryos. Nucleic Acids Res 43:D751–5 Rafiqi AM, Lemke S, Ferguson S, Stauber M, Schmidt-Ott U (2008) Evolutionary origin of the amnioserosa in cyclorrhaphan flies cor- relates with spatial and temporal expression changes of zen. Proc Natl Acad Sci U S A 105:234–239 Davis GK, Patel NH (2002) Short, long, and beyond: molecular and embryological approaches to insect segmentation. Annu Rev Entomol 47:669–699 Rafiqi AM, Lemke S, Schmidt-Ott U (2011) The scuttle fly Megaselia abdita (Phoridae): a link between Drosophila and Mosquito devel- opment. Cold Spring Harb Protoc 2011, pdb.emo143 Handler AM, McCombs SD, Fraser MJ, Saul SH (1998) The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly. Proc Natl Acad Sci U S A 95:7520–7525 Rafiqi AM, Park C-H, Kwan CW, Lemke S, Schmidt-Ott U (2012) BMP- dependent serosa and amnion specification in the scuttle fly Megaselia abdita. Development 139:3373–3382 Horn C, Wimmer EA (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210:630–637 Rebollo E, Karkali K, Mangione F, Martín-Blanco E (2014) Live imaging in Drosophila: the optical and genetic toolkits. Methods 68:48–59 Horn C, Jaunich B, Wimmer EA (2000) Highly sensitive, fluorescent transformation marker for Drosophila transgenesis. Dev Genes Evol 210:623–629 Ritter R (1890) Die Entwicklung der Geschlechtsorgane und des Darmes bei Chironomus. Z Wiss Zool 50:408–427 Dev Genes Evol (2015) 225:179–186 186 Sander K (2000) Chironomid embryology in the 19th century. In Late 20th Century Research on Chironomidae (ed. Hofftrichter, O., pp. 1–16. Aachen: Shaker Verlag Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G et al (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18:81–84 St Johnston D (2013) Using mutants, knockdowns, and transgenesis to investigate gene function in Drosophila. Sander K (2000) Chironomid embryology in the 19th century. In Late 20th Century Research on Chironomidae (ed. Hofftrichter, O., pp. 1–16. Aachen: Shaker Verlag References WIREs Dev Biol 2:587–613 Wiegmann BM, Trautwein MD, Winkler IS, Barr NB, Kim J-W, Lambkin C, Bertone M A, Cassel BK, Bayless KM, Heimberg AM et al (2011) Episodic radiations in the fly tree of life. 108, 5690–5695. Stauber M, Taubert H, Schmidt-Ott U (2000) Function of bicoid and hunchback homologs in the basal cyclorrhaphan fly Megaselia (Phoridae). Proc Natl Acad Sci U S A 97:10844–10849 Strobl F, Stelzer EHK (2014) Non-invasive long-term fluorescence live imaging of Tribolium castaneum embryos. Development 141:2331– 2338 Wotton KR, Jimenez-Guri E, García Matheu B, Jaeger J (2014) A staging scheme for the development of the scuttle fly Megaselia abdita. PLoS ONE 9, e84421
https://openalex.org/W2064698174	https://www.scielo.br/j/jvb/a/vq3v8nzzVFt47mdBDrMJSzp/?lang=en&format=pdf	English	null	Life saving surgery for ruptured pseudo aneurysm of external iliac artery: case report	Jornal Vascular Brasileiro	2,010	cc-by	2,301	Vascular Surgery Unit, Anaesthesia Department, Radiology and Orthopedic Surgery Department of Armed Forces Hospital, Southern Region, Saudi Arabia. 1 Consultant in Vascular Surgery, Armed Forces Hospitals, Southern Region (AFHSR), Saudi Arabia. 2 Consultant in Anaesthesia and Intensive Care Unit AFHSR, Saudi Arabia; Assistant Professor, Anaesthesia Zagazig University, Zagazig, Egypt. 3 Consultant in Anaesthesia and Intensive Care Unit, AFHSR, Saudi Arabia; Assistant professor, Anaesthesia Ain Shams University, Cairo, Egypt. 4 Professor of Diagnostic Radiology, Mansoura University, Mansoura, Egypt. 5 Specialist in Orthopedics, Germany; Consultant Orthopedic and Spinal Surgery, AFHSR, Saudi Arabia. No conflicts of interest declared concerning the publication of this article. Received on Feb 18, 2009 Accepted on Jan 8, 2010 J Vasc Bras. 2010;9(4):241-244. Resumo A incidência de pseudoaneurisma após a artroplastia total de quadril é extremamente rara. O mecanismo mais comum de lesão vascular deve-se ao trauma direto durante o procedimento cirúrgico, e os casos mais relatados são de apresentação aguda. Relatamos um caso incomum de ruptura de pseudoaneurisma e controle de hemorragia intraoperatória com risco de morte da artéria ilíaca externa em um paciente do sexo masculino, de 68 anos, com artroplastia total do quadril deslocada, planejada para remoção, ocorrendo 2 anos depois da última cirurgia de quadril, no Hospital das Forças Armadas, região sul da Arábia Saudita. Este caso destaca a importância do pronto reconhecimento da hemorragia intraoperatória com risco de morte para salvar a vida e o membro do paciente. Palavras-chave: Falso aneurisma; artroplastia; hemorragia. Cirurgia para salvamento de vida após ruptura de pseudoaneurisma da artéria ilíaca: relato de caso Raafat Shalabi1, Wagih Ouda Ahmed2, Samer Mounir Soliman3, Ahmed Yousof Kandeel4, Khaled M Abstract The incidence of pseudo aneurysm after total hip arthroplasty is extremely rare. The most common mechanism of vascular injury is due to direct trauma during the operative procedure, and the most reported cases are acute in presentation. We reported an unusual case of ruptured pseudo aneurysm and control of life-threatening intra-operative hemorrhage of the external iliac artery in a male patient, 68 years old, with displaced total hip arthroplasty (THA), planned for removal, occurring 2 years after the last hip surgery, in Armed Forces hospital, Southern region, Saudi Arabia. This case highlights the importance of prompt recognition of life-threatening intra-operative hemorrhage to save the patient’s life and the limb. Keywords: Aneurysm, false; arthroplasty; hemorrhage. CASE REPORT CASE REPORT Ruptured pseudo aneurysm of external iliac artery - Shalabi R et al. J Vasc Bras 2010, Vol. 9, Nº 4 Ruptured pseudo aneurysm of external iliac artery - Shalabi R et al. 242 and there were no palpable swellings or bruit in the groin. Blood tests showed 9.5 g/dL of hemoglobin, white blood cell count of 7.7/mm and a normal coagulation screen. A radio graph of the right hip showed pelvic bone with displacement of the prosthesis out of its cup into the medial pelvis (Figure 1). Doppler ultrasound scan of the right leg showed expan ded, non-compressible femoral vein, suggestive of deep ve nous thrombosis (DVT). The venogram confirmed iliofemo ral DVT. A retrievable inferior vena cava filter was inserted (Figure 2). The patient was started on low molecular heparin prior to the removal of the THR. Through a standard lateral approach and after opening the wound and the trail to take out the prosthetic femur from its socket in the acetabulum, uncontrollable bleeding was encountered. Immediate control of hemorrhage was achieved by packing. In the meantime, the vascular surgeon was contacted and transfusion of blood was started. and there were no palpable swellings or bruit in the groin. Blood tests showed 9.5 g/dL of hemoglobin, white blood cell count of 7.7/mm and a normal coagulation screen. A radio graph of the right hip showed pelvic bone with displacement of the prosthesis out of its cup into the medial pelvis (Figure 1). Doppler ultrasound scan of the right leg showed expan ded, non-compressible femoral vein, suggestive of deep ve nous thrombosis (DVT). The venogram confirmed iliofemo ral DVT. A retrievable inferior vena cava filter was inserted (Figure 2). The patient was started on low molecular heparin prior to the removal of the THR. Through a standard lateral approach and after opening the wound and the trail to take out the prosthetic femur from its socket in the acetabulum, uncontrollable bleeding was encountered. Immediate control of hemorrhage was achieved by packing. In the meantime, the vascular surgeon was contacted and transfusion of blood was started. Bleeding was noticed from behind the acetabular prosthetic cup. A Foley’s urinary catheter (size 16) was inflated behind the cup and surrounded by gauze pa cks. The pressure and the pulse rate began to improve and the patient became controlled. The orthopedic sur geons started to take out the femoral prosthesis, which was toughly adhered to its surrounding. Case report A 68-year-old Saudi male patient was admitted for re moval of infected total right hip replacement (THR), which was revised in 2000. A chronic hip sinus has developed follo wing the revision surgery with continuous oozing, which, on occasions, was bloody. However, the culture swabs from the exudates were negative. Gradually, symptoms of pain and decreased hip motion have developed. He noticed right leg swelling prior to his admission to our hospital. He had other systemic medical diseases (diabetes mellitus and hyperten sion). During examination, he had normal temperature and non-pitting edema in the right leg. Distal pulses were present Vascular injury secondary to hip surgery is uncommon in that the reported incidence of major vascular injury after surgical procedures on the hip is only 0.25%1. The develo pment of a pseudo aneurysm after total hip arthroplasty (THA) is an extremely rare complication. Most reported cases are acute in onset and are usually due to direct trauma during the operative procedure1. We reported an unusual case of ruptured pseudo aneurysm and control of life-thre atening intra-operative hemorrhage of the external iliac ar tery in a patient with displaced THA, planned for removal, occurring two years after the last hip surgery. Ruptured pseudo aneurysm of external iliac artery - Shalabi R et al. Ruptured pseudo aneurysm of external iliac artery - Shalabi R et al. Discussion Vascular complications associated with THA are re markably rare, making diagnosis and treatment of such sequelae extremely challenging for surgeons who are not familiar with their management (as evidenced by the high rate of limb loss, 70%). Pseudo aneurysms are usually asymptomatic and detected incidentally during surgery or radiographic study, unless infection, local compression on neurovascular structures or rupture oc cur1. The common causes of pseudo aneurysms include trauma, tumor, infection, vasculitis and inflammation, atherosclerosis, infarction, and various iatrogenic com plications, such as those from surgery and angiography2. The mechanism of vascular injury, in most cases, are due to direct trauma during the operative procedure, such as perforation of vessels by retractors, osteotomes, powered reamers, screws, cement or even maneuvers to dislocate hip1. Figure 4 – Right external iliac artery grafted. direct lesion of the arterial wall. Although the acetablar component used in this patient did not have sharp cutting flutes, we believe that the arterial lesions might have been similarly caused by repetitive direct trauma from either the acetablar cup or the implanted femoral head. Rupture of pseudo aneurysms of iliac artery usually demands immediate surgical repair, but surgery invol ves high mortality and morbidity risks, especially for debilitated patients in the emergency setting4. In mana ging these aneurysms, there is a very high periopera tive mortality rate (33 to 50% in emergency surgery; 7 to 11% in elective procedures)5,6. A systematic, planned operative approach is necessary to reduce morbidity and mortality. Injuries causing delayed symptoms are of three types and give rise to symptoms appearing between a few days and several years after the operation: a) pain in the hip caused by pressure of a pseudo aneurysm;f b) ischemic symptoms in the affected limb due to impai red blood flow or distal microembolization; c) severe hemorrhage when extracting a hip prosthesis. The etiology is either a too large volume of cement with intrapelvic spiculae causing thermal damage or erosion of the artery or an intrapelvic dislocation of the socket with pressure and angulations of the artery3. Ruptured pseudo aneurysm of external iliac artery - Shalabi R et al. Infra-inguinal exploration of the proximal part of the common femoral artery and a retroperitoneal exploration of the right ex ternal iliac artery were carried out. No apparent bleeding was seen. The clamping of the external iliac artery was carried out and the acetabular cup was removed from the acetabular space. A false capsule and granulation tissues were seen avulsed from the surroundings. After dissecting circumferentially the external iliac artery and the proximal part of the right common femoral artery, a posterior wall rounded hole was seen. After examining it and the lumen of the artery, there was an intimal dis section with old subintimal dissection of 10-20 mm pro ximal and distal to the arterial hole. An interposition of 6 mm Dacron graft was carried out by the use of 5/0 no absorbable prolene stitches for anastomosis.h Figure 1 – Right total hip arthroplasty displaced. The pulse regained palpable at the right posterior ti bial and dorsalis pedis arteries. The patient’s postopera tive course was uneventful until the fifth postoperative day. The patient started to complain of lower abdominal pain. Abdominal and pelvic ultrasonography was carried out, which revealed massive retroperitoneal hematoma. Patient’s hemodynamics and hemoglobin level did not change. Computerized tomography with contrast showed a big retroperitoneal hematoma communicating with the la teral wound of the removed prosthesis (Figure 3). The graft was intact and no signs of abnormality were seen associated Figure 1 – Right total hip arthroplasty displaced. Figure 2 – Computerized tomography with contrast showing inferior vena cava filter. Figure 3 – CTA. Right hip hematoma. Figure 2 – Computerized tomography with contrast showing inferior vena cava filter. Figure 3 – CTA. Right hip hematoma. Figure 3 – CTA. Right hip hematoma. Ruptured pseudo aneurysm of external iliac artery - Shalabi R et al. J Vasc Bras 2010, Vol. 9, Nº 4 243 with it (Figure 4). The patient was observed for two weeks until the skin stapler clips were removed. The follow-up was carried out monthly for over six months. The right pedal pulses were well palpable and there were no signs of graft infection or thrombosis. Figure 4 – Right external iliac artery grafted. Conclusion Rupture of pseudo aneurysms of iliac artery usually demands immediate surgical repair. Awareness of this rare complication, prompt diagnosis and immediate treatment are key factors in saving the lives of such patients.h Unlike most reported cases, our patient did not deve loped symptoms of pseudo aneurysm since his last surgery. The sequence of the most likely events began with the screw in the acetablar cup at the time of the last surgery, two years ago. The patient developed iliofemoral DVT from the com pression of the external iliac artery pseudo aneurysm. This case demonstrated that a pseudo aneurysm can manifest as an acute presentation secondary to direct in jury during a surgical procedure. It can appear late and be caused by repetitive trauma from arthroplasty components. If a pseudo aneurysm is suspected, an angiogram should be performed, followed by appropriate treatment as soon as possible. Rengsen et al.1 reported an injury to the external iliac artery from an acetablar cup, which resulted in formation of a pseudo aneurysm. They believed that the threaded acetablar cup with sharp cutting flutes might have caused Ruptured pseudo aneurysm of external iliac artery - Shalabi R et al. J Vasc Bras 2010, Vol. 9, Nº 4 244 References Correspondence: Raafat Shalabi, MD Armed Forces Hospital, Southern Region, Saudi Arabia Tel: +966-566874773, +966-7-2500001 ext.: 2552 Fax: +966-7-2571699 E-mail: raafatshalabi@hotmail.com Author contributions Conception and design: RS, WOA, SMS, AYK, KMAA Analysis and interpretation: RS, WOA, SMS, AYK, KMAA Data collection: RS, WOA, SMS, AYK, KMAA Writing the article: RS, WOA, SMS, AYK, KMAA Critical revision of the article: RS, WOA, SMS, AYK, KMAA Final approval of the article: RS, WOA, SMS, AYK, KMAA Statistical analysis: RS, WOA, SMS, AYK, KMAA Overall responsibility: RS, WOA, SMS, AYK, KMAA Obtained funding: RS, WOA, SMS, AYK, KMAA All authors have read and approved the final version of the article submitted to J Vasc Bras. Correspondence: Raafat Shalabi, MD Armed Forces Hospital, Southern Region, Saudi Arabia Tel: +966-566874773, +966-7-2500001 ext.: 2552 Fax: +966-7-2571699 E-mail: raafatshalabi@hotmail.com 1. Rengsen P, Abbas AA, Choon SK, Tai CC. Pseudoaneurysm of ex ternal iliac artery following septic loosening of total hip arthroplas ty. MOJ. 2007;1:42-4. 2. Huang WY, Huang CY, Chen CA, Hsieh CY, Cheng WF. Ruptured pseudoaneurysm of the external iliac artery in an advanced cer vical cancer patient treated by endovascular covered stent place ment. J Formos Med Assoc. 2008;107:348-51. Author contributions Conception and design: RS, WOA, SMS, AYK, KMAA Analysis and interpretation: RS, WOA, SMS, AYK, KMAA Data collection: RS, WOA, SMS, AYK, KMAA Writing the article: RS, WOA, SMS, AYK, KMAA Critical revision of the article: RS, WOA, SMS, AYK, KMAA Final approval of the article: RS, WOA, SMS, AYK, KMAA Statistical analysis: RS, WOA, SMS, AYK, KMAA Overall responsibility: RS, WOA, SMS, AYK, KMAA Obtained funding: RS, WOA, SMS, AYK, KMAA All authors have read and approved the final version of the article submitted to J Vasc Bras. 3. Bergqvist D, Carlsson AS, Ericsson BF. Vascular complications after total hip arthroplasty. Acta Orthop Scand. 1983;54:157-63. 4. Sanada J, Matsui O, Arakawa F, et al. Endovascular stent-grafting for infected iliac artery pseudoaneurysms. Cardiovasc Intervent Radiol. 2005;28:83-6. 5. Richardson JW, Greenfield LJ. Natural history and management of iliac aneurysms. J Vasc Surg. 1988;8:165-71. 6. Brunkwall J, Hauksson H, Bengtsson H, Bergqvist D, Takolander R, Bergentz SE. Solitary aneurysms of the iliac arterial system: an esti mate of their frequency of occurrence. J Vasc Surg. 1989;10:381-4.
https://openalex.org/W2124419982	https://europepmc.org/articles/pmc4228421?pdf=render	English	null	Path2Models: large-scale generation of computational models from biochemical pathway maps	BMC systems biology	2,013	cc-by	15,206	© 2013 Büchel 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. RESEARCH ARTICLE Open Access Path2Models: large-scale generation of computational models from biochemical pathway maps Finja Büchel1,2†, Nicolas Rodriguez1,3†, Neil Swainston4†, Clemens Wrzodek2†, Tobias Czauderna5, Roland Keller2, Florian Mittag1,2, Michael Schubert1, Mihai Glont1, Martin Golebiewski6, Martijn van Iersel1, Sarah Keating1, Matthias Rall2, Michael Wybrow7, Henning Hermjakob1, Michael Hucka8, Douglas B Kell4,9, Wolfgang Müller6, Pedro Mendes4,10,11, Andreas Zell2, Claudine Chaouiya12, Julio Saez-Rodriguez1, Falk Schreiber5,13, Camille Laibe1, Andreas Dräger2,14 and Nicolas Le Novère1,3* Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Abstract Background: Systems biology projects and omics technologies have led to a growing number of biochemical pathway models and reconstructions. However, the majority of these models are still created de novo, based on literature mining and the manual processing of pathway data. Results: To increase the efficiency of model creation, the Path2Models project has automatically generated mathematical models from pathway representations using a suite of freely available software. Data sources include KEGG, BioCarta, MetaCyc and SABIO-RK. Depending on the source data, three types of models are provided: kinetic, logical and constraint-based. Models from over 2 600 organisms are encoded consistently in SBML, and are made freely available through BioModels Database at http://www.ebi.ac.uk/biomodels-main/path2models. Each model contains the list of participants, their interactions, the relevant mathematical constructs, and initial parameter values. Most models are also available as easy-to-understand graphical SBGN maps. Conclusions: To date, the project has resulted in more than 140 000 freely available models. Such a resource can tremendously accelerate the development of mathematical models by providing initial starting models for simulation and analysis, which can be subsequently curated and further parameterized. Keywords: Modular rate law, Constraint based models, Logical models, SBGN, SBML * Correspondence: lenov@babraham.ac.uk †Equal contributors 1European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge, UK 3Babraham Institute, Babraham Research Campus, Cambridge, UK Full list of author information is available at the end of the article Background result in changes in the concentration, state or location of chemical entities. Pathways aim at providing a detailed representation of this biochemical reality, based on obser- vations of the reactions. As such, the elucidation of bio- chemical pathways is being dramatically sped up with the efforts of molecular biology and biochemistry research, and particularly with the recent appearance of high- throughput omics technologies. Since the discovery of the set of biochemical transforma- tions known as the Embden-Meyerhof-Parnas glycolysis pathway in the early twentieth century, the concepts of pathways and networks have become useful and ubiqui- tous tools in the understanding of biochemical processes. Biochemical pathways provide a qualitative representation of chains of molecular interactions and chemical reactions that are known to take place in cells. Such interactions The definition of biochemical pathways is largely arbi- trary, as in practice they are interlinked and interdepend- ent in the functioning cell. Nevertheless, it is convenient to partition these pathways into different types such as signaling pathways, metabolic networks, gene regulatory networks, etc. With the growing number and complexity of biochemical pathways, a number of public databases * Correspondence: lenov@babraham.ac.uk †Equal contributors 1European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge, UK 3Babraham Institute, Babraham Research Campus, Cambridge, UK Full list of author information is available at the end of the article Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 2 of 19 Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Quantitative methods for modeling biological net- works require accurate knowledge of the biochemical re- actions, their stoichiometric and kinetic parameters, and in the case of metabolic pathway modeling [11], initial concentrations of metabolites [12] and enzymes [13]. In many cases, such experimentally derived parameters are unavailable. This has led to the development of several qualitative approaches, based on influence networks ra- ther than process descriptions. Examples are logical modeling in multiple variants, from Boolean or multi- valued networks [14-16] to discrete algebra [17] and dif- ferential equations [18], Petri nets [19] and predicate logic [20]. Qualitative models typically refer to regulatory or signaling networks, and are based on the definition of an influence or signal-flow graph, rather than the depic- tion of consumption and production of pools of entities. These methods have proven useful in recent years in the interpretation of data from perturbation experiments, phosphoproteomics and gene expression studies [21]. Background SBML has recently been extended to support such lo- gical models, which can be encoded with the newly in- troduced Qualitative Models package for SBML Level 3 (henceforth abbreviated as the SBML qual package [22]) and represented in the Activity Flow language of SBGN. have attempted to catalog them and provide access to their computational representation. These well-curated resources include MetaCyc [1], KEGG [2], the Nature Pathway Interaction Database (PID) [3], Reactome [4] and WikiPathways [5]. While such resources remain extremely useful, they pro- vide purely qualitative, static, representations of molecular interactions. Although such representations can be used in the context of experimental data mapping and interpret- ation [6], they fail to provide a quantitative understanding of cellular mechanisms. A key to the understanding of bio- logical processes is to go beyond mere accumulation of ob- servations, even on the large scale as in multi-omics data collection, and to move towards their quantitative predic- tion. This understanding can in turn lead to the alteration of biological processes, for instance through pharmaceutical intervention, and even to the design of entirely novel pro- cesses in the fields of metabolic engineering and synthetic biology. Accordingly, over the last decade and a half, the in- creased availability of quantitative experimental data has motivated scientists to develop predictive and quantitative representations of pathways and entire networks in the form of computational models. Computational models rely on mathematical frame- works to describe the structures and behaviors of systems. A model consists of variables, functions and constraints. Different types of models exist, such as kinetic models, lo- gical models, rule-based models, multi-agent models, stat- istical models and many more. In contrast to most pathways, which seek to provide detailed representations of biochemical knowledge, models can be more abstract representations of the reality, depending on the needs of the modeler, the experimental data available and the inves- tigation being undertaken. Models can therefore exhibit different levels of granularity for the variables and different degrees of precision for the mathematical functions. Computational models of biochemical systems are shared through databases such as BioModels Database [7] and the CellML repository [8], with their storage and exchange relying heavily on the adoption of standard formats such as the Systems Biology Markup Language (SBML [9]) and the Systems Biology Graphical Notation (SBGN [10]). Background In addition to curated pathway databases, the availabil- ity of well-annotated entire genomes, together with methods for reconstructing and constraining large-scale biochemical networks, has led to the reconstruction of comprehensive metabolic pathways, including all enzymes known to be encoded by an organism. The development of these genome-scale metabolic network reconstructions, and their analysis through constraint-based modeling ap- proaches, is becoming increasingly widespread in driving the understanding of metabolism in a diverse range of organisms. The number of such genome-scale metabolic reconstructions published over the last ten years has grown considerably, with over 50 such reconstructions re- cently reported [23], covering a range of single- and multi- cellular organisms. Metabolic reconstructions attempt to provide a com- putational and mathematical representation of the meta- bolic capabilities of the cell. Reconstructions have been used in a number of research topics including metabolic engineering, genome-annotation, evolutionary studies, network property analysis, and interpretation of omics datasets [24]. The development of genome-scale meta- bolic reconstructions typically involves a labor-intensive, manual process, with timescales of up to two years re- ported for their production [25]. While it is recognized that the development of high-quality metabolic recon- structions requires significant curation, and is dependent upon manual [26-30] or semi-automated literature min- ing [31,32], there have been notable recent steps towards semi-automation of the reconstruction process, which Different types of models can be generated from path- way databases. Biochemistry, and in particular metabol- ism, is very often represented using process descriptions. Processes are the biochemical reactions and transport pro- cesses between compartments that transform nominally homogeneous pools of biochemical entities into other pools of entities. In process descriptions, a pathway is a bi- partite graph formed of the biochemical entities and the processes that consume or produce them. Models based on process descriptions can be encoded with the elements of SBML Core and represented in the Process Description language of SBGN [10]. Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 3 of 19 aim to reduce the number of tasks that must be per- formed manually. Information Required In the Annotation of Models (MIRIAM) specification [33]. In practice, this means that all components of the models (metabolites, genes, enzymes, reactions, etc.) are tagged with unambiguous identifiers from publicly available, third party databases. The models can therefore be easily queried, compared, merged and ex- panded, and are immediately amenable to integration with experimental data [34]. Background The resulting models are made publicly available through BioModels Database [7] and can be used as starting point for further development. Traditionally, computational models have been pains- takingly (and manually) built from primary information obtained from the literature and from dedicated experi- ments. Because of the increasing size and complexity of these models, this approach is no longer sustainable. Mod- elers have therefore begun to build models directly based on data imported from pathway databases. However, until recently, this has mostly been done on a tedious case-by- case basis and repeated separately by different researchers because the results were not shared in a consistent fashion. The Path2Models project attempts to mitigate this often duplicated initial modeling step by generating computa- tional models from pathways on a large scale, applying con- sistent, community-developed and well-supported data formats, and to make the results available to the commu- nity as a whole. Workflow from biochemical pathways to computational models In order to generate computational models from bio- logical pathways on a large scale, a software pipeline composed of several steps that can be run sequentially or in parallel was developed (Figure 1). The pathways must first be converted from their original format to a standard computer-readable format, which will be used through- out all subsequent steps of the pipeline. This work de- scribes the conversion of pathway information from KEGG, MetaCyc, and BioPAX [35] into SBML models, lacking both mathematics and numerical values. These preliminary networks were then processed to annotate, merge, extend and complete them with mathematical expressions where possible. All software modules utilized in this work are freely distributed, and readers can re-use them on their own or within their own workflows. This manuscript therefore describes the conversion of pathway information to computational models in a con- sistent and high-throughput manner. The Path2Models project has generated three types of models: quantitative, kinetic models of metabolic pathways; qualitative, logical models of non-metabolic (primarily signaling) pathways; and genome-scale metabolic reconstructions. The models are generated in SBML, and in many cases are augmented with visual representations in the form of SBGN docu- ments. All of the models share a consistent format and are semantically annotated according to the Minimum Figure 1 Workflow leading from pathway descriptions to computational models. From the pathway databases on the left, information is extracted and encoded in SBML. Mathematical features, such as kinetic rate equations and flux bounds, are then added to each model, along with a graphical description. The completed models are all distributed through the BioModels Database. See Methods for a detailed explanation of each step. Figure 1 Workflow leading from pathway descriptions to computational models. From the pathway databases on the left, information is extracted and encoded in SBML. Mathematical features, such as kinetic rate equations and flux bounds, are then added to each model, along with a graphical description. The completed models are all distributed through the BioModels Database. See Methods for a detailed explanation of each step. Figure 1 Workflow leading from pathway descriptions to computational models. From the pathway databases on the left, information is extracted and encoded in SBML. Mathematical features, such as kinetic rate equations and flux bounds, are then added to each model, along with a graphical description. The completed models are all distributed through the BioModels Database. Workflow from biochemical pathways to computational models See Methods for a detailed explanation of each step. Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 4 of 19 maps, in order to provide defined graphical representa- tions of all models (Figure 2). Three parallel pipelines of data processing were imple- mented: 1) kinetic metabolic models represented by pro- cesses were encoded in SBML Level 3 Core format, enriched with modular rate-laws and depicted using SBGN Process Descriptions; 2) qualitative metabolic and non- metabolic (mostly signaling) pathways, represented as influ- ence diagrams, were encoded in SBML using the Level 3 qual package, in a form ready for logical modeling and depicted using SBGN Activity Flows; 3) genome-scale me- tabolism reconstructions were similarly encoded in SBML, in a format amenable to constraint-based modeling. Reconstructions of metabolic networks were com- pleted by the addition of experimentally determined rate laws and parameter values from the SABIO-RK database [36]. SABIO-RK is a reaction-kinetics database that con- tains experimentally obtained rate laws for a large collec- tion of (bio-) chemical reactions, including measured parameter values and experimental conditions, such as the pH value or the temperature, under which the rate was measured [37]. It was therefore desirable to extract as much information from SABIO-RK as possible and relevant. For all reactions that lacked corresponding en- tries in SABIO-RK, the kinetic rate laws were inferred ab initio (see Methods). At the moment, the SABIO-RK database mainly focuses on a selection of relevant model organisms, for which many rate laws can already be ex- tracted (see Figure 3), for instance, 12% for Homo sapi- ens, 10% for Rattus norvegicus, and 8% for Escherichia coli. Across the full range of organisms we considered, 6204 reactions (0.22%) could be equipped with rate laws from SABIO-RK. Generation of quantitative kinetic process models from metabolic pathways The metabolic pathways distributed by KEGG are de- scribed in terms of processes, and formed the basis of the process-based reconstructions. 112 898 maps describing up to 154 metabolic pathways in 1 514 organisms were converted into process description models encoded in SBML Level 3 Core. The resulting SBML documents were converted into SBGN Process Descriptions (PD) Figure 2 SBGN Process Description map of a pathway, cutout of the pathway and parts of the SBML file describing the reactions shown in the cutout. Process Description map of a pathway, cutout of the pathway and parts of the SBML file describing the reactions tout. Figure 2 SBGN Process Description map of a pathway, cutout of the pathway and parts of the SBML file describing the reactions shown in the cutout. Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 5 of 19 Page 5 of 19 Figure 3 Rate equations from SABIO-RK for models from selected organisms. Figure 3 Rate equations from SABIO-RK for models from selected organisms. Generation of qualitative models from signaling pathways From the KEGG pathway database, 27 306 maps de- scribing 167 non-metabolic pathways in 1 514 organisms were converted into influence maps models encoded with the SBML Level 3 qual package. transition (for instance, a phosphorylation can increase or decrease the activity of a protein). In those cases, the sign attribute was initially set to unknown for the input element of the corresponding transition. Whenever possible, the KEGG pathways were augmented with interaction informa- tion imported from the BioCarta pathways distributed by the Nature Pathway Interaction Database (PID) [3]. PID provides human pathways in the BioPAX format Level 3, which specifies a ControlType attribute for each interaction. The ControlType attribute determines whether the inter- action represents activation or inhibition. With the add- itional information from the PID, it was possible to extend 35 human pathways. Generation of qualitative models from signaling pathways From the KEGG pathway database, 27 306 maps de- scribing 167 non-metabolic pathways in 1 514 organisms were converted into influence maps models encoded with the SBML Level 3 qual package. Prior to our use to convert non-metabolic pathways, no attempt had been made to encode pathway models using the SBML qual syntax. Generation of quantitative kinetic process models from metabolic pathways We uncovered several as- pects of the package specification that caused problems when applied to actual pathways and the project pro- vided a valuable concrete situation to help resolve these issues. For example, the information available originally permitted the description of interaction graphs but was not sufficient to define logical rules specifying the effects of combined interactions. This led to the introduction of a sign attribute for indicating whether a given inter- action has a positive, negative or unknown effect. This can then be used as a constraint to parameterize a lo- gical model further. The project therefore accelerated the development and finalization of the SBML Level 3 qual specification. Genome-scale metabolic reconstructions Genome-scale metabolic reconstructions of 2 630 organ- isms were generated through extraction of pathway data from the KEGG and MetaCyc databases using an up- dated version of the pre-existing software libAnnota- tionSBML and the SuBliMinaL Toolbox [38,39]. All reconstructions contain data from KEGG, and many of these have been augmented with data from MetaCyc for the corresponding organism. In each case, MNXref was used to reconcile metabolite and reaction identifiers across the different data resources [40]. As well as KEGG relations sometimes consist exclusively of the sub- types phosphorylation, dephosphorylation, glycosylation, ubiquitination, or methylation. These relations cannot be interpreted in terms of positive or negative influences on a Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 6 of 19 providing mapping of KEGG and MetaCyc identifiers, MNXref also applies a default metabolite formula and charge state according to an assumed pH of 7.3, and ensures mass and charge balancing of reactions where pos- sible. Furthermore, MNXref provides mapping to additional identifiers, which have been extracted and incorporated into the collection of genome-scale reconstructions. As such, as well as ensuring consistent metabolite and reaction identifiers across all 2 630 reconstructions, all models also contain identifier cross references to numerous commonly used resources, including BiGG [41] and the Model SEED [42], further enhancing their interoperability. Figure 4 Workflow indicating the SuBliMinaL Toolbox modules that were linked to produce draft metabolic models from the source data. KEGG extract and MetaCyc extract produce MIRIAM-annotated SBML representations of the contents of KEGG and MetaCyc, respectively. Metabolite and reaction ids are reconciled through reference to the MNXref namespace, unifying the metabolites to an assumed intracellular pH of 7.3, and mass and charge balancing reactions where possible. The Merge module merges the individual reconstructions from KEGG and MetaCyc, to which a limited growth medium and transport reactions are added, along with gene-protein relationships (GPRs) and flux bounds. The models are then formatted to allow for their analysis with the COBRA Toolbox and then released as draft models that represent the union of the information held in both KEGG and MetaCyc. A minimal growth medium (consisting of a single car- bon source, glucose), appropriate transport reactions, and 30 common biomass components were specified in each model, including all 20 amino acids, RNA and DNA nucleotide precursors, glycogen and ATP (see Methods). Genome-scale metabolic reconstructions KEGG extract and MetaCyc extract produce MIRIAM-annotated SBML representations of the contents of KEGG and MetaCyc, respectively. Metabolite and reaction ids are reconciled through reference to the MNXref namespace, unifying the metabolites to an assumed intracellular pH of 7.3, and mass and charge balancing reactions where possible. The Merge module merges the individual reconstructions from KEGG and MetaCyc, to which a limited growth medium and transport reactions are added, along with gene-protein relationships (GPRs) and flux bounds. The models are then formatted to allow for their analysis with the COBRA Toolbox and then released as draft models that represent the union of the information held in both KEGG and MetaCyc. source data. KEGG extract and MetaCyc extract produce MIRIAM-annotated SBML representations of the contents of KEGG and MetaCyc, respectively. Metabolite and reaction ids are reconciled through reference to the MNXref namespace, unifying the metabolites to an assumed intracellular pH of 7.3, and mass and charge balancing reactions where possible. The Merge module merges the individual reconstructions from KEGG and MetaCyc, to which a limited growth medium and transport reactions are added, along with gene-protein relationships (GPRs) and flux bounds. The models are then formatted to allow for their analysis with the COBRA Toolbox and then released as draft models that represent the union of the information held in both KEGG and MetaCyc. This resource allows biologists to store, search, retrieve and display mathematical models. One of the main qual- ities of the repository lies in its contents: all are distributed in standard formats and using a free license, allowing easy re-use. The models generated by the project have been made publicly available from BioModels Database since release 22 under the name “Path2Models” [47]. The size of the distribution of all these models is presented in Figure 7. A new branch in the model-processing pipeline The full results of this study are provided in a defini- tive list of all models produced in Additional file 1: Table S1. The results can also be viewed as a phylogenetic tree, generated by the Integrated Tree Of Life (iTOL) web ap- plication [45], at [46] (see Figures 5 and 6). This resource allows biologists to store, search, retrieve and display mathematical models. One of the main qual- ities of the repository lies in its contents: all are distributed in standard formats and using a free license, allowing easy re-use. Genome-scale metabolic reconstructions The models generated by the project have been made publicly available from BioModels Database since release 22 under the name “Path2Models” [47]. The size of the distribution of all these models is presented in Figure 7. A new branch in the model-processing pipeline Genome-scale metabolic reconstructions A default biomass objective function was added, containing these components, with the intention of facilitating subsequent analysis and curation. The models were then formatted such that they could be an- alyzed with a range of SBML-compatible software tools, including the COBRA Toolbox [43,44]. Figure 4 de- scribes the workflow that was used in the automated re- construction process. The resulting 2 630 models range in size from the smal- lest, Candidatus Tremblaya princeps PCVAL, containing 131 metabolites and 63 metabolic reactions, to Homo sapi- ens, with 3 270 metabolites and 3 416 metabolic reactions. All models were analyzed for their ability to synthesize each defined biomass precursor from the minimum growth medium, taking into account reaction directionalities speci- fied in KEGG and/or MetaCyc where available. Of these, only the model of Drosophila melanogaster was able to synthesize all specified 30 biomass components. The Homo sapiens model was incapable of synthesizing the amino acids cysteine, histidine, isoleucine, leucine, lysine, methio- nine, threonine, tryptophan and valine. Of these, all but cysteine are known essential amino acids. Additionally, the model is unexpectedly able to synthesize phenylalanine, an essential amino acid. Nevertheless, these analysis results in- dicate that the draft model is largely predictive of the amino acid essentiality, with the anomalies of cysteine and phenyl- alanine synthesis pathways providing starting points for manual curation. Figure 4 Workflow indicating the SuBliMinaL Toolbox modules that were linked to produce draft metabolic models from the source data. KEGG extract and MetaCyc extract produce Figure 4 Workflow indicating the SuBliMinaL Toolbox modules that were linked to produce draft metabolic models from the source data. KEGG extract and MetaCyc extract produce MIRIAM-annotated SBML representations of the contents of KEGG and MetaCyc, respectively. Metabolite and reaction ids are reconciled through reference to the MNXref namespace, unifying the metabolites to an assumed intracellular pH of 7.3, and mass and charge balancing reactions where possible. The Merge module merges the individual reconstructions from KEGG and MetaCyc, to which a limited growth medium and transport reactions are added, along with gene-protein relationships (GPRs) and flux bounds. The models are then formatted to allow for their analysis with the COBRA Toolbox and then released as draft models that represent the union of the information held in both KEGG and MetaCyc. Figure 4 Workflow indicating the SuBliMinaL Toolbox modules that were linked to produce draft metabolic models from the source data. Access to the resulting knowledge base BioModels Database is the reference repository of compu- tational models of biological interest encoded in SBML. Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 7 of 19 Figure 5 Phylogenetic tree illustrating all 2 630 genome-scale metabolic models. The tree is color coded, indicating the presence of archaea, bacteria and eukaryota in the collection. Analysis results of each model are displayed, with bars indicating the number of metabolic reactions, metabolites, makeable metabolites and makeable biomass components in blue, red, purple and green respectively. In this illustration, the bars have been scaled for ease of visualization. Figure 5 Phylogenetic tree illustrating all 2 630 genome-scale metabolic models. The tree is color coded, indicating the presence of archaea, bacteria and eukaryota in the collection. Analysis results of each model are displayed, with bars indicating the number of metabolic reactions, metabolites, makeable metabolites and makeable biomass components in blue, red, purple and green respectively. In this illustration, the bars have been scaled for ease of visualization. essentially the same as for building an individual model from the same data. However, instead of independent scientists enacting this procedure again and again as the needs arise, the initial data processing is performed in bulk. Scientists can then focus on the more interesting tasks of adapting the models to their questions, adding initial conditions and parameter values, and running simulations to answer biological questions in the organ- isms and/or pathways in which they are interested. was created in order to accommodate those models, as they are not expected to go through the usual manual cur- ation and annotation phases. A dedicated search infra- structure for the Path2Models branch was provided with release 23. Figure 8 presents the relative populations of the different topics, as compiled from the Gene Ontology annotation of the models. The Path2Models branch of BioModels Database is not considered to be a frozen re- source, and improved versions will be released as they are made available. The added value provided by the initial models to such research activities largely depends on the quality of those models. True errors, such as erroneous reactions, can produce misleading results. Incompleteness increases the need for completion and refinement. Incorrect syn- tax makes it more difficult to re-use the initial models with existing software tools. In the end, all of these Discussion Automatically generated models are only a starting point The workflow described here enables the automatic gen- eration of a large number of computational models from existing pathway data resources. The procedure is Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 8 of 19 Figure 6 A zoomed in view of the eukaryotic branch of the phylogenetic tree of Figure 5. The online iTOL web application version of the tree, available at [40], allows for zooming, searching and visualization of the tree and its associated statistics. Figure 6 A zoomed in view of the eukaryotic branch of the phylogenetic tree of Figure 5. The online iTOL web application version of the tree, available at [40], allows for zooming, searching and visualization of the tree and its associated statistics. homology, which can lead to missing reactions and there- fore disconnected graphs. issues translate into greater workload and time loss for the user. However, the quality of the models produced by the workflow crucially depends on the accuracy and completeness of the sources of information. If the path- way data are incorrect, there is little that an automatic conversion system can do beyond checking for feasible stoichiometries, mass and charge conservation and the like. Similarly, if some biological information is missing, the pathway-to-model workflow cannot easily create it. An example of this is information about compartmentalization. If the localization of the pathway nodes is not speci- fied in the initial data, the resulting models will have a single compartment containing all molecular species. Systematic generation of genome-scale metabolic reconstructions from existing data resources While the generation of genome-scale metabolic recon- structions typically relies upon time-consuming and manual efforts, techniques are being introduced which attempt to automate at least part of the process. One such approach to semi-automated reconstruction of such networks is that of the Model SEED [42]. This method provides a web-based resource for the gener- ation of genome-scale metabolic reconstructions from assembled genome sequences. It has resulted in the gen- eration of 130 (reported) reconstructions of a range of bacterial species, and has the potential for generating many more. While an approach that allows for the auto- mated generation of reconstructions directly from the genome will clearly grow in importance given the ever- increasing volume of sequencing data, it is also clear that existing, curated data resources such as MetaCyc and KEGG still provide a great deal of biochemical know- ledge that can be exploited in the metabolic reconstruc- tion process. Many reconstruction projects take existing pathway databases such as these as a starting point, and indeed, recently introduced software tools such as the Figure 7 presents the size of the models produced by the project, in terms of number of state variables and number of mathematical relationships (i.e., reactions and transitions). The whole genome reconstructions present similar distributions for variables and relationships (Figure 7A). The situation is similar to the curated branch of BioModels Database (Figure 7D), which fea- tures models capable of numerical simulation. In con- trast, the individual metabolic pathways (Figure 7C) are severely underdetermined, with many more variables than relationships. A possible reason for this is that en- tities in KEGG pathways are inferred by gene/enzyme Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 9 of 19 Page 9 of 19 Figure 7 Distribution of the models generated by the project according to their size, in terms of the number of molecular species (blue) and the number of mathematical relationships – i.e. reactions, transitions, rules etc. (salmon) in each class. A-C: the whole genome reconstructions, qualitative models, and chemical kinetic models. D-E: the curated and non-curated literature-based branches of the BioModels Database. Figure 7 Distribution of the models generated by the project according to their size, in terms of the number of molecular species (blue) and the number of mathematical relationships – i.e. reactions, transitions, rules etc. (salmon) in each class. A-C: the whole genome reconstructions, qualitative models, and chemical kinetic models. Systematic generation of genome-scale metabolic reconstructions from existing data resources D-E: the curated and non-curated literature-based branches of the BioModels Database. Figure 7 Distribution of the models generated by the project according to their size, in terms of the number of molecular species (blue) and the number of mathematical relationships – i.e. reactions, transitions, rules etc. (salmon) in each class. A-C: the whole genome reconstructions, qualitative models, and chemical kinetic models. D-E: the curated and non-curated literature-based branches of the BioModels Database. Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 10 of 19 Figure 8 Relative sizes of the different classes of models, based on their main Gene Ontology (GO) annotations. The GO terms annotating the SBML Model element for each model generated by the project were collected, and clustered to generate groups of models covering (what are considered therefrom to be) the same domain of biology. Figure 8 Relative sizes of the different classes of models, based on their main Gene Ontology (GO) annotations. The GO terms annotating the SBML Model element for each model generated by the project were collected, and clustered to generate groups of models covering (what are considered therefrom to be) the same domain of biology. RAVEN Toolbox [48] have followed the examples set by the SuBliMinaL Toolbox [39] and KEGGtranslator [49] in automating the generation of models from KEGG. approaches [51]. The modular rate laws suggested by Liebermeister et al. [52] have been specifically derived for cases in which more precise information remains elusive. This work describes the first example in which an auto- mated model reconstruction tool has been systematically applied to a wide range of organisms on such a scale. The result of this is the largest collection of genome-scale metabolic reconstructions to date. Due to their common formatting, use of identifiers and semantic annotations, the collection provides both a useful starting point for subsequent manual and semi-automated curation, and, as can be seen in the phylogenetic tree of Figure 5, a framework upon which metabolism can be systematically compared across species. Each modular rate law can be used in three different modes or versions, which increase in complexity from the explicit (cat), through the Haldane-compliant (hal), to the Wegscheider-compliant (weg) version. These ver- sions determine the form of the numerator in the equa- tion (see Methods). A parsimonious approach was chosen in this work, where only as much complexity as necessary was introduced. Systematic generation of genome-scale metabolic reconstructions from existing data resources Therefore, the most simple cat version of these rate laws was selected for all reversible reactions, even if this equation might not guarantee thermodynamic correctness. If the models created by this approach are used as the basis for subsequent calibration by experimental data, use of the cat version has two im- portant advantages: (i) it contains a small number of pa- rameters with uncertain values; and (ii) it has a low complexity in comparison to the hal or the weg version, with consequences on runtime. It should be noted that Liebermeister et al. have suggested an algorithm for transforming the parameter values of complex versions of the modular rate laws to the nearest simple form. It is possible to compute thermodynamically correct cat-pa- rameters based on randomly selected weg-parameters through an intermediate step involving hal-parameters. However, application of this method would also require that all rate laws are re-created before and after param- eter estimation. Complementing pathway models with kinetic information Some aspects of the procedure described here compare with the work of Li and colleagues [50]. For instance, both their workflow and ours extract kinetic data from SABIO-RK. However, the aim of Li et al. was to provide full models, including parameterization and initial condi- tions. Their workflow could therefore plug in down- stream of Path2Models’ workflow; starting from models containing tentative rate-laws rather than stoichiometric reactions alone. Even for the most extensively investigated organism, Homo sapiens, kinetic data is only available for 12.2% of its known metabolic reactions. Much less information is available for other organisms. It should be noted that despite the wealth of pathways and reactions gathered in databases such as KEGG or MetaCyc, they could still not claim to be comprehensive. The model presented here can therefore only reflect the knowledge available today in a re-usable form. Since kinetic equations (and parameters) have not been experimentally determined, there is a great interest in the application of generic Since the modular rate laws can only be applied to reversible metabolic reactions, it was therefore necessary to select further generic rate equations for the large- scale approach described in this work. It can be hoped that the percentage of experimentally determined rate laws will increase in the future, but generic rate Page 11 of 19 Page 11 of 19 Büchel et al. Scaffold of logic models from KEGG signaling pathways g g g p y As mentioned above, the automatically generated models are only partially parameterized. In the case of KEGG sig- naling pathways for which no mechanistic details are provided, the models (with qual constructs) contain only topological relationships together with interaction signs. No logical rules specify the effects of (combined) interac- tions, and these models should be seen as scaffolds to be further parameterized before use in simulation. This can be done either by considering default, yet biologically meaningful, logical functions (e.g., requiring the presence of at least one activator and absence of all inhibitors) [53], by doing further manual refinement of the model (e.g., by literature mining), or by using dedicated experi- mental data to identify the functions [54]. Conclusion All the software building blocks used in this project are freely available and can be used to build similar work- flows. For instance, new modules can be used to read pathway information from other databases, as was shown for the entire PID [65]. As more sets of models are produced, they will be added to BioModels Database, where they will be easily retrievable and accessible. The availability of models in standard formats facilitates their import, comparison, merging and re-use. Automated de- velopment of models on the large scale will become cru- cial as automatic generation of pathways from genomics and metagenomics becomes common practise. Ready- made models will also be accurate starting points for the development of mechanistic models of whole cell models [66] where manual reconstruction is hardly an option. Several simulation tools now support the SBML Level 3 qual package, including GINsim [55], CellNOpt [56] and the Cell Collective platform [57]. CellNOpt provides a pipeline to generate logical rules by pruning a general scaffold with all possible rules so as to find the submodel that best describes the data. This can be done using vari- ous formalisms [58] of increasing detail, depending of the data at hand. The Cell Collective platform includes Bio- Logic Builder to facilitate the conversion of biological knowledge into a computational model [59]. GINsim provides complementary features that allow performing multiple analyses of logical models using powerful algo- rithms [60]. Therefore, relying on a combined use of these tools, one could use the Path2Models qualitative models by training them against data of, for instance, a cell type of interest, and subsequently analyzing the resulting models. Systematic generation of genome-scale metabolic reconstructions from existing data resources BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 laws will still be required to complete the quantitative models. used. This allowed us to generate layouts without node overlaps and with improved readability while still pre- serving the overall structure of the map. Nevertheless, some open questions remain, such as the occasional presence of oversized labels in contrast to the uniform size of the glyphs, and long edges between glyphs. The impact of the latter issue could be reduced in subsequent versions by additional cloning of glyphs, involving the an- notated multiplication of symbols representing the same entity, thus allowing this entity to be located at different points of the map. Generation of SBML Level 3 Core from KEGG metabolic pathways KEGG groups (which mostly correspond to complexes or gene families) were translated to species with all con- tained elements specified in the SBML notes and annota- tion. A human-readable list of contained gene symbols was added to the notes. A machine-readable term from a controlled vocabulary with a BioModels.net biology quali- fier is encoded by was used to denote all group members. The generation of pathway models from KEGG informa- tion was performed with KEGGtranslator [49,67]. Each KGML entry was translated to an SBML Level 3 species (SBML Core) and an SBO term [68] was assigned (see Table 1). Each KGML reaction was translated to an SBML reaction (SBML Core). In addition to all substrates, prod- ucts and catalyzing enzymes, this includes information about the reversibility of the reaction and the stoichiom- etry of each participant. Each reaction was checked against the KEGG API’s reaction definition and missing reaction components and reaction modifiers (i.e., enzymes) were added to the model. The layout of each node (position, width and height) was also stored in the model, using the SBML Layout extension [69]. During the translation, en- zymes that are contained in the orthologous template pathway, but have no instance in the current organism were removed from the model. Furthermore, for the meta- bolic translations, all nodes that do not correspond to physical instances of compounds or gene products were removed (i.e., pathway-reference nodes). Generation of kinetics models for the metabolic networks The program SBMLsqueezer [72,73] was used to fetch kinetic equations from SABIO-RK. For all cases when a corresponding entry for a reaction in the model could be found in SABIO-RK, the rate law and kinetic parameters (including SBML values and UnitDefinition objects) were extracted. Corresponding entries within the SABIO-RK database were identified using the MIRIAM-compliant annotations of reactions within each model. SABIO-RK returns an SBML document that may contain several rate equations for the same reaction, depending on experimen- tal conditions. For every rate law found in SABIO-RK, a correspondence was established between its species and compartments and those involved in the reaction of the query model. Functions and units defined by SABIO-RK that are referenced within the rate law of interest were also added to the model. In some cases such a matching was not possible. In these situations, the algorithm tries to add another rate law from SABIO-RK that matches the search criteria to the current reaction. Methods KEGG th KEGG pathways and the KEGG Markup Language For the construction of quantitative kinetic models and qualitative models, the content of the KEGG PATHWAY database was obtained through its FTP site prior to 1 July 2011. Generic, reference pathways and organism- specific pathways for 1 515 specie were downloaded, all encoded in the KEGG Markup Language (KGML). These files mainly consist of entries, describing proteins and compounds of a pathway, and interactions between them. The interactions are subdivided into reactions and rela- tions. Reactions correspond to biochemical reactions in- volving compounds and enzymes. Relations are used in the case of signaling pathways to specify protein-protein interactions. Layout information is given only for entries (i.e., nodes). Furthermore, each organism-specific path- way is derived from a reference pathway map. This in- volves adding organism-specific identifiers and setting the color (green) of enzymes that have protein instances in the current organism. Enzymes that have no known in- stance in an organism-specific pathway are retained in the map (albeit, while being colored differently) and keep their orthology identifier. This retention of absent en- zymes is due to the focus of KGML files on visual Creation of SBGN maps applying constraint-based layout SBGN provides a uniform and unambiguous graphical representation of biological knowledge. Providing models represented using this standard graphical format there- fore facilitate visual human understanding. Some tools provide translation of SBML files into SBGN maps. How- ever, to improve readability of such maps an appropriate layout of its elements is necessary. Here the initial posi- tions of the model elements, extracted from the KEGG database graphical pathway representations, were used to produce layout of the SBGN maps. Although many gen- eral layout algorithms have been proposed in the last three decades [61,62], almost none of them support add- itional constraints such as predefined positions and spatial relationships that would be necessary to preserve the essence of the original KEGG maps. Therefore a constraint-based layout approach [63] in conjunction with orthogonal object-avoiding edge routing [64] was Page 12 of 19 Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 biology qualifier [71] has version was used. Otherwise, BioModels.net biology qualifier is was used. representation of pathways rather than computational modeling. Completion and post-processing steps are therefore required to generate correct models from the KGML files [67]. Methods KEGG th Additional information was stored in SBML notes, in- cluding a human-readable description (i.e., the full name), synonyms (different gene symbols, compound la- bels, etc.), pathways, and for small molecules, links to im- ages of chemical compounds (hosted by KEGG and ChEBI), Chemical Abstract Service (CAS) numbers, chemical formula and molecular weight. Construction of the genome-scale metabolic recon- structions was performed through access of the publicly accessible KEGG web services, and was therefore applied to a more recent version of April 2013. Generation of SBML Level 3 Core from KEGG metabolic pathways Generation of SBML Level 3 Core from KEGG metabolic pathways The algorithm re- tains the order of rate laws as given by the search results from SABIO-RK. For the remaining reactions, either SABIO-RK could not find a rate equation or it was not possible to match species and compartments returned by SABIO-RK to the ones in the query model. The models were augmented with Identifiers.org URI [70] cross-references to the following resources: 3DMET, ChEBI, DrugBank, Enzyme Nomenclature (EC code), Ensembl, Gene Ontology, GlycomeDB, HGNC, KEGG (gene, glycan, reaction, compound, drug, pathway, orthology), LipidBank, NCBI Gene, OMIM, PDBeChem, PubChem, Taxonomy, UniProt. Furthermore, every spe- cies, qualitative species, reaction and transition was assigned the ECO-code ECO:0000313 meaning “a type of imported information that is used in an automatic as- sertion”. If multiple identifiers from the same database could be assigned to a single element, BioModels.net All missing rate laws were generated with the program SBMLsqueezer. To create ab initio kinetic laws for revers- ible enzyme-catalyzed reactions, the Common Modular (CM) rate law of Liebermeister et al. [52] was used. The explicit cat form was selected because it requires fewer in- dependent parameters than the Haldane- (hal [74]) and Wegscheider-compliant (weg [75]) CM forms, described in more detail below. The CM rate law can be used for any kind of reversible enzyme-catalyzed metabolic reac- tion whose precise mechanism remains unknown. This is the case if rate laws are automatically created for all reac- tions in KEGG. In their work on the CM rate law, Table 1 KGML entry type and corresponding mapping to SBO term KGML entry type SBO identifier SBO name compound SBO:0000247 simple chemical enzyme SBO:0000252 polypeptide chain gene SBO:0000252 polypeptide chain ortholog SBO:0000252 polypeptide chain group SBO:0000253 non-covalent complex map SBO:0000552 reference annotation Table 1 KGML entry type and corresponding mapping to SBO term Page 13 of 19 Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 13 of 19 Liebermeister et al. also proposed four additional modular rate laws that all cover certain special cases. otherwise. For non-enzymatic reactions, the generalized mass action rate law [78] has been used. Effects of inhibi- tors or activators using the prefactor terms suggested by Liebermeister and Klipp were included. Just like the con- venience rate law this equation can also be applied for arbi- trary numbers of reactants and products and is therefore well suited for the automatic creation of unknown kinetic equations. A common denominator characterizes all modular rate laws. Generation of SBML Level 3 Core from KEGG metabolic pathways The precise structure of the denominator term de- pends on the number and type of involved modulators, such as inhibitors or stimulators, as well as the number of reactants and products. Each modular rate laws can be used in three different modes or versions: the explicit (cat), Haldane-compliant, and Wegscheider-compliant. These versions determine the form of the numerator in the equation. The cat version has the smallest number of parameters. Its numerator resembles the mass action rate law, but with each reacting species divided by its corresponding Michaelis constant. Equation (1) displays the cat version of the CM rate law with modulation function f that includes activations, inhibitions and ef- fects of catalysts: In order to keep the kinetic equations simple, a list of ions and small molecules to ignore when creating kinetic equations was defined. This is necessary to reduce the complexity of rate laws where their contribution would actually be limited (Table 3). For gene-regulatory processes, the generalized version of Hill’s equation [79] was selected. For species that are annotated as genes (SBO term identifier is a derivative of gene; SBO:0000), the boundaryCondition in the SBML def- inition of the species was set to true. This means that the concentration of genes is seen as a constant pool that can- not be influenced by reactions. Finally, in case of zeroth order reactions (i.e., reactions without any reactant or re- versible reactions without any product), zeroth order ver- sions of the generalized mass-action rate law were used. vr Rr; Pr; Mr; k → ¼ f Rr; Pr; Mr; k → kþ r Π i∈Rr Si ½ Kri hrnir−k− r Π i∈Pr Si ½ Kri hrnir Π i∈Rr 1 þ Si ½ Kri hrnir þ Π i∈Pr Si ½ Kri hrnir−1 ð1Þ ð1Þ Rr, Pr, and Mr denote the index sets for reactants, products and modifiers in the rth reaction, nir gives the stoichiometric coefficient for the ith reactant, and vector k contains all parameters, such as the Michaelis constant Kri and the cooperativity factors hr. Multiplying the rate law with a well-defined prefactor function f allows the influence of modifiers, such as non-competitive inhib- ition to be included. The values of all new parameters were set to 1.0. The compartment sizes and species amounts or concentrations were also initialized with 1.0. Generation of SBML Level 3 Core from KEGG metabolic pathways If no substance, time, and volume units were defined in previous steps, the default substance unit was set to mole, time unit to second, and volume unit to litre. The units of all newly generated parameter objects were derived in order to ensure consistency of the overall models. This means that upon derivation, the units of reaction rates are all specified in substance per time. To this end, the SBML hasOnlySub- stanceUnits attribute was set to true if it was undefined before, and species quantities that were given in concen- tration units were multiplied by the size of their contain- ing compartment (within the kinetic equation) in order to obtain substance units for all species, irrespective if these were initially defined in concentration or substance units. As mentioned above, modular rate laws are only defined for reversible enzyme-catalyzed reactions. Table 2 summa- rizes the selected rate laws for irreversible reactions. In sim- ple cases, the well-described Henri-Michaelis-Menten equation and the random-order ternary-complex mechan- ism were selected as the default rate law [76]. For arbitrary irreversible enzyme-catalyzed reactions, convenience rate laws [77] were created. These used the simpler thermo- dynamically dependent form when the stoichiometric matrix of the reaction system has full column rank, and the more complex thermodynamically independent form In order to facilitate the interpretation of the equa- tions, units, and parameter objects created by this pro- cedure, all elements were annotated with appropriate terms from SBO and the Unit Ontology [80]. Table 2 Rate-laws for irreversible reactions Type of irreversible reaction Rate law non-enzyme reaction Generalized mass action rate law uni-uni enzyme reaction Henri-Michaelis-Menten equation bi-uni enzyme reaction Random-order ternary-complex mechanism bi-bi enzyme reaction Random-order ternary-complex mechanism arbitrary enzyme reaction Convenience rate law Table 2 Rate-laws for irreversible reactions Type of irreversible reaction Rate law non-enzyme reaction Generalized mass action rate law uni-uni enzyme reaction Henri-Michaelis-Menten equation bi-uni enzyme reaction Random-order ternary-complex mechanism bi-bi enzyme reaction Random-order ternary-complex mechanism arbitrary enzyme reaction Convenience rate law Table 2 Rate-laws for irreversible reactions Development and implementation of SBML Level 3 Qual package Development and implementation of SBML Level 3 Qual package Level 3 of SBML introduced the concept of modularity, with a Core package, shared by all, and domain-specific packages that add representational features on top of the core. The qual package is designed to provide SBML with the ability to encode qualitative models, such as logical models, or qualitative Petri-net models. The variables and the transformations of the models encoded in qual differ from species and reactions as defined in SBML Core. Page 14 of 19 Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Table 3 Small molecules and ions with negligible impact on reaction velocities Name Formula KEGG identifier Water H2O C00001 Zinc cation Zn2+ C00038 Copper(II) Cu2+ C00070 Calcium cation Ca2+ C00076 Hydron H+ C00080 Cobalt ion(II) Co2+ C00175 Potassium cation K+ C00238 Hydrogen H2 C00282 Nickel Ni C00291 Hydrochloric acid HCl C01327 Hydrogen selenide H2Se C01528 Iron(II) ion Fe2+ C14818 Iron(III) ion Fe3+ C14819 Table 3 Small molecules and ions with negligible impact on reaction velocities relations. These relations describe enzyme-enzyme relations, protein-protein interactions, interactions of transcription factors and genes, protein-compound in- teractions and links to other pathways. The KEGG speci- fication defines 16 different subtypes to describe the nature of the relations in more detail [83]. SBML qual describes relations as Transitions. Transitions consist of Input, Output, and Term objects. In contrast to KGML, SBML qual specifies the kind of relation in the attribute sign of the Input, instead of using type and subtype attri- butes for the relation. The sign attribute can take the values positive when the qualitativeSpecies linked to the input stimulates the transition, negative when it inhibits the transition, dual when the effects can go in both di- rections (depending upon the context), and unknown. Before converting the KEGG pathway to SBML qual, the pathway relations were further enriched with BioCarta information distributed by the Nature Pathway Interaction Database [3], which provides human pathways in BioPAX Level 3 format. To this end, for each KEGG relation, a search for a corresponding BioCarta interaction was per- formed. Then, the relation was assigned to a new subtype depending on the BioCarta-ControlType attribute that can be activating or inhibiting. Qualitative models typically represent discrete levels of activities that are involved in transformations that cannot always be described as processes (consuming from and producing to pools of elements). Development and implementation of SBML Level 3 Qual package To represent those con- cepts, QualitativeSpecies and Transition elements have been defined, together with their attributes and sub- elements. Briefly, a QualitativeSpecies encodes a variable representing a quantity or activity associated with an en- tity (e.g., gene, protein, but also phenomenological entity such as external condition, cell size, etc.) that can take discrete values (Boolean or multi-valued, e.g., in {0,1,2}). A Transition element encodes the rules governing the evolution of its Output node depending on the state of its Input nodes, both Input and Output nodes each refer- encing a particular QualitativeSpecies whilst providing additional information relating to the Transition. As most of the software packages used in this project were written in Java, JSBML [81] was chosen to implement the first library support for the SBML qual package. JSBML is a community-driven project to create a pure Java applica- tion programming interface (API) for reading, writing, and manipulating SBML files. It is an alternative to the Java interface provided in the C++ version, libSBML [82]. For the conversion from KGML to SBML qual, the subtypes activation and expression are translated to the value positive. The subtypes inhibition and repression are translated to the value negative. All other subtypes are translated to the value unknown. The value dual is assigned if a KEGG relation has both an activating as well as an inhibiting subtype. In addition to the sign at- tribute, the Input object is assigned an SBO term that further specifies the semantics based on subtype trans- lated (see Table 4). Genome-scale metabolic reconstructions The genome-scale metabolic reconstructions were gen- erated by applying a software pipeline based on modules of the SuBliMinaL Toolbox [39] and libAnnotationSBML [38] to all organisms in KEGG, release 66 (April 2013), accessed via the resource’s web services interface. Many models were augmented with metabolic pathway infor- mation extracted from MetaCyc (version 17.0, March 2013), extending a previous approach that was applied to Arabidopsis thaliana [84]. In the cases of both KEGG and MetaCyc, this metabolic pathway information in- cluded metabolites, metabolic reactions and catalytic en- zymes. Metabolites and reactions were reconciled with MNXref [40], and enzymes were specified with UniProt identifiers where possible. Generation of SBML Level 3 Qual from KEGG signaling pathways Generation of SBML Level 3 Qual from KEGG signaling pathways Generation of SBML Level 3 Qual from KEGG signaling pathways The overall generation of SBML qualitative maps from KGML files was performed with KEGGtranslator [49,67] using an approach similar as used for kinetic models. Each KGML entry was translated to an SBML Level 3 Qualitative Species (qual package) and each KGML rela- tion was translated in an SBML Transition (qual package). The models do not contain any definitions of intracellu- lar compartments. However, extracellular and intracellular compartments are specified, and a minimal extracellular growth medium was applied to all models, along with In KGML, all interactions between two or more en- tities that are not molecular reactions are named KEGG Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 15 of 19 Table 4 KGML subtypes and the corresponding SBML Qual sign attributes and SBO identifiers KGML subtype SBML Qual sign SBO identifier SBO name activation positive SBO:0000170 stimulation inhibition negative SBO:0000169 Inhibition expression positive SBO:0000170 stimulation repression negative SBO:0000169 inhibition indirect effect unknown SBO:0000344 molecular interaction state change unknown SBO:0000168 control binding/association unknown SBO:0000177 non-covalent binding dissociation unknown SBO:0000177 non-covalent binding missing interaction unknown SBO:0000396 uncertain process phosphorylation unknown SBO:0000216 phosphorylation dephosphorylation unknown SBO:0000330 dephosphorylation glycosylation unknown SBO:0000217 glycosylation ubiquitination unknown SBO:0000224 ubiquination methylation unknown SBO:0000214 methylation Table 4 KGML subtypes and the corresponding SBML Qual sign attributes and SBO identifiers KGML subtype SBML Qual sign SBO identifier The Systems Biology Graphical Notation necessary transport reactions that allow for its uptake. The medium contains: α-D-Glucose, β-D-Glucose, am- monium, sodium, potassium, magnesium, calcium, sulphate, chlorate, phosphate, protons, water, carbon dioxide and oxygen. Furthermore, default transport reactions have been added to allow for the transport of all intracellular metabolites into the extracellular space. The Systems Biology Graphical Notation [10] is a set of standard graphical languages for representing biological processes and interactions. The Process Description (PD) language allows scientists to represent chemical kinetics models, with pools of molecular entities consumed and produced by reactions. The Activity Flow (AF) language allows scientists to represent influence diagrams, in which entity activities inhibit or stimulate other entity activities. Commonly used biomass components were applied to each model, containing the 20 most common amino acids, the nucleotide precursors of RNA and DNA, glycogen and ATP, along with a default biomass reaction consisting of all 30 of these components. Generation of SBML Level 3 Qual from KEGG signaling pathways BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Page 16 of 19 with layout information from KEGG: if these reaction partners were not available, the reactions were placed at the top of the map, otherwise the reactions were placed near to reaction partners with layout information. For macromolecules representing enzymes, initial positions were computed taking into account the positions of cor- responding substrates, products and reaction glyphs. For simple chemicals representing secondary compounds, initial positions were computed such that these elements were grouped into substrates and products and placed close to the process glyph that represents the reaction. The automatic re-layout of the maps was done using a constrained-based approach [63] with orthogonal edge routing [64] for connections. Based on layout informa- tion stored in the model, geometric constraints were de- fined to preserve horizontal and vertical alignments, containment, as well as relative order of glyphs. Orthog- onal object-avoiding edge routing was performed for all edges except the ones connecting glyphs representing secondary compounds and the corresponding process glyphs. The resulting edge routes are similar to those in the KEGG images available online. Edge nudging (mov- ing apart overlapping parallel edges) was then applied to ensure that the edge routes conform to the SBGN layout rules. models. This separate branch was necessary because these automatically generated models are not expected to go through the normal curation and annotation phases, which are mainly manual processes. The schema of the database (which is used to store metadata about the models) had to be extended. The models themselves are stored in the file system. A custom structure has been devised in order to ensure acceptable access time (as too many files in a given folder puts a lot of stress on the file system). The resulting new branch is sufficiently generic to be able to store models coming from other similar projects. A generic sys- tem of categories was also created, in order to classify the models and provide a simple method for their browsing. This is currently used to handle the three main categories (metabolic, non-metabolic and whole genome metabolism) as well as the various sub-categories (such as Photosyn- thesis or Caffeine metabolism which have models for sev- eral organisms). A model display facility was developed, providing access to information about the model, including the an- notation of the model element and its associated notes. Generation of SBML Level 3 Qual from KEGG signaling pathways No attempt to tailor the biomass components to the organism was performed, and as such, clear anomalies such as the inclusion of glycogen in bacteria and plants remain. However, the removal of such terms, and the amendment of the biomass function itself, is a simple task for manual curation. All models were analyzed with the COBRA Toolbox [43] to deter- mine whether they were able to synthesize the biomass components, with the results provided in Additional file 1: Table S1. Generation of SBGN PD maps from SBML Level 3 Core The generation of SBGN Process Description (PD) maps from SBML Level 3 Core and their subsequent auto- matic layout was performed with SBGN-ED [86]. Each SBML entry was translated to the corresponding SBGN PD glyph based on SBO terms (see Table 2). The original positions of the KGML elements, which were stored using the SBML Layout package, were used as initial po- sitions for the SBGN PD glyphs. For each reaction, arcs to the corresponding reaction glyph connected the reac- tion partners. The types of the arcs, reflecting consump- tion, production or catalysis, were also set using SBO terms. Simple chemicals without a previously stored position or with more than one connection, along with all macromolecules with more than one connection, were cloned so that they appeared multiple times in the diagram, each with a connection to just a single element. The results of these steps were SBGN PD maps with valid structure but incomplete layout. The final layout of the maps was computed as a subsequent step. The genome-scale metabolic reconstructions described in this work adhere to the existing dialect that is com- patible with the COBRA Toolbox. That is, fields such as formula are represented in the SBML notes, and flux bounds are specified under reaction kineticLaw elements. However, as uptake of the newly proposed SBML Flux Balance Constraints package [85] increases, subsequent releases of the genome-scale metabolic reconstructions will also support this extension. All source code and the compiled software applica- tion for generating genome-scale models is available in Additional file 2. For process glyphs representing reactions not con- tained in the original KEGG pathway, initial positions were calculated based on availability of reaction partners Büchel et al. Generation of SBML Level 3 Qual from KEGG signaling pathways The model page offers the possibility to download the model (encoded in SBML) as well as its graphical repre- sentation (in PNG, SVG and SBGN-ML). A link to an online form provides a convenient way for users to re- port any issues they may encounter. The results of these steps were SBGN PD maps with a compact SBGN-conforming layout similar to the original KEGG layout. Finally, the maps were exported as SBGN- ML [87] and PNG image files, and stored in the BioMo- dels Database. Finally, a tool was developed to automatically submit a large number of models. It is able to read the models, per- form several checks and customize model files (mainly at the level of the notes and annotations of the model elem- ent) to ensure greater consistency, extract all the informa- tion necessary for their display, and store both metadata and models in the database and file system. Generation of SBGN AF maps from SBML Qual Analogous to SBGN Process Description, SBGN Activity Flow (AF) maps were generated by parsing glyph loca- tions and size information from the original KEGG layout via the SBML Layout extension in the generated qualitative model files. Glyph and arc types were set on the basis of SBO terms. Glyphs having multiple positions in the original layout were added to the map only once at the best fitting position of the pre-defined set. Over- lapping glyphs were spaced out using libvpsc [88] from the Adaptagrams project [89]. PNG renderings of the SBGN-ML files were created using PathVisio [90]. Several methods have been created for browsing the data. One can start from the list of all represented organisms, followed by individual pathways, such as Photosynthesis or Caffeine metabolism, and the display of a selected model. Alternatively, one can start with the three main categories of models (metabolic, non-metabolic, and whole genome metabolism), followed by the kind of models available in this category, then choose an organism and finally access the display of one model. In addition, a dedicated search engine is provided, allowing users to retrieve models based on textual queries. It relies on an index (generated using Lucene, http://lucene.apache.org/core/) of the content of all the models. A query expansion mechanism allows searches using Gene Ontology term names. Extension of BioModels database to support the distribution of models In order to distribute the models produced by the project, several changes to the database software infrastructure were required. In order to manage models encoded in SBML Level 3 and using several SBML packages, the in- frastructure has been upgraded to use the latest version of JSBML. The underlying pipeline (handling all models from their submission to their release) has been extended, and a new branch was created in order to accommodate the Three archives (one per main category) of all the models are available for downloading from the EBI’s FTP servers. Availability of supporting data All models generated by the project are availaible from BioModels Database [40]. Page 17 of 19 Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Büchel et al. BMC Systems Biology 2013, 7:116 http://www.biomedcentral.com/1752-0509/7/116 Acknowledgements S d P k l d g NS and PM acknowledge support from the European Union FP7 project UNICELLSYS (grant number: 201142). MvI and PM received financial aid from the EU project BioPreDyn (ECFP7-KBBE-2011-5 Grant number 289434). MH and SK acknowledge support from the US National Institute of General Medical Sciences (grant number GM070923). CW, FB, FM, RK, AD, and AZ are grateful for financial support by the Federal Ministry of Education and Research (BMBF, Germany) in the projects Virtual Liver Network (grant number 0315756) and National Genome Research Network (NGFN-Plus, grant number 01GS08134). AD thanks the EU for funding his Marie Curie International Outgoing Fellowship within FP7 (project AMBiCon, 332020). PM acknowledges support from the US National Institute of General Medical Sciences (grant number GM080219) and the BBSRC (grant number BB/J019259/1). MvI, FB, FM, MS, NR received dedicated support from the EMBL-EBI. NS also thanks Ben Morris of the University of North Carolina at Chapel Hill for generously and freely making available his code for converting the NCBI Taxonomy flat files into a Newick tree, which was used to generate the phylogenetic tree of genome-scale models. MG and CL acknowledge support from the Innovative Medicines Initiative Joint Under- taking under grant agreement no. 115156. 10. Le Novère N, Hucka M, Mi H, Moodie S, Schreiber F, Sorokin A, Demir E, Wegner K, Aladjem MI, Wimalaratne SM, Bergman FT, Gauges R, Ghazal P, Kawaji H, Li L, Matsuoka Y, Villéger A, Boyd SE, Calzone L, Courtot M, Dogrusoz U, Freeman TC, Funahashi A, Ghosh S, Jouraku A, Kim S, Kolpakov F, Luna A, Sahle S, Schmidt E, et al: The systems biology graphical notation. Nat Biotechnol 2009, 27:735–741. 11. Smallbone K, Messiha HL, Carroll KM, Winder CL, Malys N, Dunn WB, Murabito E, Swainston N, Dada JO, Khan F, Pir P, Simeonidis E, Spasić I, Wishart J, Weichart D, Hayes NW, Jameson D, Broomhead DS, Oliver SG, Gaskell SJ, McCarthy JE, Paton NW, Westerhoff HV, Kell DB, Mendes P: A model of yeast glycolysis based on a consistent kinetic characterisation of all its enzymes. FEBS Lett 2013, 587:2832–2841. characterisation of all its enzymes. FEBS Lett 2013, 587:2832–2841. 12. Brown M, Dunn WB, Dobson P, Patel Y, Winder CL, Francis-McIntyre S, Begley P, Carroll K, Broadhurst D, Tseng A, Swainston N, Spasic I, Goodacre R, Kell DB: Mass spectrometry tools and metabolite-specific databases for molecular identification in metabolomics. Analyst 2009, 134:1322–1332. Additional files Mathematical Sciences, California Institute of Technology, Pasadena, CA 91125, USA. 9School of Chemistry, The University of Manchester, Manchester M13 9PL, UK. 10School of Computer Science, The University of Manchester, Manchester M13 9PL, UK. 11Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, USA. 12Instituto Gulbenkian de Ciência (IGC), Oeiras P-2780-156, Portugal. 13Institute of Computer Science, University of Halle-Wittenberg, Halle, Germany. 14Present address: University of California, San Diego, Bioengineering Department, La Jolla, CA 92093-0412, USA. 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Authors’ contributions AD coordinated the work done by CW, FB, FM, RK, MR at ZBIT, contributed to JSBML including Layout and qual package, implemented an algorithm for unit derivation, and generated kinetic laws, parameters and units ab initio. AZ supervised the researchers at ZBIT. FB, FM and MvI contributed to the development of the SBML qual implementation. FB further contributed to the translation of signaling models to SBML and augmented them with additional information from BioCarta. CW contributed the source KGML models, implemented the metabolic and signaling conversions from KGML to SBML and generated the initial SBML models. He contributed to the implementation of multiple SBML extensions that are used within the scope of the manuscript. MR and RK implemented the SABIO-RK search. NS generated the genome-scale metabolic models, responded to reviewers’ comments and edited the manuscript. PM and DBK assisted with the generation of genome-scale metabolic models. TC, MW and FS dealt with the representation of the models as SBGN PD maps and their automatic layout. MS generated SBGN AF-ML and graphical renderings of the SBML qual models. CC contributed to the discussions on SBML qual usage. NR contributed to the development of JSBML. CL, MG and NR contributed to BioModels Database. SK finalized the SBML qual specification. MH contributed to JSBML and the SBML qual specification. JSR helped to initiate the project, and contributed to the discussions on SBML qual usage. NS had the original idea to automate the creation of models from pathways. Competing interests The authors declare they have no conflicts of interests. The authors declare they have no conflicts of interests. Acknowledgements S d P k l d All authors would like to dedicate this paper to the memory of Isabel Rojas, creator of the SABIO-RK database, who passed away in July 2013. Mathematical Sciences, California Institute of Technology, Pasadena, CA 91125, USA. 9School of Chemistry, The University of Manchester, Manchester M13 9PL, UK. 10School of Computer Science, The University of Manchester, Manchester M13 9PL, UK. 11Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, USA. 12Instituto Gulbenkian de Ciência (IGC), Oeiras P-2780-156, Portugal. 13Institute of Computer Science, University of Halle-Wittenberg, Halle, Germany. 14Present address: University of California, San Diego, Bioengineering Department, La Jolla, CA 92093-0412, USA. Competing interests Received: 19 July 2013 Accepted: 23 October 2013 Published: 1 November 2013 Received: 19 July 2013 Accepted: 23 October 2013 Published: 1 November 2013 Author details 1E M l Ebrahim A, Lerman JA, Palsson BØ, Hyduke DR: COBRApy: COnstraints-Based Reconstruction and Analysis for Python. BMC Syst Biol 2013, 7:74. 45. 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https://openalex.org/W4307628432	https://zenodo.org/record/7275886/files/7302_abstract_yarime.pdf	English	null	The Smart City as a Field of Innovation: Effects of Public-Private Data Collaboration on the Innovative Performance of Small and Medium-Sized Enterprises in China	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,455	Extended Abstract Data is increasingly considered to be a key component in stimulating innovation (Cockburn, Henderson, and Stern, 2019). Numerous promising possibilities have been opened up by rapidly emerging, data-intensive technologies, including the Internet of Things (IoT) and artificial intelligence. The analysis and interpretation of big data are critical in the growth of technology firms in terms of AI training and computing capabilities (Allam and Dhunny, 2019). Small and medium-sized enterprises (SMEs), with their limited resources internally, particularly face a serious challenge of implementing innovation that depends upon data. The smart city provides an important opportunity for creating data-driven innovation. Significant amounts of data are increasingly available from various sources through sophisticated devices and equipment scattered in smart cities. Many smart city projects across the globe provide rich opportunities for SMEs to explore data-driven innovation (Bresciani, Ferraris, and Del Giudice, 2018). China, in particular, has recently been active in collecting and utilizing various kinds of data in smart cities. The availability of and access to data help to improve the software development of firms in China, where massive amounts of data resources are held by the government (Beraja, Yang, and Yuchtman, 2022). y g j g In the process of smart city development, there are also many tasks that are complementary to each other, including connecting databases, building online platforms that connect different data coming from different data sources, operating online platforms, and providing products and services to citizens. These diverse kinds of tasks involved in smart city projects initiated by local governments have brought about new business opportunities for innovative SMEs in China. To implement the policies of encouraging the development of SMEs by the central government, municipal governments have introduced policies that give priority to SMEs in participating in smart city projects (Ministry of Industry and Information Technology, 2016). Those companies that have access to the data held by government agencies are expected to benefit from utilizing the rich data for creating innovative products and services. There were few empirical studies conducted, however, to examine how data are actually managed and provided in smart cities and how they affect companies’ innovative activities. It remains unclear how public agencies and private enterprises collaborate on data and how that influences the innovation performance of SMEs in China. In smart cities, different types of public-private collaboration are involved, including hardware purchase, platform building, platform operation, and data analysis. Data for Policy 2022 Conference, Hong Kong, Seattle, and Brussels, December 2022 Data for Policy 2022 Conference, Hong Kong, Seattle, and Brussels, December 2022 The Smart City as a Field of Innovation: Effects of Public-Private Data Collaboration on the Innovative Performance of Small and Medium-Sized Enterprises in China Xiaohui Jiang1, Masaru Yarime1* 1 Division of Public Policy, The Hong Kong University of Science and Technology E-mail: yarime@ust.hk Keywords: Smart city, China, Data-driven innovation, Small and medium-sized enterprise, Public-private collaboration Extended Abstract It is not yet well-understood how these different types of collaboration influence the innovative performance of SMEs. In this study, we intend to address how data are managed through collaboration between the government and companies in smart cities and how the mode of collaboration influences firms’ performance on innovation. By focusing on the case of SMEs in China, this Data for Policy 2022 Conference, Hong Kong, Seattle, and Brussels, December 2022 research aims to shed light on what kinds of data are available and used in smart cities and how the government and enterprises collaborate on data to facilitate innovation. research aims to shed light on what kinds of data are available and used in smart cities and how the government and enterprises collaborate on data to facilitate innovation. The analysis of this study utilizes data on more than eight million contracts extracted from the official procurement database of the government. Data on companies are assembled with regard to the registered capital, industry, software products, and patents in 1990-2021 from the Tianyancha website. A panel data is established with key characteristics of SMEs, software and patents outputs, and their record on obtaining different government contracts annually. The government contracts are divided into three categories, namely, data analysis, platform building, and equipment supply, based on keyword identifications. To deal with the unbalance between the treatment group (the companies that obtained government contracts) and the control group, we use propensity score matching (one-to-one nearest neighbor matching) to narrow down the sample size of the control group to that of the treatment group. Then we apply the traditional difference-in-difference (DID) and DID with multiple time periods (Callaway & Sant’Anna, 2021) methods to examine whether there are significant differences in innovative outputs of software products and patents before and after the companies receive government contracts. We also compare how the innovation performance of companies differs based on the types of contracts these companies obtain. That makes it possible to identify what kinds of data collaboration would be effective in improving the innovative performance of SMEs. Our preliminary analysis of average treatment effect suggests that obtaining the government contracts, especially research contracts and platform building type of contracts, can effectively help improve the innovation performance when comparing between the control and treatment group. After checking the common support graph and doing a balance test, the outcome is solid. Extended Abstract In general, based on the average treatment effect on the treated group, innovation performance, as measured by the number of patents and software productions, has shown a significant increase for those companies that obtain a research type of contract, whereas the increase is smaller for the platform building contractor and slight for the equipment purchase contractor. Government purchase of equipment that contains data-intensive technologies promotes the use of these products in smart cities. That, however, does not involve any substantive exchange or transfer of data possessed by the government and would not significantly contribute to stimulating innovation at firms. g g y g This study will have useful implications for establishing public-private collaboration on data for facilitating innovation in smart cities. Specifically, government procurement should also be viewed as a policy tool for improving China's technological innovation. According to our findings, research and platform development can successfully assist firms in improving their innovation performance. As a result, the government should grant enterprises access to additional data resources and data management related tasks if they want to see more industry structural transformation, as stated in the government documents. References Allam, Z. and Z. A. Dhunny (2019). "On big data, artificial intelligence and smart cities." Cities, 89, 80-91. Allam, Z. and Z. A. Dhunny (2019). "On big data, artificial intelligence and smart cities." Cities, 89, 80-91. Beraja, Martin, David Y. Yang, and Noam Yuchtman (2022). "Data-intensive Innovation and the State: Evidence from AI Firms in China." Working Paper, January 11. the State: Evidence from AI Firms in China. Working Paper, January 11. Bresciani, S., A. Ferraris, and M. Del Giudice (2018). "The management of organizational ambidexterity through alliances in a new context of analysis: Internet of Things (IoT) smart city projects." Technological Forecasting and Social Change, 136, 331-338. Bresciani, S., A. Ferraris, and M. Del Giudice (2018). "The management of organizational ambidexterity through alliances in a new context of analysis: Internet of Things (IoT) smart city projects." Technological Forecasting and Social Change, 136, 331-338. Callaway, B., & Sant’Anna, P. H. (2021). Difference-in-differences with multiple time. periods. Journal of Econometrics, 225(2), 200-230. Callaway, B., & Sant’Anna, P. H. (2021). Difference-in-differences with multiple time. periods. Journal of Econometrics, 225(2), 200-230. Cockburn, Ian M., Rebecca Henderson, and Scott Stern (2019). "The Impact of Artificial Intelligence on Innovation: An Exploratory Analysis," Agrawal, Ajay, Joshua Gans, and Avi Goldfarb, eds. The Economics of Artificial Intelligence: An Agenda. Chicago: Cockburn, Ian M., Rebecca Henderson, and Scott Stern (2019). "The Impact of Artificial Intelligence on Innovation: An Exploratory Analysis," Agrawal, Ajay, Joshua Gans, and Avi Goldfarb, eds. The Economics of Artificial Intelligence: An Agenda. Chicago: Cockburn, Ian M., Rebecca Henderson, and Scott Stern (2019). "The Impact of Artificial Intelligence on Innovation: An Exploratory Analysis," Agrawal, Ajay, Joshua Gans, and Avi Goldfarb, eds. The Economics of Artificial Intelligence: An Agenda. Chicago: Data for Policy 2022 Conference, Hong Kong, Seattle, and Brussels, December 2022 The University of Chicago Press. The University of Chicago Press. Ministry of Industry and Information Technology (2016). "The "Plan for Promoting the Development of Small and Medium-sized Enterprises (2016-2020)" was officially released, and promoting entrepreneurship and innovation will become a key task." Ministry of Industry and Information Technology, Beijing, China, June 28. The University of Chicago Press. Ministry of Industry and Information Technology (2016). "The "Plan for Promoting the Development of Small and Medium-sized Enterprises (2016-2020)" was officially released, and promoting entrepreneurship and innovation will become a key task." Ministry of Industry and Information Technology, Beijing, China, June 28.
https://openalex.org/W3080151387	https://www.phisci.info/jour/article/download/3083/2880	Russian	null	Outlines of a New World Order	Filosofskie nauki	2,020	cc-by	6,256	* Статья подготовлена при поддержке гранта РФФИ № 20-511-00003 Бел_а «Философский, методологический и междисциплинарный анализ перспектив и рисков развития цифровой реальности». Новый технологический уклад: социокультурные основания DOI: 10.30727/0235-1188-2020-63-5-7-27 Оригинальная исследовательская статья Original research paper DOI: 10.30727/0235-1188-2020-63-5-7-27 Оригинальная исследовательская статья Original research paper Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) 7 * The research was supported by the grant of the Russian Foundation for Ba- sic Research (RFBR), project no. 20-511-00003 Бел_а “Philosophical, Method- ological, and Multidisciplinary Analysis of Prospects and Risks of the Develop- ment of Digital Reality.” Аннотация В статье исследуются трансформации мирового уклада в контек- сте развития общественно-экономических отношений и научно- технологического прогресса. Показано, что по мере научно- технического развития, являющегося главным фактором глобальных трансформаций, менялись общественно-экономические отношения, формы государственности, системы управления. Отмечается, что с начала Нового времени философскую основу мирового уклада стали определять рыночные отношения, стимулировавшие интенсивный научно-технологический прогресс и развитие финансово-кредитной системы. Собственно капитализм изначально рассматривался как служение. По мере развития принципиально изменяется функция денег, превращающихся из ресурса в инструмент управления. Это привело к нарастанию дифференциации социально-экономического развития стран, а также к расслоению общества. Попытка построе- ния однополярного мира в результате экономической глобализации дала обратный результат. Исчерпание возможностей капиталистиче- ских моделей социально-экономического развития на фоне научно- технологического прогресса привело к каскаду экономических, по- литических и военных кризисов. Наблюдаемые тенденции позволяют говорить о том, что сбывается прогноз Д. Белла о переходе к постин- дустриальному обществу, т.е. обществу, где приоритетом является Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) Новый технологический уклад... повышение качества жизни на основе новых технологий. Поэтому на повестку дня выходит определение контуров нового мирового укла- да. Новый мировой (постиндустриальный) уклад сформируется в результате гуманитарно-технологической революции, суть которой заключается в синхронном развитии науки, технологий и культуры, ориентированном на удовлетворение потребностей человека. Осно- ву постиндустриального технологического уклада составят конвер- гентные технологии. При этом направления и траектория развития человечества будут определяться уровнем взаимодействия общества, бизнеса и власти. Однако, как показывает исторический опыт, в насто- ящее время отсутствуют механизмы эффективного контроля власти и бизнеса со стороны общества. Поэтому делается вывод, что на данном этапе необходим консенсус, который по крайней мере обеспечит рав- новесное состояние и глобальную безопасность, а в идеале – придаст новый импульс развитию человечества. Новый технологический уклад... Ключевые слова: мировой уклад, глобальные процессы, техноло- гический уклад, гуманитарно-технологическая революция, постин- дустриальное общество, бизнес, власть, деньги, информация. Иванов Владимир Викторович – доктор экономических наук, член-корреспондент РАН, заместитель президента РАН, руководитель Информационно-аналитического центра «Наука» РАН. ivanov@presidium.ras.ru https://orcid.org/0000-0002-9823-8767 Для цитирования: Иванов В.В. Контуры нового мирового уклада // Философские науки. 2020. Т. 63. № 5. С. 7–27. DOI: 10.30727/0235-1188-2020-63-5-7-27 V.V. Ivanov Russian Academy of Science, Moscow, Russia Abstract The article discusses the transformations of the world order in the context of the development of socio-economic relations and scientific and techno- logical progress. It is shown that in the course of scientific and technologi- * The research was supported by the grant of the Russian Foundation for Ba- sic Research (RFBR), project no. 20-511-00003 Бел_а “Philosophical, Method- ological, and Multidisciplinary Analysis of Prospects and Risks of the Develop- ment of Digital Reality.” 8 В.В. ИВАНОВ. Контуры нового мирового уклада ур р у cal development, which is the main factor of global transformations, there are the changes of social and economic relations, forms of statehood, and management systems. It is noted that since the beginning of the New Age, the philosophical basis of the world order began to be determined by market relations, which stimulated intensive scientific and technological progress and the development of the financial and credit system. Capital itself was originally seen as a service. As capitalism evolves, the function of money changes fundamentally, turning from a resource into a mean of manage- ment. This led to an increase in the differentiation of the socio-economic development of countries as well as to the stratification of society. An at- tempt to build a unipolar world as a result of economic globalization gave the opposite result. Exhaustion of the possibilities of capitalism models to provide socio-economic development in spite of scientific and technological progress, led to a cascade of economic, political, and military crises. The observed trends prove Daniel Bell’s forecast about the transition to a post- industrial society, that is, to a society where the priority is to improve the quality of life based on new technologies. Therefore, defining the contours of a new world order is on the agenda. The new (post-industrial) world’s way of life will be formed as a result of the humanitarian and technological revolution, the essence of which is the synchronous development of sci- ence, technology, and culture, focused on meeting human needs. The basis of the post-industrial technological paradigm will be convergent technolo- gies. Moreover, the directions and trajectory of human development will be determined by the level of interaction between society, business, and government. However, as historical experience shows, there are currently no mechanisms for effective control of government and business by society. For citation: Ivanov V.V.(2020) Outlines of a New World Order. Rus- sian Journal of Philosophical Sciences = Filosofskie nauki. Vol. 63, no. 5, рр. 7–27. DOI: 10.30727/0235-1188-2020-63-5-7-27 Abstract Therefore, it is concluded that at this stage, a consensus is needed, which at least will ensure a balance and global security, and ideally will give a new impetus to the development of mankind. cal development, which is the main factor of global transformations, there are the changes of social and economic relations, forms of statehood, and management systems. It is noted that since the beginning of the New Age, the philosophical basis of the world order began to be determined by market relations, which stimulated intensive scientific and technological progress and the development of the financial and credit system. Capital itself was originally seen as a service. As capitalism evolves, the function of money changes fundamentally, turning from a resource into a mean of manage- ment. This led to an increase in the differentiation of the socio-economic development of countries as well as to the stratification of society. An at- tempt to build a unipolar world as a result of economic globalization gave the opposite result. Exhaustion of the possibilities of capitalism models to provide socio-economic development in spite of scientific and technological progress, led to a cascade of economic, political, and military crises. The observed trends prove Daniel Bell’s forecast about the transition to a post- industrial society, that is, to a society where the priority is to improve the quality of life based on new technologies. Therefore, defining the contours of a new world order is on the agenda. The new (post-industrial) world’s way of life will be formed as a result of the humanitarian and technological revolution, the essence of which is the synchronous development of sci- ence, technology, and culture, focused on meeting human needs. The basis of the post-industrial technological paradigm will be convergent technolo- gies. Moreover, the directions and trajectory of human development will be determined by the level of interaction between society, business, and government. However, as historical experience shows, there are currently no mechanisms for effective control of government and business by society. Therefore, it is concluded that at this stage, a consensus is needed, which at least will ensure a balance and global security, and ideally will give a new impetus to the development of mankind. Keywords: world order, global processes, technological order, humani- tarian and technological revolution, post-industrial society, business, power, money, information. Vladimir V. Ivanov – D.Sc. Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) Новый технологический уклад... Все исторические системы обладают ограниченным сроком жизни и в конце концов должны уступить свое место другим системам-преемницам. И Валлерстайн Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) 0. 63(5) Новый технологический уклад... Все исторические системы обладают ограниченным сроком жизни и в конце концов должны уступить свое место другим системам-преемницам. И. Валлерстайн Постановка задачи История развития человечества, по сути, есть история развития общественно-экономических отношений и научно- технологического прогресса. По мере научно-технического развития менялись общественно-экономические отношения, государства, система управления. Процесс трансформаций выглядит следующим образом: получение новых знаний – их освоение (образование) − создание новых технологий и про- дукции – изменение системы отношений. В настоящее время Мир находится в процессе перехода к новому состоянию. На повестку дня выходит определение контуров нового мирового уклада. Abstract in Economy, Corresponding Member of the Russian Academy of Sciences, Deputy President of the Russian Academy of Sciences (RAS), Head RAS Information and Analytical Center “Nauka.” ivanov@presidium.ras.ru https://orcid.org/0000-0002-9823-8767 Vladimir V. Ivanov – D.Sc. in Economy, Corresponding Member of the Russian Academy of Sciences, Deputy President of the Russian Academy of S i (RAS) H d RAS I f ti d A l ti l C t “N k ” Sciences (RAS), Head RAS Information and Analytical Center “Nauka.” ivanov@presidium.ras.ru https://orcid.org/0000-0002-9823-8767 For citation: Ivanov V.V.(2020) Outlines of a New World Order. Rus- sian Journal of Philosophical Sciences = Filosofskie nauki. Vol. 63, no. 5, рр. 7–27. DOI: 10.30727/0235-1188-2020-63-5-7-27 For citation: Ivanov V.V.(2020) Outlines of a New World Order. Rus- sian Journal of Philosophical Sciences = Filosofskie nauki. Vol. 63, no. 5, рр. 7–27. DOI: 10.30727/0235-1188-2020-63-5-7-27 9 Трансформация мирового уклада: от религиозного к административному L’état, c’est moi (Государство – это я) Людовик XIV Христианская религия более 2000 лет является одним из главных институтов, оказывающих существенное, если не ре- шающее, влияние на формирование мирового уклада. Христи- анство можно считать первым системным глобализационным институтом, распространившим свое влияние практически на все территории, до которых дошла цивилизация. О роли церкви можно судить по булле Папы Бонифация VIII (1302 г.) «Unam Sanctam», в которой говорилось, что во власти Папы находятся два меча: «один, подчиняющийся другому, светский – духовному… Духовная власть, правда, передана че- ловеку, но она не человеческая, а божеская, и кто не повинуется ей, противится воле господней и подлежит принудительному 10 В.В. ИВАНОВ. Контуры нового мирового уклада спасению», «…короли должны служить церкви по первому приказанию папы (ad nutum). Папе принадлежит право карать светскую власть за всякую ошибку. Светской власти это право не дано» [Лозинский 1986, 151]. Следует заметить, что речь не идет о каком-то конкретном государстве, а о принципиальных взаимоотношениях светской и религиозной власти. Роль церкви наглядно просматривается в процедурах цере- монии королевского посвящения. Так, например, во Франции, начиная с 759 г., коронацию проводили высшие представители Церкви в присутствии знати. При этом король подтверждал свою приверженность Церкви. При вступлении на престол в 877 г. Людовика Заики ему было подано прошение от лица Церкви, в котором, в частности, говорилось: «…лучшие люди Церкви вверяют тебе свои кано- нические привилегии и вменяют законные ленные права, так как король своей властью обязан исключительно епископству и Церкви, преданным ему». На это последовал ответ: «Обещаю Вам и подтверждаю только для Вас и нашей Церкви быть вер- ным каноническим привилегиям и своей законной обязанности сохранять и защищать предъявленное право, так как король своей властью един с епископством и Церковью, верен им и буду продолжать защищать их» [Польская 2004]. Несмотря на то, что христианство имело много различных, зачастую противоречивых, направлений, в целом именно религия в течение многих веков носила надгосударственный характер и составляла основу мирового уклада, экономическую базу которого составляли феодальные отношения. В XVII в. на смену концепции «смешанной монархии», объеди- нявшей аристократические и монархические методы управления, пришла теория единого и неделимого суверенитета, который в монархическом государстве всецело принадлежал монарху. Тем самым ответственность монарха была определена не только пе- ред Богом, но и перед своими подданными. Монархи перешли от царствования к управлению [Всемирная история… 2013, 482]. Классическим примером происходящих трансформаций ста- ла Франция XVII в. В результате проведенных реформ, обуслов- ленных противостоянием административной и судебной власти, 11 Филос. науки / Russ. J. Philos. Sci. 2020. Трансформация мирового уклада: от религиозного к административному 63(5) Новый технологический уклад... сформировалась административная система управления при личном правлении короля Людовика XIV. Королю полностью подчинялись парламент и судебная система. Новый технологический уклад... При этом король резко сократил численность государствен- ных министров, которыми лично управлял. Представители аристократии и духовные лица, входившие в близкий круг советников короля, были заменены на чиновников, вышедших из административного аппарата. В России заметное ослабление влияния церкви на си- стему госуправления происходит в ходе реформ Петра I [Костомаров 2004, 640]. В 1700 г. был упразднен Патриарший приказ. Все проводившиеся в нем мирские дела, связанные пре- жде всего с экономической деятельностью, были переданы в другие ведомства, а духовные дела были поручены временно на- значенному блюстителю. Монахам и монахиням было запрещено вмешиваться в управление вотчинами – все доходы от вотчин направлялись в специально созданный в 1701 г. Монастырский приказ. Также были определены государственные средства, вы- деляемые на содержание монахов и монахинь. Таким образом церковь была переведена на государственное обеспечение. В результате трансформаций церковь во многом утратила свое влияние на государственное управление, но сохранила воспитательные и образовательные функции. Основу миро- вого уклада, сформировавшегося в Новое время, составила административная система управления, многие черты которой сохранились и до наших дней. Капитализм – это прежде всего и главным образом историческая социальная система. Капитализм – это прежде всего и главным образом историческая социальная система. Капитализм: от служения к тоталитаризму Задача предприятия – производить для потребления, а не для наживы или спекуля- ции. А условия такого производства – …чтобы продукты эти служили на пользу народу, а не только одному производителю. Г. Форд Капитализм – это прежде всего и главным образом историческая социальная система. И. Валлерстайн Капитализм – это прежде всего и главным образом историческая социальная система. И. Валлерстайн 12 В.В. ИВАНОВ. Контуры нового мирового уклада В.В. ИВАНОВ. Контуры нового мирового уклада Философскую основу следующего мирового уклада опреде- лил капитализм, стимулировавший интенсивный научно- технологический прогресс и развитие финансово-кредитной системы. При этом произошла замена базовых ценностей. Если основу религиозной парадигмы составляли духовные принципы, то капитализм изначально рассматривался как служение [Форд 2015]. Но по мере развития приоритеты изме- нились: «Экономика капитализма… управляется рациональ- ным стремлением к максимальному увеличению накопления» [Валлерстайн 2018, 130]. Трансформация базовых положений мирового уклада в XVII−XXI вв. выглядит следующим образом: нравственные ценности – абсолютизм и капитализм как служение – капита- лизм как механизм бесконечного накопления капитала. Обратим внимание на то обстоятельство, что на рынке суще- ствуют только два потребителя конечной продукции и услуг: человек и государство. Человеку товары и услуги нужны для удовлетворения собственных потребностей. При этом часть своих доходов человек передает государству для осуществления общегосу- дарственных функций – оборона, безопасность, образование, здравоохранение и т.д. В этом плане государство также можно рассматривать как потребителя конечной продукции и услуг, необходимых для выполнения его функций. Что касается бизнеса, то ситуация представляется иным об- разом. Продукция, которую приобретает бизнес, предназначена не для конечного потребления, а для организации производства товаров и услуг. Так, например, компьютер, купленный чело- веком, является конечной продукцией, поскольку служит для удовлетворения потребностей. Тот же компьютер, но установ- ленный в офисе, служит для организации производственного процесса, т.е. является элементом технологической цепочки, обеспечивающей выпуск конечной продукции. Бизнес не име- ет собственных денег – все деньги у него заемные. Именно на этом построена кредитно-денежная система. В этом свете следует обратить особое внимание на изменение функций денег. Известны пять функций денег (cм., например: 13 Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) Новый технологический уклад... [Геращенко 2009]): деньги как средство обращения, деньги как средство платежа, деньги как средство накопления, деньги как средство кредитования, мировые деньги. р р р Главной институциональной структурой финансово- кредитной системы являются банки, которые изначально соз- давались для хранения денег. Эта деятельность также стоит определенных средств, и кроме того, временно свободные деньги одного владельца можно дать взаймы кому-то другому для реализации конкретной бизнес-идеи. Однако для этого не- обходимо, чтобы заемщик не только вернул заимствованные деньги, но также обеспечил издержки по их хранению, банков- ские риски, а также учел интересы владельца, чьим реальным ресурсом он воспользовался. Капитализм – это прежде всего и главным образом историческая социальная система. И. Валлерстайн Естественно, что банки закла- дывают в ссудный процент ставку, которая гарантированно застрахует их от всяких негативных последствий и кроме того обеспечит получение прибыли. По сути, роль банков сводится к торговле деньгами. С течением времени в банках сосредотачивается значитель- ный капитал, которым они могут распоряжаться по собствен- ному усмотрению. Это принципиально изменяет функцию денег: из ресурса они превращаются в инструмент управления. С одной стороны, наличие значительных финансовых ресур- сов позволяет развивать бизнес. С другой, тормозить его в случае необходимости, например, для устранения конкурента. Но самое главное − банки имеют возможность использовать финансовые ресурсы для влияния на власть. Международная финансовая система стала основой эконо- мической глобализации, а ее основным признаком – свободное движение капитала. Как отмечал Д. Сорос, «возможность сво- бодного движения финансового капитала подрывает способ- ность национального государства контролировать экономику. Глобализация финансовых рынков ведет к упразднению государства всеобщего благосостояния.., поскольку люди, нуждающиеся в социальном обеспечении, не могут покинуть свою страну, а финансовый капитал, задействованный в системе социального страхования, − может» [Сорос 2004, 17]. Таким образом государство исключается из участия в экономическом 14 В.В. ИВАНОВ. Контуры нового мирового уклада процессе. Согласившись с этим, и общество, и государство сра- зу теряют возможность демократического развития, поскольку все управление переходит на уровень глобальных финансовых структур, что в свою очередь формирует жесткий, формальный и сверхтоталитарный механизм управления. Таким образом, в начале XXI в. заложены основы форми- рования глобальной тоталитарной системы, базирующейся на финансовых и информационных рычагах управления. Наше «общество потребления» XX в. является функцией науки и ее техники. И. Валлерстайн Наше «общество потребления» XX в. является функцией науки и ее техники. И. Валлерстайн Процессы глобальных трансформаций и формирования но- вых общественно-экономических формаций проходят на фоне развития науки и технологий. При этом об уровне развития в конкретный период времени можно судить по набору техно- логий − технологическому укладу [Глазьев 1993]. Получение новых знаний о закономерностях развития При- роды, Человека и Общества − предмет фундаментальной науки. Эти исследования проводятся во всем возможном спектре на- правлений познания и как правило, не имеют жестко заданных приоритетов. Одной из разновидностей фундаментальных научных исследований являются ориентированные исследо- вания, т.е. фундаментальные исследования, целью которых является получение фундаментальных знаний для создания новых технологий. Технология – это предмет прикладных исследований и разра- боток. Под технологией следует понимать последовательность операций, в основе которых лежит новое знание, приводящее к достижению заранее заданной цели в виде создания новых видов продукции или услуг. Можно показать, что научно-технический прогресс подчи- няется следующим законам: 1. Коммерческая ценность результатов фундаментальных научных исследований постоянно повышается. 1. Коммерческая ценность результатов фундаментальных научных исследований постоянно повышается. 15 Новый технологический уклад... 2. Стоимость технологий и наукоемкой продукции постоянно снижается. 3. Технологии не могут противоречить законам природы. 4. Распространение знаний и технологий не имеет границ. Новые знания и технологии приводят к новым способам организации производства, созданию качественно новых ви- дов продукции, следовательно, – и новых рынков. При этом технологические прорывы могут приводить к формированию новой системы социально-экономических отношений. Напри- мер, считается общепризнанным, что первая промышленная революция, произошедшая в результате изобретения паровой машины, дала импульс к переходу к капиталистическим фор- мам взаимоотношений. Последующие вторая, третья и четвертая промышленные революции [Винер 1958; Рифкин 2015; Шваб 2017] открывали новые возможности для создания новых технологий, направ- ленных на обеспечение жизнедеятельности человека (табл. 1). Таблица 1 Научно промышленные революции Таблица 1 Научно-промышленные революции Период Базовая технология Распределение энергии Топливо/ технологии Конец XVIII – начало XX вв. Паровая машина Локальные источники энергии Природное сырье с минимальной переработкой Конец XIX – начало XX вв. ДВС + электричество Производство Сеть Потребление Природные энергоносители / промышленная переработка Конец XX – начало XXI вв. ВИЭ + ИКТ Производство Потребление Сеть Силы природы / Высокотехно- логичные преобразователи Начало XXI в. Цифровая экономика SMART GRID Электрическая энергия Гуманитарно-технологическая революция После 2009 г. Технологии обеспечения жизнедеятельности: энергетика, материалы, ИКТ, науки о жизни и биотехнологии SMART GRID Электрическая энергия 16 В.В. ИВАНОВ. Контуры нового мирового уклада В.В. ИВАНОВ. Контуры нового мирового уклада Конкуренция капиталистической и социалистической систем сыграла важнейшую роль в научно-технологическом прогрессе, получившем мощный импульс в ходе Второй мировой войны. Очевидно, что в середине XX века человечеству не требовались дополнительные источники энергии − необходимые потребности вполне обеспечивались природными ресурсами. Также не было потребности в развитии ракетной техники. Однако для обеспече- ния победы в войне требовались новые виды оружия, что и стало причиной реализации Манхэттенского проекта США и Атомного проекта СССР, а также работ по освоению космического простран- ства и развитию цифровой вычислительной техники. Однако гонка вооружений не могла в полном объеме решить задачи социально-экономического развития, поскольку рынок вооружений достаточно ограничен и в основном контролиру- ется государственными структурами. В то же время рынок гражданской продукции представляет значительно больший интерес и по мере развития постоянно расширяется. Человек является потребителем конечной продукции и услуг, необхо- димых для удовлетворения собственных потребностей, а госу- дарство, наряду с обеспечением безопасности, должно решать и вопросы социального развития, для чего также требуется соответствующая продукция. Появление новых технологий и видов продукции сформи- ровали новые потребности и новые рынки. Так, например, рынка космических услуг не существовало до запуска в 1957 г. первого искусственного спутника Земли. Однако появившиеся перспективы использования космического пространства не только сформировали новые задачи для науки, но и кроме собственно рынка запуска космических аппаратов, создали новые рынки наукоемкой продукции и услуг (навигация, теле- коммуникации и т.д.). По мере развития, технологии стали неотъемлемой частью среды обитания человека [Дергачева 2015], что выдвинуло в повестку дня проблему техногенной безопасности − экологии технологий [Иванов 2011; Иванов 2015]. Следует также отметить, что создание и распространение но- вых технологий меньше подвержено внешнему регулированию, 17 Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) Новый технологический уклад... чем экономическая деятельность. Например, если в середине прошлого века только две страны имели ядерное оружие (США и СССР), то в настоящее время в «ядерном клубе» насчитыва- ется уже 10 стран, несмотря на принятые международные акты, направленные на нераспространение ядерного оружия. В целом же научно-технологическое развитие несет в себе значительные риски, обусловленные тем, что на начальной стадии познания и разработки новых технологий далеко не всегда можно дать ответ, насколько новая технология будет способствовать повышению качества жизни и не приведет ли она к негативным последствиям. Эта ситуация может быть описана следующей моделью (рис. 1). Результаты фундаментальных научных исследова- ний являются достоянием всего человечества. На базе фун- даментальных результатов создаются технологии, которые в свою очередь распространяются в культурном простран- стве. В.В. ИВАНОВ. Контуры нового мирового уклада Культурная среда условно может быть разделена на два сегмента: позитивный – ориентированный на развитие человечества, и консервативный – не ставящий целью гло- бальное развитие. В зависимости от того, в какой культурный сектор попадает технология, определяются и направления ее использования. При этом в случае попадания в консер- вативный сектор существуют высокие риски использования технологий в ущерб развитию. В этом плане показателен пример атомной энергии, первое практическое применение которой было осуществлено для бомбардировки в 1945 г. японских городов Хиросима и Нагасаки. Таким образом, разрыв между культурным и технологическим уровнем мо- жет приводить к непредсказуемым последствиям вплоть до глобальных катастроф. В настоящее время остаются малоисследованными угрозы и риски цифровых технологий, которые, как принято считать, составляют основу четвертой промышленной революции (IR-4) [Шваб 2017]. При ближайшем рассмотрении тезис о том, что Мир вступает в IR-4, выглядит не так однозначно. Прежде всего надо заметить, что в основе своей концепции К. Шваб использовал работу Н. Винера [Винер 1958], впервые изданную 18 В.В. ИВАНОВ. Контуры нового мирового уклада Рис. 1. От знаний к развитию Рис. 1. От знаний к развитию Рис. 1. От знаний к развитию в Лондоне в 1954 г., в которой изложены взгляды на промыш- ленную революцию на базе вычислительных систем. в Лондоне в 1954 г., в которой изложены взгляды на промыш- ленную революцию на базе вычислительных систем. Развитию цифровой техники человечество обязано фун- даментальным открытиям, прежде всего в области физики, энергетики, новых материалов и математики, позволившим создавать воспроизводительные вычислительные системы. Что же касается интересов общества, то это требует отдель- ного рассмотрения, поскольку пока нет однозначного ответа на принципиальные вопросы: повысило ли производительность труда создание нового сегмента реальной экономики – произ- водства многофункциональных цифровых систем? Повлияло ли массированное внедрение цифровых технологий на повы- шение качества жизни или только обеспечило новые источники прибыли? Какие риски и угрозы влекут за собой цифровые технологии? Ответы на эти вопросы требуют проведения спе- циальных комплексных исследований. Рассматривая перспективы развития цифровых технологий, необходимо особое внимание обратить на следующие возмож- ные риски и угрозы: – подчинение человека искусственному интеллекту, – лишение человека права на личное пространство и личную жизнь, – деградация человека за счет снижения интеллектуальной и физической нагрузки, – деградация человека за счет снижения интеллектуальной ф й – деградация человека за счет снижения интеллектуальной и физической нагрузки, и физической нагрузки, – тоталитарная система управления. 19 19 Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) Новый технологический уклад... Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) Новый технологический уклад... Наблюдаемые глобальные тенденции показывают, что на- чинает сбываться прогноз Д. Белла [Белл 1999] о переходе к постиндустриальному обществу. Этот переход будет осущест- вляться в рамках гуманитарно-технологической революции (ГТР) [Иванов 2017; Контуры... 2019; Иванов, Малинецкий 2019], суть которой заключается в синхронном развитии науки, техно- логий и культуры в направлении удовлетворения потребностей человека. В итоге ГТР сформируется постиндустриальный тех- нологический уклад (ПТУ) (табл. 2), основу которого составят технологии энергетики, материаловедения, жизнеобеспечения, а также информационные технологии, объединенные в техно- логические секторы [Иванов 2015, 78−84]. Всего можно выделить три типа технологических секторов. Таблица 2 Структура постиндустриального технологического уклада (ПТУ) Фундаментальные научные исследования Приоритеты социально- экономического развития Ядро технологического уклада Технологический сектор Базовые технологии Безопасность Жилье и ЖКХ Здравоохранение Образование Продовольствие Транспорт Энергетика Экология Управление ТС-1 Биотехнологии Лазерные технологии Нанотехнологии Ядерные технологии ТС-2 ИКТ Космические технологии Социальные технологии Технологии природопользования Энергетика ТС-3 NBICS-технологии ТС-1. Технологический сектор представляет собой совокуп- ность технологий, в основе которых лежат общие фундамен- тальные научные принципы. К ним относятся: ядерные техно- логии, лазерные технологии, нанотехнологии, биотехнологии. 20 В.В. ИВАНОВ. Контуры нового мирового уклада Например, в основе лазерных технологий лежит явление коге- рентного излучения, а собственно сектор лазерных технологий включает в себя широкий спектр от медицинских инструментов для проведения микрохирургических операций до мощных технологических систем обработки материалов. ТС-2. Технологический сектор как совокупность технологий, направленных на решение сходных задач, но различными ме- тодами, представляет собой множество технологий, базирую- щихся на различных исходных законах природы, но направ- ленных на решение одной задачи. Примерами ТС-2 являются социальные технологии (здравоохранение, образование и т.д.), информационные технологии, нетрадиционные источники энергии. Наиболее интенсивно развиваются информационно- коммуникационные технологии (ИКТ), которые вместе с энергетикой создают технологический фундамент постинду- стриального общества. Собственно энергетика представляет собой широкий спектр технологий – от добычи полезных ископаемых до создания альтернативных источников энергии. ТС-3. Технологический сектор как совокупность технологий, созданных в результате конвергенции наук. Технологический сектор третьего типа представляет собой совокупность тех- нологий, созданных на основе исследований, проводимых на стыке наук. В настоящее время все большее распространение получают нано-био-инфо-когнитивные технологии (NBIC). В результате ГТР сформируется новый мировой уклад, базирующийся на новых технологиях повышения качества жизни (постиндустриальный технологический уклад), новой культуре и новой экономической парадигме, суть которой составит переход от «человека для экономики» к «экономике для человека». Новый мировой уклад: возможные переходы Можно считать установленным, что новый мировой уклад будет формироваться на основе качественно новых технологий, ориентированных на повышение качества жизни. Вследствие 21 Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) Новый технологический уклад... высоких темпов распространения новых технологий главным объектом конкуренции на глобальном пространстве становится человеческий потенциал. Это обусловлено тем обстоятельством, что именно человек является единственным генератором про- рывных идей, которые в дальнейшем находят свое практическое применение. Поэтому государство, обладающее наилучшим человеческим потенциалом, имеет бесспорное преимущество, поскольку может создавать передовые технологии как для коммерческого использования, так и для обеспечения глобаль- ного военного превосходства, что позволяет активно влиять на мировой порядок. Картина новой реальности может быть выражена словами Э. Тоффлера: Мы мчимся к полностью иной структуре власти, которая соз- дает мир, разделенный не на две, а на три четко определенные, контрастирующие и конкурентные цивилизации. Первую из них символизирует мотыга, вторую – сборочная линия, третью – ком- пьютер. Измените все эти социальные, технологические и культурные эле- менты одновременно – и вы получите не переход, а преображение; не просто новое общество, но начало – как минимум – полностью новой цивилизации. Однако ввести на планете новую цивилизацию и ожидать мира и спокойствия – это верх политической наивности. У каждой цивили- зации есть свои экономические (не говоря уже о политических и во- енных) требования [Тоффлер 2005, 51]. Очевидно, что мировой уклад будет устанавливать огра- ниченное число институтов. Традиционный набор выглядит следующим образом: Общество, Власть и Бизнес. Но в новой реальности, по-видимому, в качестве системообразующего ин- ститута мирового уклада следует рассматривать информацию, которая сейчас, как уже отмечалось, превратилась в неотъем- лемый элемент среды обитания. Тогда распределение функций между основными институ- тами будет выглядеть следующим образом. Общество формирует требования к дальнейшему развитию, исходя из главного принципа – повышения качества жизни. 22 В.В. ИВАНОВ. Контуры нового мирового уклада Общество формирует Общественный заказ (идеологию), и для его реализации устанавливает систему власти. Власть (государство) устанавливает через систему законо- дательства механизмы и правила реализации Общественного заказа, доводит их до бизнеса через информационные каналы и контролирует реализацию, в т.ч. через правоохранительную и судебную систему. Бизнес обеспечивает практическую реализацию Обществен- ного заказа. При этом качество реализации определяется двумя основными параметрами – качеством жизни (по отношению к ведущим странам) и позицией страны в глобальном про- странстве, ее возможностями по установлению правил игры на глобальных рынках. Главным инструментом влияния Бизнеса на процессы являются финансы. Информация обеспечивает доведение до общества новых знаний, в том числе через систему образования, оповещение о текущем состоянии дел и возможных перспективах. Новый мировой уклад: возможные переходы Информа- ция, как и фундаментальная наука, не имеет границ и может рассматриваться в данном случае как основной интегрирую- щий институт. Таким образом, информация превращается из ресурса в инструмент управления, что мы уже наблюдали на примере денег. Тезис «кто владеет информацией – тот владеет миром» получает новое практическое значение. Исходя из этого, ключевым вопросом формирования нового мирового уклада становится определение взаимоотношений между Обществом-Властью-Бизнесом-Информацией. у ф р Рассмотрим в общем виде возможные сценарии формирова- ния нового мирового уклада Монополия на информацию принадлежит власти. В этом случае развитие пойдет по жестко заданным алгоритмам, будет осуществлен тотальный контроль над обществом и бизнесом. Что, как известно из практики, в итоге приведет к торможению развития и распаду системы. Монополия права на информацию принадлежит бизнесу. В этом случае бизнес через влияние на общество будет фор- мировать общественный заказ, в том числе на власть и новые потребности. Но при этом надо учитывать, что «исторический 23 Филос. науки / Russ. J. Philos. Sci. 2020. 63(5) Новый технологический уклад... капитализм – это социальная система, в которой именно те, кто действует по ее правилам, оказывают решающее влияние на социальное целое и задают некие условия. Это такая социальная система, в которой поле действия этих правил (закон стоимости) увеличивалось; те, кто навязывал эти правила, становились все менее склонны к социальному компромиссу; эти правила все более и глубже проникали в социальную ткань, несмотря на то, что общественное противодействие им становилось все сильнее и организованнее» [Валлерстайн 2018]. В итоге реализации этого сценария произойдет передел существующей системы глобального взаимодействия, в ре- зультате которого власть от политических структур и системы международных отношений перейдет к транснациональным финансово-промышленным альянсам. При этом государства, хотя формально сохранят свои границы и государственные институты, по факту будут полностью зависеть от этих альян- сов. Система образования, воспитания, распределения благ сформирует новую многоуровневую глобальную социальную структуру, не допускающую перехода с уровня на уровень. Поскольку основной идеей бизнеса является получение прибыли и накопление капитала, то все вопросы социального развития будут сведены к решению этой задачи. Ориентация на финансовую прибыль, прежде всего за счет финансовых спекуляций, приводит к усложнению доступа к финансовым ресурсам и к возникновению неравенства как внутри отдельной страны, так и на глобальном пространстве [Сорос 2004; Сти- глиц 2016], а также к эскалации политических, экономических кризисов и военных конфликтов. При этом нельзя исключить возможности глобального конфликта с использованием ядерно- го оружия в силу расширения доступа к этому вооружению. Монополия на информацию принадлежит обществу. В.В. ИВАНОВ. Контуры нового мирового уклада Поэтому следует ожидать достижения консенсуса основных глобальных институтов, обеспечивающих глобальное разви- тие, что по крайней мере обеспечит равновесное состояние и глобальную безопасность, а в идеале – даст новый импульс развитию человечества. Новый мировой уклад: возможные переходы В этом случае общество формирует заказ на дальнейшее развитие, а также создает условия для его выполнения. Однако, как по- казывает исторический опыт, в настоящее время отсутствуют механизмы эффективного контроля власти и бизнеса со сто- роны общества. Научные подходы к решению этой проблемы также не разработаны. 24 В.В. ИВАНОВ. Контуры нового мирового уклада ЦИТИРУЕМАЯ ЛИТЕРАТУРА Белл 1999 – Белл Д. Грядущее постиндустриальное общество. Опыт социального прогнозирования. – М.: Academia, 1999. Белл 1999 – Белл Д. Грядущее постиндустриальное общество. Опыт социального прогнозирования. – М.: Academia, 1999. Валлерстайн 2018 – Валлерстайн И. Исторический капитализм. Капиталистическая цивилизация. – М: УРСС, ЛЕНАНД, 2018. Винер 1958 – Винер Н. Кибернетика и общество. – М.: Издательство иностранной литературы, 1958. Всемирная история… 2013 – Всемирная история в 6 т. Т. 3. Мир в Новое раннее время. – М.: Наука, 2013. Геращенко 2009 – Геращенко В.В. Россия и деньги. Что нас ждет? – М.: Астрель: Русь-Олимп, 2009. Глазьев 1993 – Глазьев С.Ю. Теория долгосрочных технологических укладов. – М.: ВлаДар, 1993. Дергачева 2015 – Дергачева Е.А. Концепция социотехноприродной глобализации: междисциплинарный анализ. – М.: ЛЕНАНД, 2015. Иванов 2011 – Иванов В.В. Технологическое пространство и эколо- гия технологий // Вестник РАН. 2011. Т. 81. № 5. С. 414–418. Иванов 2015 – Иванов В.В. Инновационная парадигма ХХI. – М.: Наука, 2015. Иванов 2017 – Иванов В.В. Глобальная гуманитарно-технологическая революция: предпосылки и перспективы // Инновации. 2017. № 6. С. 3–8. Иванов, Малинецкий 2019 – Иванов В.В., Малинецкий Г.Г. Философ- ские основания гуманитарно-технологической революции // Фило- софские науки. 2019. Т. 62. № 4. С. 76–95. ф у Костомаров 2004 – Костомаров Н.И. Русская история в жизнеопи- сании ее главнейших деятелей. – М.: ЭКСМО, 2004. Контуры… 2018 – Контуры цифровой реальности: Гуманитарно- технологическая революция и выбор будущего / под ред. 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London: Verso (Russian translation: Moscow: URSS, Lenand, 2018). Weiner N. (1950) The Human Use of Human Beings: Cybernetics and So- ciety. Boston: Houghton Mifflin (Russian translation:Moscow: Izdatel’stvo inostrannoy literatury, 1958). 27
https://openalex.org/W4362477783	https://figshare.com/articles/journal_contribution/Supplementary_Figure_2_from_Novel_Immunotherapy_for_Malignant_Melanoma_with_a_Monoclonal_Antibody_That_Blocks_CEACAM1_Homophilic_Interactions/22499481/1/files/39957861.pdf	English	null	Supplementary Figure 6 from Novel Immunotherapy for Malignant Melanoma with a Monoclonal Antibody That Blocks CEACAM1 Homophilic Interactions	null	2,023	cc-by	58	B ic lysis 30 40 Control RNA KD#1 KD#2 * * KD#2 % Specifi 0 10 20 KD#2 TIL014 JKF6 B ic lysis 30 40 Control RNA KD#1 KD#2 * * KD#2 % Specifi 0 10 20 KD#2 TIL014 JKF6 B ic lysis 30 40 Control RNA KD#1 KD#2 * * % Specifi 0 10 20 TIL014 JKF6
https://openalex.org/W2892648808	http://www.spatial-economics.com/images/spatial-econimics/2010_4/SE.2010.4.082-105.Devaeva.pdf	Russian	null	Commodity Markets in Northeast Asia:Directions for Exports from the Russian Far East	Prostranstvennaâ èkonomika	2,010	cc-by	9,367	Пространственная Экономика 2010. ¹ 4. С. 82—105 Пространственная Экономика 2010. ¹ 4. С. 82—105 УДК 339.9(5-012) Е. И. Деваева, Т. Е. Котова1 © Деваева Е. И., Котова Т. Е., 2010 Статья подготовлена при поддержке проектов ДВО РАН № 09-I-П24-01, № 09-I-00Н-01. Д , , Статья подготовлена при поддержке проектов ДВО РАН № 09-I-П24-01, № 09-I-00Н-0 Товарные рынки Северо-Восточной Азии: ориентиры для экспорта Дальнего Востока России2 Рассмотрены динамические и структурные изменения, происходящие на рынках рыбо- и лесопродукции, продукции ТЭК в странах Северо- Восточной Азии. Показаны позиции России на отдельных товарных рынках Китая, Японии и Республики Корея. Конъюнктура товарных рынков, производство, потребление, экспорт, импорт, товарные ниши, Северо-Восточная Азия, Китай, Республика Ко- рея, Япония, Россия, российский Дальний Восток. В условиях прогрессирующей глобализации мировой экономики и формирования центров экономической активности, в том числе и на субре- гиональном уровне, долгосрочные перспективы экономического развития Дальнего Востока России во многом будут зависеть от степени его вовлечен- ности в процесс международного разделения труда. Реальность получения экономикой Дальнего Востока долгосрочных выгод от участия в этом про- цессе напрямую будет связана с изменениями параметров внешней среды, в частности, динамикой и структурой спроса со стороны сопредельных стран Северо-Восточной Азии (СВА) на продукцию базовых отраслевых комплек- сов региона, и прежде всего, рыбохозяйственного, лесопромышленного и топливно-энергетического. Это обусловлено, во-первых, тем, что Китай, Япония и Республика Корея традиционно являются основными внешними 82 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ ПЭ № 4 2010 рынками для российского Дальнего Востока, и во-вторых, продукция ре- сурсных отраслей традиционно составляет основу российского, в том числе дальневосточного, экспорта в эти страны1 (см.: [1; 2]). В настоящее время на рынки стран СВА направляется 83,3% произво- димой на российском Дальнем Востоке деловой древесины, 74,1% добытой нефти, 38,7% выловленной рыбы и морепродуктов, 3,8% угля, 2,4% нефте- продуктов2. При этом большая часть экспортных потоков лесопродукции (около 87%) с территории Дальнего Востока замыкается на китайский ры- нок, экспорт нефти ориентирован в основном на рынки Республики Корея (46,3%) и Японии (41,6%), рыбопродукции — Китая (56,7%) и Республики Корея (36,6%), угля — Японии (57,9%) и Китая (31,6%), нефтепродуктов — Японии (57,3%) и Китая (31,5%). Высокий спрос со стороны стран СВА на продукцию ресурсного секто- ра экономики Дальнего Востока России позволил региону занять довольно устойчивые позиции в импорте этих стран отдельных видов продукции. Наи- более весомая роль принадлежит российскому Дальнему Востоку в форми- ровании объемов потребления рыбы свежемороженой (25,4%), деловой дре- весины (15,4%) и морепродуктов (12,9%). Что касается присутствия Дальнего Востока России на рынке топливно- энергетических ресурсов (ТЭР) стран СВА, то до настоящего времени его роль в структуре импорта энергетических ресурсов этих стран не столь за- метна: на долю дальневосточной продукции в импорте СПГ, нефти сырой и угля стран СВА приходится соответственно 3; 2,4 и 0,2%. 1 За счет регионального вывоза формируется 45% российского экспорта деловой древе- сины в страны СВА, 57% нефти, 98% рыбы и морепродуктов. В российском экспорте сжи- женного природного газа (СПГ) в восточноазиатском направлении Дальний Восток является монополистом (100%). Товарные рынки Северо-Восточной Азии: ориентиры для экспорта Дальнего Востока России2 Вместе с тем долго- срочные перспективы развития международного торгово-экономическо- го сотрудничества российского Дальнего Востока со странами СВА будут в дальнейшем тесно связаны именно с топливно-энергетическим комплексом экономики региона. 2 Объемы экспорта оценивались по данным таможенной статистики. Топливно-энергетические ресурсы В последние годы происходит существенное повышение доли стран СВА в мировом потреблении продукции ТЭК. Рост использования энергоносителей в СВА обусловлен, главным образом, быстрым ростом эко- номики Китая. Так, за период 1995—2008 гг. потребление нефти в Китае вы- росло в 2,3 раза, угля — в 2 раза, в то время как общемировой рост соответ- ственно составил 120,6 и 145,7% . 83 Е. И. Деваева, Т. Е. Котова ПЭ № 4 2010 Растущие потребности КНР в энергоресурсах являются основной при- чиной подъема конъюнктуры на мировом рынке угля. Несмотря на попытки КНР снизить темпы роста потребления угля, он по-прежнему занимает ли- дирующую позицию в структуре валового энергопотребления. Если в 2000 г. на этот вид топлива приходилось около 64% всего потребления первичных энергоресурсов в Китае, то к настоящему времени — 70,2%. Начиная с 2003 г., в условиях бурного экономического роста, а также в результате принятия правительственной программы перевода части тепло- вых электростанций с нефти на уголь, потребление твердого топлива в элек- троэнергетике Китая стало заметно возрастать. На этот же период пришлось стремительное развитие китайской сталелитейной промышленности, по- влекшее за собой увеличение потребления угля для нужд металлургии, вслед- ствие чего Китай стал наращивать объемы его импорта. В 2008 г. страна импортировала 40,7 млн т угля, при этом 79,1% всего объ- ема поставок обеспечивали страны Восточной Азии (Вьетнам — 41,4%, Ин- донезия — 28,4, КНДР — 6,2%) и Россия (1,9%). В течение последнего десятилетия, вне зависимости от конъюнктуры не- фтяных цен на международных рынках, в Китае происходило существенное увеличение спроса на нефть и нефтепродукты, который почти на 2/3 удов- летворялся за счет импорта (табл. 1). За период 2000—2008 гг. объемы импор- та сырой нефти возросли в 2,5 раза (до 178,9 млн т). К настоящему времени по импорту нефти Китай уступает только США и Японии. Одновременно рост потребления нефти именно в этой стране выступает фактором давления на рынок и повышение цен. Возрастающие потребности в жидком топливе обусловлены быстрым, преимущественно экстенсивным, экономическим ростом и увеличением численности населения КНР. На современном этапе Китай планомерно проводит политику диверси- фикации источников поставки нефти и поиска новых торговых партнеров. В перспективе ожидается, что Китай станет самым крупным в мире потреби- телем нефти1. К 2030 г. спрос на нее возрастет до 740—750 млн т, в том числе до 75% потребностей китайской экономики будет удовлетворяться за счет импорта (540—550 млн т) [11]. В отличие от нефти природный газ в Китае до настоящего времени не нашел широкого применения. 1 В последние годы Китай интенсивно наращивает потенциал нефтепереработки с целью увеличения нефтеперерабатывающих мощностей до 8,8 млн баррелей в сутки к 2011 г. (в 2008 г. — 6,4 млн баррелей в сутки) и снижения отрицательного сальдо в торговле нефтепродуктами. Топливно-энергетические ресурсы Так, если в мире в среднем доля газа в структуре энерго- потребления составляет 24,1%, то в Китае — всего 3,6%. К числу проблем, сдер- живающих широкое использование газа в Китае, относятся: точечный характер газификации, отсутствие единой национальной системы транспортировки и снабжения, слабая законодательная база и фрагментация рынка газа. 1 В последние годы Китай интенсивно наращивает потенциал нефтепереработки с целью увеличения нефтеперерабатывающих мощностей до 8,8 млн баррелей в сутки к 2011 г. (в 2008 г. — 6,4 млн баррелей в сутки) и снижения отрицательного сальдо в торговле нефтепродуктами. 84 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ ПЭ № 4 2010 Таблица 1 Производство и потребление первичных топливно-энергетических ресурсов в Китае Показатель 1995 2000 2005 2008 2008 / 2000, % Уголь, млн т производство 1361 998 2205 2793 279,9 сальдо торговли 27 53 46 5 9,4 видимое потребление* 1334 945 2159 2788 295,0 Нефть сырая, млн т производство 149,0 162,6 180,8 189,7 116,7 сальдо торговли —12,0 —60,0 —118,7 —175,2 292,0 видимое потребление* 161,0 222,6 299,5 364,9 163,9 Природный газ, млрд м3 производство 17,9 27,2 49,3 76,1 279,8 сальдо торговли 0,2 2,7 2,5 —4,6 —170,4 видимое потребление* 17,7 24,5 46,8 80,7 329,4 Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источники: составлено на основе данных [3; 7; 13]. Производство и потребление первичных топливно-энергетических ресурсов в Китае Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источники: составлено на основе данных [3; 7; 13]. Собственные запасы природного газа в КНР относительно невелики и оцениваются в 2,5 трлн м3. В 2008 г. объемы добычи природного газа соста- вили 76,1 млрд м3, импорта (как в газообразном, так и в сжиженном виде) — 4,6 млрд м3. Поскольку Китай в последние годы сталкивается с проблемой изменения энергобаланса в сторону высококачественных энергоносителей — возобнов- ляемой энергии, природного газа и нефти, правительство Китая планирует к 2030 г. довести объем потребления газа до 270—280 млрд м3. Вторым после Китая крупнейшим потребителем энергоресурсов в СВА является Япония. В отличие от Китая Япония обладает незначительным за- пасом энергетических ресурсов: 0,044 млрд баррелей доказанных запасов нефти, 0,74 трлн кубических футов природного газа и 355 млн т извлекаемых запасов угля. За исключением ядерной энергетики, новых и возобновляемых источников энергии, а также небольшого объема внутреннего производства ископаемого топлива, большая часть потребностей Японии в энергии удов- летворяется за счет импорта (табл. 2). За период 1995—2008 гг. Топливно-энергетические ресурсы импорт ка- менного угля увеличился с 126,2 до 191,7 млн т, нефти — с 267 до 202,4 млн т, сжиженного природного газа — с 42,9 до 69,3 млрд т (свыше 40% мировой торговли СПГ). 85 Е. И. Деваева, Т. Е. Котова № 4 2010 Таблица 2 Потребление первичных топливно-энергетических ресурсов в Японии Показатель 1995 2000 2005 2008 2008/ 2000, % Уголь, тыс. т производство 6,3 2,5 0,9 1,2 48,0 сальдо торговли —126,2 —145,3 —180,8 —191,7 131,9 видимое потребление* 132,4 147,7 181,7 192,9 130,6 Нефть сырая, млн т производство 0,9 0,8 0,9 1,0 125,0 сальдо торговли —266,9 —212,0 —210,3 —202,4 95,5 видимое потребление* 267,8 212,8 211,3 203,4 95,6 Природный газ, млрд м3 производство 0,0 0,0 0,0 0,0 — сальдо торговли —57,9 —72,3 —78,6 —93,7 129,6 видимое потребление* 57,9 72,3 78,6 93,7 129,6 Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источники: составлено на основе данных [3; 7; 13]. Таблица 2 Потребление первичных топливно-энергетических ресурсов в Японии Т Потребление первичных топливно-энергетических ресурсов в Японии Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источники: составлено на основе данных [3; 7; 13]. Одним из наиболее потребляемых энергетических ресурсов в Японии яв- ляется нефть, хотя доля последней в структуре энергопотребления страны снизилась к настоящему времени до 44% (в 1970 г. — 80%), что стало возмож- ным благодаря успешной реализации политики повышения энергоэффектив- ности и расширению использования ядерной энергии и природного газа. Основными поставщиками сырой нефти на японский рынок являются страны Ближнего Востока (Саудовская Аравия, ОАЭ, Иран, Катар, Кувейт), на долю которых приходится 87,5% общего объема нефтяных поставок, Ин- донезия — 3,0 и Россия — 2,4%. В связи с замедлением темпов роста экономики и увеличением объемов использования альтернативных источников энергии в Японии прогнозирует- ся снижение потребления нефти. К 2030 г. правительством Японии в рамках реализации политики, направленной на снижение доли нефти в структуре энергопотребления, планируется увеличить использование атомной энергии и природного газа, доля которого в энергобалансе страны должна возрасти до 20—23%. Япония занимает второе место по объему нефтеперерабатывающих мощ- ностей в Восточной Азии после Китая (4,7 млн баррелей нефти в день). В последние годы на фоне сокращения объемов внутреннего потребления в Японии наблюдается ситуация товарного перепроизводства нефтепродук- тов. 1 Крупнейшим нефтеперерабатывающим предприятием Республики Корея является за- вод корпорации SK в г. Ульсан, перерабатывающий 817 тыс. баррелей в день, который является вторым по величине НПЗ в мире. В Республике Корея также находится третий по величине НПЗ в мире, перерабатывающий 650 тыс. баррелей в день — завод г. Йосу, принадлежащий компании GS Caltex. Топливно-энергетические ресурсы Данное обстоятельство, в свою очередь, стимулирует нефтеперерабаты- вающие компании к активному поиску внешних рынков сбыта своей про- 86 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ дукции (особенно в Китае), что в долгосрочной перспективе может усилить позиции Японии как одного из крупнейших экспортеров нефтепродуктов в Восточной Азии. Значительную долю в структуре энергетического баланса Японии по- прежнему занимает уголь. Основными потребителями угля в Японии яв- ляются сталелитейная промышленность и производители кокса (44%), электроэнергетика (42%), прочие отрасли промышленности (12%), комму- нально-бытовое хозяйство (2%). В географической структуре импорта твер- дого топлива лидируют Австралия (63,3%), Индонезия (13%), Канада (7,6%), Китай (7,3%), Россия (5,4%). Поставки газа в Японию осуществляются преимущественно из стран Юго- Восточной Азии (ЮВА) (47,3%) и Ближнего Востока (24,4%). На сегодняшний день на долю природного газа в энергобалансе Японии приходится 16,6%. В настоящее время японские потребители в основном используют природный газ, импортируемый в сжиженном виде (СПГ), незначительные объемы до- бываются внутри страны (префектуры Ниигата, Хоккайдо и др.). Несмотря на существенное увеличение объемов потребления природного газа (за последнее десятилетие оно возросло на 30%), его дальнейший рост сдерживается слабым развитием газопроводной сети (всего 2100 км). Действующие терминалы по приему СПГ (свыше 20 единиц) не связаны между собой, газ с них преимуще- ственно поступает на расположенные рядом теплоэлектростанции. Наряду с Японией крупнейшим потребителем СПГ в СВА является Ре- спублика Корея (2-е место в мировом импорте СПГ). На протяжении послед- них десятилетий эта страна занимала одно из ведущих мест в мире по росту потребления первичных энергоносителей, что было обусловлено стабильно высоким темпом ее экономического развития. Высокая энергоемкость ВВП Республики Корея определяется наличием в структуре промышленного про- изводства сталелитейной, судостроительной и других энергоемких отраслей промышленности, являющихся основой экономики страны. Основная доля в суммарном энергопотреблении приходится на промышленность (42%) и электроэнергетику (20%). Собственная обеспеченность природными топливно-энергетическими ресурсами Республики Корея самая низкая среди стран региона СВА. Ко- эффициент зависимости Республики Корея от импорта энергоносителей, определяемый как отношение чистого импорта к общему объему потребле- ния энергоресурсов, составляет 97%. Основным видом энергоресурсов, используемых в Республике Корея, яв- ляется нефть сырая, на долю которой в структуре энергопотребления прихо- дится 43%. За период 1995—2008 гг. объем поставок нефти на рынок страны возрос с 84,3 млн т до 104,7 млн т. Около 80% объемов импорта сырой нефти 87 Е. И. Деваева, Т. Е. Котова ПЭ4 ПЭ № 4 2010 приходилось на страны Ближнего Востока. Удельный вес стран Восточной Азии в суммарном объеме импорта сырой нефти находился на уровне 6%, России — 4%. В текущем десятилетии объемы импорта нефти Республики Корея пре- вышали потребление, так как страна реэкспортировала около четверти вво- зимой нефти и нефтепродуктов в основном в страны Восточной Азии. В на- стоящее время рынок характеризуется избытком нефтеперерабатывающих мощностей (около 2,6 млн баррелей в день)1 и является крупнейшим среди стран СВА экспортером нефтепродуктов. В современной структуре энергопотребления Республики Корея вторую позицию после нефти занимает уголь (свыше 27%). Собственные запасы угля в стране невелики (80 млн т), что предопределяет незначительный объ- ем его добычи в 2,8 млн т/год (табл. 3). Объем импорта угля в 2008 г. составил 97,4 млн т, что вдвое превышает уровень 1995 г. В географической структуре импорта твердого топлива лидирующие позиции принадлежали Австралии (33%), Индонезии (28,7%) и Китаю (22,6%). На долю России приходилось 7,2% общего объема поставок. Таблица 3 Потребление первичных топливно энергетических ресурсов Таблица 3 Потребление первичных топливно-энергетических ресурсов в Республике Корея Показатель 1995 2000 2005 2008 2008/ 2000, % Уголь, тыс. т производство 5,7 4,2 2,8 2,8 66,7 сальдо торговли —43,8 —63,8 —76,8 —97,4 152,7 видимое потребление* 49,6 68,0 79,6 100,1 147,2 Нефть сырая, млн т производство 10,6 0,0 0,0 0,0 — сальдо торговли —84,3 —101,4 —103,1 —104,5 103,1 видимое потребление* 94,9 101,4 103,1 104,5 103,1 Природный газ, млрд м3 производство 0,0 0,0 0,0 0,0 — сальдо торговли —10,2 —21,0 —33,7 —39,7 189,0 видимое потребление* 10,2 21,0 33,7 39,7 189,0 Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источники: составлено на основе данных [3; 7; 13]. Потребление первичных топливно-энергетических ресурсов в Республике Корея Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источники: составлено на основе данных [3; 7; 13]. Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источники: составлено на основе данных [3; 7; 13]. 1 Крупнейшим нефтеперерабатывающим предприятием Республики Корея является за- вод корпорации SK в г. Ульсан, перерабатывающий 817 тыс. баррелей в день, который является вторым по величине НПЗ в мире. В Республике Корея также находится третий по величине НПЗ в мире, перерабатывающий 650 тыс. баррелей в день — завод г. Йосу, принадлежащий компании GS Caltex. 88 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ В перспективе Республика Корея планирует импортировать уголь из ЮАР, несмотря на географическую удаленность и высокие ставки фрахта. Переориентация на южноафриканский рынок обусловлена ограниченными возможностями наращивания объемов поставок со стороны традиционных для Республики Корея экспортеров (Китая, Австралии, Индонезии и Рос- сии) вследствие роста потребления угля на внутреннем рынке этих стран. Газовый рынок Республики Корея практически полностью зависит от импорта СПГ. В 2008 г. Республика Корея импортировала 39,7 млрд м3 газа. Основными поставщиками являлись Катар (31,4%), Малайзия (24,1%), Оман (18,7%) и Индонезия (14,7%). В меньшем количестве газ в страну поставляли производители из Египта (4,4%), Брунея (2,3%) и Австралии (1,6%). В настоящее время среди стран СВА корейская экономика в наибольшей степени ориентирована на использование природного газа. Наличие разви- той внутренней газотранспортной сети, соединяющей береговые терминалы СПГ с основными центрами потребления газа в стране, позволяет широко использовать природный газ не только для производства электроэнергии, но и в промышленном и жилищно-коммунальном секторах экономики. Доля газа в балансе потребления энергоресурсов страны увеличилась за период 1995—2008 гг. с 6,2 до 14,9% и, по прогнозным оценкам, достигнет к 2030 г. 21% [11]. В долгосрочной перспективе импорт первичных топливно-энергетиче- ских ресурсов для Республики Корея, равно как для Китая и Японии, будет экономически неизбежен. Общей тенденцией в обозначенный период для стран СВА станет значительное увеличение потребления природного газа и снижение доли нефти в энергопотреблении. Основными потребителями сжиженного газа будут оставаться Япония и Республика Корея. К 2020 г. в Японии прогнозируется увеличение потребления природного газа до 110—115 млрд м3, к 2030 г. — 125—130 млрд м3. Спрос Японии на при- родный газ будет полностью удовлетворяться за счет импорта. К настоящему времени Японией уже заключен ряд соглашений о долгосрочных поставках СПГ, совокупный объем которых должен составить порядка 15 млн т (ме- сторождения Австралии (Иксис), Индонезии (Абади) и Восточного Тимора). Дополнительно сжиженный природный газ будет импортироваться из Рос- сии (с месторождений Сибири и Дальнего Востока). Согласно прогнозам Центра исследования проблем энергетики стран АТР (APERC), рост потребления природного газа в Республике Корея к 2020 г. может приблизиться к 60—70 млрд м3. До 2030 г. объем потребления природного газа может достичь 80—85 млрд м3. Учитывая, что заключенные к настоящему времени контракты способны покрыть лишь часть этого объ- ема, а также наличие в мире свободных незаконтрактованных мощностей по 89 Е. И. Деваева, Т. Е. Котова ПЭ4 производству СПГ, Республика Корея может заключить долгосрочные кон- тракты на его импорт с Россией. ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ Что касается Китая, то, по мнению экспертов APERC, объем потребле- ния природного газа к 2020 г. будет находиться в пределах 165—170 млрд м3, к 2030 г. — 265—270 млрд м3. В целом спрос на газ в СВА возрастет к 2020 г. до 345—350 млрд м3, к 2030 г. — до 475—480 млрд м3, за счет импорта будет удовлетворяться до 70% потребностей в голубом топливе. Потребление нефти в ведущих странах СВА с учетом прогнозируемых темпов роста валового внутреннего продукта к 2030 г. может достичь 1120— 1130 млн т в год, причем зависимость стран СВА от импорта сырой нефти возрастет по сравнению с 2008 г. с 71,6 до 82%. Более медленными темпами в сравнении с нефтью и газом в странах СВА будет расти потребление угля. Использование угля будет все больше концен- трироваться в сфере производства энергии, где он останется доминирующим видом топлива. Спрос на уголь в энергетическом секторе будет расти парал- лельно с ожидаемым повышением цен на газ. По оценкам APERC, объем по- требления угля к 2020 г. в странах СВА составит 4,4 млрд т, к 2030 г. — 5,3 млрд т, при этом основной объем (свыше 90%) будет приходиться на Китай. Таким образом, Северо-Восточная Азия в перспективе будет оставаться одним из крупнейших потребителей энергетического сырья в мире, что име- ет особенно важное значение для России и ее восточных регионов. К 2030 г. объемы дальневосточного экспорта продукции ТЭК в восточноазиатском на- правлении могут возрасти в 3,7 раза относительно 2008 г. (до 34,0 млрд долл. в ценах 2008 г.), в результате чего продукция ТЭК составит основу дальнево- сточного экспорта (62% от общего объема регионального экспорта). Лесная продукция Неопределенными остаются долгосрочные перспективы относи- тельно наращивания объемов дальневосточного экспорта лесопродукции в страны СВА. В настоящее время крупнейшим импортным рынком лесной продукции является Китай (52,5%), в товарной структуре импорта которого доминируют круглые лесоматериалы (из США и России), целлюлоза и цел- люлозно-бумажные изделия. В последнее десятилетие благодаря расширению внутреннего спроса и росту масштабов инвестиций высокими темпами в Китае развивается дере- вообрабатывающая и целлюлозно-бумажная промышленность. Особое вни- мание обращает на себя стремительное наращивание объемов производства древесных плит, бумаги и картона (табл. 4). Увеличение объемов производства древесных плит (в 5,5 раза за период 90 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ 1995—2008 гг.) связано, главным образом, с развитием внутреннего рынка недвижимости, увеличением объемов строительства и производства мебе- ли, ростом уровня жизни населения. В 2008 г. в структуре выпуска древесных плит на долю фанеры приходилось 45,3%, ДВП — 36,4%, ДСП — 14,4%. По- рядка 90% листовых материалов потреблялось внутри страны, главным об- разом в строительстве, во внутренней отделке помещений и в производстве мебели (в том числе предназначенной для экспорта). Таблица 4 Производство и потребление лесных товаров в КНР Товарная позиция 1990 1995 2000 2005 2008 2000/ 1990, % 2008/ 2000, % Производство Деловая древесина, млн м3 91,2 101,2 96,0 94,7 95,8 105,3 99,8 Пиломатериалы, млн м3 23,6 25,6 7,3 18,8 29,3 30,9 401,4 Древесные плиты, млн м3 3,0 14,5 19,2 55,6 79,9 640,0 416,1 шпон, млн м3 0,0 0,1 0,4 3,1 3,1 — 775,0 фанера, млн м3 1,3 8,1 10,7 26,0 36,2 823,1 338,3 ДСП, млн м3 0,5 4,4 3,0 5,8 11,5 600,0 383,3 ДВП, млн м3 1,2 1,8 5,2 20,6 29,1 433,3 559,6 Целлюлоза, млн т 20,4 36,2 30,3 67,0 99,8 148,5 329,4 Бумага и картон, млн т 17,4 28,5 34,7 60,4 83,7 199,4 241,2 Видимое потребление* Деловая древесина, млн м3 98,1 106,6 111,0 124,9 133,2 113,1 120,0 Пиломатериалы, млн м3 24,6 27,9 12,0 25,6 37,1 48,8 309,2 Древесные плиты, млн м3 5,3 19,2 24,2 52,5 72,3 456,6 298,8 шпон, млн м3 0,1 0,6 1,1 3,3 3,2 в 11 р. Лесная продукция 290,9 фанера, млн м3 3,1 11,3 11,7 21,7 30,0 377,4 256,4 ДСП, млн м3 0,6 4,6 3,6 6,7 12,0 600,0 333,3 ДВП, млн м3 1,5 2,7 7,7 20,8 27,1 513,3 351,9 Целлюлоза, млн т 22,4 39,9 37,5 85,6 132,0 167,4 352,0 Бумага и картон, млн т 19,2 31,5 40,8 64,4 84,2 212,5 206,4 П Табли Производство и потребление лесных товаров в КНР Примечания: * масса из древесины или из других волокнистых целлюлозных материалов; регенерируемые бумага или картон (макулатура и отходы); ** видимое потребление = внутрен- нее производство + импорт — экспорт. Источник: составлено на основе данных [6] Примечания: * масса из древесины или из других волокнистых целлюлозных материалов; регенерируемые бумага или картон (макулатура и отходы); ** видимое потребление = внутрен- нее производство + импорт — экспорт. И [6] р р р Источник: составлено на основе данных [6]. Источник: составлено на основе данных [6]. Наряду с листовыми материалами высокие темпы роста характерны для производства и потребления целлюлозно-бумажных изделий. За период 1995—2008 гг. производство данной продукции увеличилось с 28,5 млн т до 83,7 млн т. За тот же период потребление увеличилось с 31,4 млн т до 84,2 млн т, выводя Китай на второе место в мире после США. Однако из-за технических 91 Е. И. Деваева, Т. Е. Котова ПЭ № 4 2010 и технологических ограничений китайский рынок вынужден импортировать высококачественные целлюлозно-бумажные изделия для восполнения вну- треннего спроса. В структуре потребления целлюлозно-бумажных изделий около 60% при- ходится на долю картона и оберточной и упаковочной бумаги. Это значение намного превышает среднемировой уровень и является результатом бурно- го развития промышленного сектора. В то же время в силу низкого уровня доходов населения Китай значительно меньше, чем остальная часть мира, потребляет газетной, печатной и писчей бумаги. Доля данной продукции со- ставляет 32% общего потребления целлюлозно-бумажных изделий. Высокие темпы роста производства и потребления продукции дерево- обрабатывающей и целлюлозно-бумажной отраслей, на долю которых в по- следние годы приходится порядка 60% ВДС лесопромышленного комплекса КНР [14], отражают изменение структуры производства лесотоваров, смещая акцент на производство продукции, обеспечивающей более существенный прирост добавленной стоимости, и высокую зависимость Китая от импорта необработанной древесины. В последние годы Китай занимает лидирующие позиции в мировом им- порте лесопродукции. Основу китайского импорта составляют целлюлоза (49,1%), деловая древесина (21,5%), целлюлозно-бумажные изделия (17,5%) и пиломатериалы (8,2%). Обращает на себя внимание тот факт, что в течение 1995—2008 гг. Лесная продукция на фоне увеличения в структуре лесного импорта КНР удель- ного веса целлюлозы (с 17,7 до 49,1%) и деловой древесины (с 7,9 до 21,5%) стабильно сокращалась доля листовых материалов (с 19,4 до 2%) и целлюлоз- но-бумажных изделий (с 48,7 до 17,5%). Произошедшая в указанный период трансформация товарной структуры импорта явилась следствием растущего спроса китайского рынка на древесное сырье, особенно высококачествен- ные сорта древесины, и гибкой таможенно-тарифной политики государства. Так, с 2000 г. ставки таможенного тарифа на импорт круглого леса и древес- ной целлюлозы были понижены до нулевого уровня. За период 2001—2008 гг. существенные изменения произошли в геогра- фической структуре импорта целлюлозы Китая: на фоне снижения доли стран АСЕАН (с 16,9 до 7,8% в объеме импорта целлюлозы) заметно усилили свои позиции страны ЕС (с 6,4 до 17,6%). Удельный вес России, обеспечива- ющей в 2001 г. около 13% объема импортных поставок целлюлозы на рынок Китая, к настоящему времени сократился до 6,4%. Ведущим экспортером неизменно остаются США (26,7% в 2008 г.). Крупнейшим поставщиком деловой древесины на китайский рынок яв- ляется Россия. За период 1995—2008 гг. объемы импортируемого российско- го круглого леса возросли практически в 78 раз. В результате доля России в 92 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ ПЭ4 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ структуре китайского импорта необработанных лесоматериалов увеличилась с 14,2 до 49,4%. Другими крупными экспортерами круглого леса являются страны Юго-Восточной Азии (10,4%), Габон (8%), Папуа — Новая Гвинея (8%), Новая Зеландия (4,8%) и Соломоновы острова (4,1%). В структуре импорта пиломатериалов, так же как и в импорте круглого леса, России принадлежат лидирующие позиции. В 2008 г. российские пи- ломатериалы составляли 19,5% от общего объема импорта пиломатериалов в Китае (в 1995 г. — 4,3%). В отличие от импорта в структуре экспорта лесопродукции Китая в 2008 г. доминировали товары с высокой добавленной стоимостью: мебель (39,2%), целлюлозно-бумажные изделия (27,4%), листовые материалы (17%), а также строительные и конструкционные изделия из древесины (6,2%). На долю прочих готовых изделий из древесины, к которым относятся упаковоч- ная тара, бондарные изделия, столовые и кухонные принадлежности, ин- струменты и различные декоративные изделия, приходилось немногим бо- лее 8%. В целом доля готовой продукции в лесном экспорте Китая возросла в 2008 г. по сравнению с 1995 г. с 84 до 98%. За период с 1995 по 2008 г. среднегодовые темпы роста экспорта бумаги и картона составили 52,4%. Лесная продукция Объемы экспорта мебели за этот период увели- чились в 18,4 раза, листовых материалов — 5,8 раза, в том числе фанеры — в 8,7 раза, ДВП — 8,5 раза, ДСП — в 1,6 раза. Значительный рост экспорта ли- стовых материалов позволил Китаю занять лидирующие позиции в мировом экспорте фанеры (34%) и МДФ (23,6%). В настоящее время Китай поставляет на внешний рынок 31,6% произ- веденной в стране фанеры, 16,7% древесностружечных плит, 9,3% бумаги и картона, 6,8% шпона. При этом за период 1995—2008 гг. доля экспорта в про- изводстве фанеры и ДСП увеличилась практически втрое. Основными рынками сбыта листовых материалов являются страны ЕС (22,8%), США (21,5%), страны Ближнего Востока (14,4%) и Япония (6,2%); мебели — США (41,4%), страны ЕС (20%) и Ближнего Востока (7%); цел- люлозно-бумажных изделий — США (19%), Гонконг (14,5%), страны ЕС (12,1%) и Юго-Восточной Азии (9,5%), а также Япония (8,5%). Наряду с Китаем к числу основных потребителей лесопродукции в СВА относится Япония, экономика которой испытывает дефицит деловой древе сины, поскольку лесной сектор страны покрывает лишь пятую часть вну- тренних потребностей. Пик лесозаготовок в стране приходился на 1967 г. (53,0 млн м3), и с тех пор производство древесины и других первичных лес- ных продуктов в Японии непрерывно снижается (табл. 5). К настоящему времени объем производства деловой древесины составляет 17,7 млн м3, пи- ломатериалов — 10,9 млн м3. 93 Е. И. Деваева, Т. Е. Лесная продукция Котова ПЭ4 № 4 2010 Таблица 5 Производство и потребление лесопродукции в Японии Товарная позиция 1990 1995 2000 2005 2008 2000/ 1990, % 2008/ 2000, % Производство Деловая древесина, млн м3 29,3 22,9 18,0 16,2 17,7 61,4 98,3 Пиломатериалы, млн м3 29,8 24,5 17,1 12,8 10,9 57,4 63,7 Древесные плиты, млн м3 8,6 7,0 5,6 5,4 4,6 65,1 82,1 шпон, млн м3 0,3 0,2 0,1 0,1 0,1 33,3 100,0 фанера, млн м3 6,4 4,4 3,2 3,2 2,6 50,0 81,3 ДСП, млн м3 1,0 1,3 1,3 1,2 1,1 130,0 84,6 ДВП, млн м3 0,9 1,1 1,0 0,9 0,8 111,1 80,0 Целлюлоза, млн т 25,4 26,4 29,4 33,1 33,2 115,7 112,9 Бумага и картон, млн т 28,1 29,7 31,8 29,3 28,4 113,2 89,3 Видимое потребление* Деловая древесина, млн м3 56,9 44,8 33,9 26,8 24,4 59,6 72,0 Пиломатериалы, млн м3 38,8 36,2 27,0 21,2 17,4 69,6 64,4 Древесные плиты, млн м3 12,4 13,0 11,8 11,3 9,2 95,2 78,0 шпон, млн м3 0,9 0,9 0,2 0,2 0,1 22,2 50,0 фанера, млн м3 9,3 8,8 8,2 7,9 6,2 88,2 75,6 ДСП, млн м3 1,2 1,7 1,6 1,6 1,5 133,3 93,8 ДВП, млн м3 1,0 1,7 1,6 1,6 1,4 160,0 87,5 Целлюлоза, млн т 28,8 30,3 32,3 31,6 31,6 112,2 97,8 Бумага и картон, млн т 28,3 30,2 31,9 29,9 28,3 112,7 88,7 Примечания: масса из древесины или из других волокнистых целлюлозных материалов; регенерируемые бумага или картон (макулатура и отходы); ** видимое потребление = внутрен- нее производство + импорт — экспорт. Источник: составлено на основе данных [6]. Примечания: * масса из древесины или из других волокнистых целлюлозных материалов; регенерируемые бумага или картон (макулатура и отходы); ** видимое потребление = внутрен- нее производство + импорт — экспорт. Источник: составлено на основе данных [6] Примечания: * масса из древесины или из других волокнистых целлюлозных материалов; регенерируемые бумага или картон (макулатура и отходы); ** видимое потребление = внутрен- нее производство + импорт — экспорт. р р р Источник: составлено на основе данных [6]. Одной из причин сокращения объемов производства является быстрый рост производственных затрат (стоимость трудовых ресурсов, объемы нало- говых отчислений), в то время как цена на древесину, произведенную внутри страны, снижается под давлением цен на импортное сырье. Сокращение объемов лесозаготовок привело к тому, что к настоящему времени Япония является одним из основных импортеров древесины в мире, на ее долю приходится порядка 10,5% мировой торговли деловой древеси- ной. 1 Импорт пиломатериалов из Финляндии, Швеции, Австрии, Чехословакии и Германии возник в начале 1990-х гг. и из-за большой удаленности экспортеров является своеобразным феноменом. Это объясняется перенасыщенностью европейского рынка лесоматериалов, стремлением европейских производителей приспособиться к высоким требованиям японско- го рынка древесины, а также высоким качеством европейской древесины. ПЭ №4 низкой обеспеченностью лесными ресурсами: собственные лесные ресурсы обеспечивают лишь 10% объемов потребления древесины в стране, осталь- ной спрос возмещается за счет импорта (табл. 6). Порядка 60% импортируе- мых лесных продуктов используется в строительстве, 20% — в производстве мебели, 5% — для упаковки. Таблица 6 Производство и потребление лесопродукции в Республике Корея Товарная позиция 1990 1995 2000 2005 2008 2000/ 1990, % 2008/ 2000, % Производство Деловая древесина, млн м3 1,14 1,37 1,59 2,35 2,70 139,5 169,8 Пиломатериалы, млн м3 3,90 3,44 4,54 4,37 4,37 116,4 96,3 Древесные плиты, млн м3 1,46 2,14 3,20 3,76 3,69 219,2 115,3 шпон, млн м3 0,00 0,00 0,72 0,57 0,38 — 52,8 фанера, млн м3 1,12 0,97 0,82 0,68 0,67 73,2 81,7 ДСП, млн м3 0,17 0,55 0,72 0,85 0,95 423,5 131,9 ДВП, млн м3 0,17 0,61 0,94 1,66 1,70 552,9 180,9 Целлюлоза, млн т 2,19 4,22 5,60 7,60 8,44 255,7 150,7 Бумага и картон, млн т 4,52 6,88 9,31 10,25 10,64 206,0 114,3 Видимое потребление* Деловая древесина, млн м3 11,19 9,69 8,33 8,57 7,60 74,4 91,2 Пиломатериалы, млн м3 4,39 4,42 5,26 5,13 4,92 119,8 93,5 Древесные плиты, млн м3 2,63 3,94 5,09 6,42 5,48 193,5 107,7 шпон, млн м3 0,03 0,06 0,96 0,88 0,57 в 32 р. 59,4 фанера, млн м3 1,78 2,18 1,70 1,91 1,46 95,5 85,9 ДСП, млн м3 0,57 1,03 1,20 1,60 1,45 210,5 120,8 ДВП, млн м3 0,25 0,67 1,22 2,03 2,00 488,0 163,9 Целлюлоза, млн т 4,76 7,43 9,72 11,41 11,96 204,2 123,0 Бумага и картон, млн т 4,33 6,58 7,41 7,78 8,77 171,1 118,4 Примечания: масса из древесины или из других волокнистых целлюлозных материалов; регенерируемые бумага или картон (макулатура и отходы); ** видимое потребление = внутрен- нее производство + импорт — экспорт. Источник: составлено на основе данных [6]. Таб Производство и потребление лесопродукции в Республике Корея Примечания: * масса из древесины или из других волокнистых целлюлозных материалов; регенерируемые бумага или картон (макулатура и отходы); ** видимое потребление = внутрен- нее производство + импорт — экспорт. Примечания: * масса из древесины или из других волокнистых целлюлозных материалов; регенерируемые бумага или картон (макулатура и отходы); ** видимое потребление = внутрен- нее производство + импорт — экспорт. И [6] Источник: составлено на основе данных [6]. Источник: составлено на основе данных [6]. В текущем десятилетии среднегодовые темпы прироста объемов импор- та деловой древесины составили 6,9%, пиломатериалов — 3,1%, фанеры — 11,7%, ДСП — 8,6%, ДВП — 7,2%. Лесная продукция Географическая структура импорта круглого леса представлена США (39%), Россией (23%), Канадой (16,1%), Малайзией (10,6%) и Новой Зелан- дией (7,6%). Длительное время крупнейшим поставщиком круглого леса хвойных по- род на японский рынок являлась Россия. Однако ужесточение таможенно- 94 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ тарифного регулирования в РФ экспорта необработанных лесоматериалов во второй половине 2007 г. негативно отразилось на динамике российского экспорта круглого леса, вызвав практически двукратное падение его объемов в 2008 г. В результате доля российского леса в структуре импортных поставок необработанных лесоматериалов хвойных пород снизилась с 41,5% в 2007 г. до 25,8% в 2008 г. Освободившуюся нишу заняли производители хвойного пиловочника из США, увеличив свою долю на японском рынке до 44,1%. Основной объем круглого леса лиственных пород, занимающего около 20% в структуре импортных поставок необработанных лесоматериалов, Япония ввозит из Малайзии (59,1%) и США (11,8%). Главными потребителями дело- вой древесины на японском рынке являются производители бумаги, мебели и стройматериалов. В последние годы в Японии неуклонно снижаются объемы потребления и импорта пиломатериалов. За период 2000—2008 гг. доля пиломатериалов в структуре импорта лесопродукции сократилась с 19,2 до 13,6%. В настоящее время в географической структуре импорта пиломатериалов лидирующие позиции занимают страны Северной Америки (свыше 34% пиломатериалов поступает из Канады и США), страны Западной Европы (30,6%)1 и Россия (12,3%). Наиболее высокий спрос в Японии на древесные листовые материалы предъявляется со стороны строительства. В структуре потребления листовых материалов доминирует фанера (около 70%), ввозимая в основном из Ма- лайзии, Индонезии и Китая, позиции которого в последние годы заметно усилились (в японском импорте фанеры его доля возросла с 4,7% в 2001 г. до 15,7% в 2008 г.). Позиции Китая укрепились также и на японском рынке мебели. За период 2001—2008 гг. доля Китая в импорте мебели возросла с 26 до 50%. В отличие от импорта, структура японского лесного экспорта менее ди- версифицирована и представлена целлюлозой и целлюлозно-бумажными изделиями (97% общего объема экспорта лесопродукции). Следует отметить, что за период 1995—2008 гг. объемы экспорта целлюлозы возросли практи- чески в 12 раз, а ее удельный вес в структуре лесного экспорта увеличился с 2,6 до 20,6%. Основными потребителями японской целлюлозы являются Ки- тай (79,2%) и страны ЮВА (10,5%); целлюлозно-бумажных изделий — стра- ны ЮВА (22,3%), Китай (18,2%) и США (15,2%). Республика Корея, в отличие от Китая и Японии, характеризуется самой 95 Е. И. Деваева, Т. Е. Котова ПЭ №4 В настоящее время на долю указанных то- варных позиций приходится 30,5% объема импорта лесопродукции. Свыше 31% в южнокорейском импорте лесопродукции занимает целлюлоза, 21,2% — бумага и картон. Порядка 60% импортируемых лесных продуктов использу- ется в строительстве, 20% — в производстве мебели, 5% — для упаковки. 96 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ ПЭ № 4 2010 Основными поставщиками лесопродукции на рынок Республики Корея являются Китай (17,6%), США (14,9%), Индонезия (8,6%), Канада (8%), Но- вая Зеландия (6,3%), Малайзия (6,9%). Доля России составляет 3%. Экспорт лесопродукции Республики Корея, структура которого в значи- тельной степени схожа со структурой экспорта Японии, представлен, глав- ным образом, целлюлозно-бумажными изделиями (92%). За период 1995— 2007 гг. объемы экспорта данной продукции увеличились практически вдвое. В настоящее время на экспорт направляется приблизительно треть произве- денной в стране продукции целлюлозно-бумажной промышленности. Среди стран-потребителей целлюлозно-бумажных изделий из Республи- ки Корея следует выделить США, Китай, Австралию и Японию. Экспорт в эти страны составляет в настоящее время порядка 50% объема экспорта по- ставок бумаги и картона Республики Корея. Наряду с целлюлозно-бумажны- ми изделиями на внешний рынок поставляются мебель (2,1%), целлюлоза (2,1%), листовые материалы (0,9%) и готовые изделия из древесины (0,7%). В долгосрочной перспективе, согласно оценкам FAO [24], рост спроса на лесоматериалы в странах СВА во многом будет представлять собой про- должение существующих в настоящее время тенденций и соответствовать глобальной перспективе, предполагающей существенное увеличение по- требления древесных плит и более умеренный рост потребления пиломате- риалов. Пиломатериалы и фанера по-прежнему останутся основной статьей потребления продукции из плотной древесины, хотя ожидается некоторое увеличение использования восстановленных плит вместо пиломатериалов и фанеры. Также ожидается заметный рост потребления бумаги и картона; при этом наибольшая часть волокна для их производства будет получена из вос- становленной бумаги и древесины, произведенной за счет посадок быстро- растущих видов деревьев. Основная доля роста объемов потребления лесопродукции в СВА при- дется на Китай. Согласно перспективным оценкам Государственного лес- ного управления КНР (SFA), к 2020 г. потребление фанеры сократится до 20 млн м3, что связано с уменьшением запасов тропической древесины, особенно бревен большого диаметра. Сокращение потребления фанеры по- влечет за собой рост потребления ДСП и ДВП (до 70 млн м3 в 2020 г.) [21]. Суммарный объем потребления листовых материалов к 2020 г. увеличится до 120 млн м3. К 2030 г. спрос на фанеру в Китае может составить более полови- ны мирового спроса, а доля КНР в мировом спросе на панели и упаковочную бумагу достигнет 35 и 28% соответственно [22]. 1 Следует отметить, что начиная с 1970 г. развитие пресноводной аквакультуры в Китае про- исходило со среднегодовым темпом прироста в 10,8% (в мире этот показатель составлял 7%), раз- витие марикультуры, за исключением водорослей, — с темпом прироста в 10,7% (5,9% в мире). ПЭ № 4 2010 ПЭ4 направлении городов из сельских районов страны, где древесина традицион- но используется в жилищном строительстве. Потребление пиломатериалов в качестве отделочных материалов, как ожидается, увеличится до 40—42 млн м3 к 2020 г., главным образом, вследствие роста уровня доходов населения. Так- же возрастет к 2020 г. спрос на пиломатериалы для производства мебели (до 35—37 млн м3). Спрос на бумагу и картон, как ожидается, в период до 2020 г. практически удвоится, составив 137,8 млн т. В целом предполагается, что спрос на древесину в Китае к 2020 г. увели- чится до 457—477 млн м3, при этом за счет импорта будет удовлетворяться до 33—36% совокупного объема спроса на древесное сырье (153—173 млн м3). В 2030 г. спрос Китая на импорт промышленного круглого леса может до- стигнуть 245 млн м3. В долгосрочной перспективе Китай также закрепит за собой позиции крупнейшего в регионе СВА производителя лесопродукции (общий объ- ем производства лесной продукции в Китае может достичь 952,8 млн м3 к 2030 г.). При этом КНР будет оставаться основным в регионе импортером древесного сырья и экспортером лесопродукции высокого технологического передела, что существенно ограничит возможности проникновения на рынок стран СВА дальневосточных производителей продукции деревообработки. ПЭ №4 По предварительным оценкам SFA, потребление пиломатериалов в стро- ительстве в 2010 г. составит 41—43 млн м3. Однако к 2020 г. их потребление со- кратится до 32—34 млн м3, что связано с усилением миграционных потоков в 97 Е. И. Деваева, Т. Е. Котова ПЭ № 4 2010 Рыбопродукция Преимущественно сырьевая направленность сохранится в долго- срочной перспективе и в дальневосточном экспорте рыбопродукции в стра- ны СВА. В настоящее время Китай, Япония и Республика Корея являются основными рынками производства и потребления рыбы и морепродуктов в СВА. Лидирующие позиции в производстве рыбы и морепродуктов среди стран СВА принадлежат Китаю. В результате проводимой экономической политики и благодаря сохранившимся традициям поликультурного нату- рального хозяйства Китай стал обладателем уникального рыбопромышлен- ного комплекса, в котором более половины производимого объема рыбопро- дукции представлено аква- и марикультурой1. В 2008 г. продукция рыбной отрасли Китая составила 47,5 млн т, в том чис- ле традиционный улов рыбы и морепродуктов — 14,8 млн т, продукция аква- культуры — 32,7 млн т (табл. 7). На протяжении многих лет Китай занимает доминирующие позиции в отрасли аквакультуры, с уже устоявшимся мировым экспортным рынком и беспрерывно растущим внутренним потреблением. 98 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ ПЭ № 4 2010 Таблица 7 Динамика производства, экспорта, импорта и потребления рыбопродукции в КНР, млн т Показатель 1995 2000 2005 2008 2008/ 2000, % Производство 28,5 36,2 42,7 47,5 131,2 рыболовство 12,6 14,6 14,6 14,8 101,4 аквакультура 15,9 21,5 28,1 32,7 152,1 Экспорт 0,7 1,5 2,5 2,9 193,3 Импорт 1,3 2,5 3,7 3,9 156,0 Видимое потребление* 29,0 37,2 43,9 48,5 130,7 Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источники: составлено на основе данных [6; 7]. Динамика производства, экспорта, импорта и потребления рыбопродукции в КНР, млн т Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источники: составлено на основе данных [6; 7]. К настоящему времени на долю Китая приходится 62,3% объема всей произведенной продукции аквакультуры в мире (в целом доля стран АТР в мировом объеме производства данной продукции составляет порядка 92%). В среднесрочной и долгосрочной перспективе, согласно планам по развитию рыбной отрасли страны, рыбопромышленный комплекс Китая будет напря- мую зависеть от состояния и дальнейшего развития аквакультуры. В последние годы Китай стал важнейшим игроком на мировом рынке ры- бопродукции. За период 2000—2008 гг. объемы экспорта рыбопродукции стра- ны увеличились с 3,8 до 10,4 млрд долл., импорта — с 2,4 до 5,3 млрд долл. Рост объемов экспорта рыбопродукции достигался исключительно за счет наращивания объемов поставок готовой продукции и полуфабрикатов (рис. 1). 1 «Процессинг» — производство продукции на основе использования давальческого сырь Рыбопродукция Наличие данной тенденции обусловлено развитием так называемой «процессинговой»1 торговли («processing» trade), механизм которой заключа- ется в следующем: рыба и морепродукты импортируются в страну и после определенной стадии обработки реэкспортируются для последующей прода- жи. К настоящему времени доля продукции переработки (готовая продукция и полуфабрикаты) в товарной структуре экспорта возросла до 80%. По оцен- кам Министерства сельского хозяйства КНР, к 2020 г. доля только готовой продукции в совокупном объеме экспорта продукции рыбопромышленного комплекса страны увеличится на 25% и достигнет 75—80%. В отличие от экспорта рост объемов импорта рыбопродукции происходил главным образом за счет стабильного увеличения объемов импорта сырья (рис. 1), при этом свыше 40% импортируемых Китаем рыбы и морепродуктов использовалось не для удовлетворения внутренних потребностей, а для даль- нейшей переработки с последующим экспортом. роцессинг» — производство продукции на основе использования давальческого сырья. 99 Е. И. Деваева, Т. Е. Котова ПЭ № 4 2010 Рис. 1. Динамика и структура внешней торговли рыбопродукцией КНР, млн долл. Источник: на основе данных [7]. 0,0 2000,0 4000,0 6000,0 8000,0 10000,0 12000,0 1990 1995 2000 2005 2008 1990 1995 2000 2005 2008 Экспорт Импорт Сырье Полуфабрикаты Готовая продукция Е. И. Деваева, Т. Е. Котова ПЭ4 Основным поставщиком рыбы и морепродуктов на китайский рынок яв- ляется Россия, доля которой в общем объеме импорта рыбы и морепродук- тов за период 2000—2008 гг. возросла с 17,5 до 24,5%. Усиление позиций РФ на китайском рынке рыбопродукции в последнее десятилетие происходило главным образом за счет наращивания объемов поставок рыбы мороженой. К наиболее востребованным видам российской рыбной продукции на китай- ском рынке относятся лосось, палтус, треска и сельдь. Источник: на основе данных [7]. Наиболее высокая степень зависимости от импорта рыбы и морепродук- тов среди стран Северо-Восточной Азии характерна для Японии. Данный факт обусловлен тем, что исторически рыбное хозяйство является важней- шим сектором японской экономики. Однако, несмотря на развитый наци- ональный рыбопромышленный комплекс, практически половина потреб- ности населения Японии в продуктах рыболовства в значительной степени удовлетворяется за счет импорта. В последние годы индекс самообеспечен- ности рыбной продукцией в Японии находится на уровне 70% (абсолютный максимум в динамике данного показателя наблюдался в 1964 г. — 113%). Следует отметить, что среди стран СВА Япония характеризуется самым либеральным таможенным регулированием в части импорта рыбопродук- ции. Однако, несмотря на относительно низкие значения импортного та- рифа, Япония продолжает сохранять импортные квоты на рыбные товары, размеры которых определяются спросом японского рынка на данный товар и предложением со стороны японских поставщиков. 1 В текущем десятилетии объем среднегодового потребления свежей рыбы (в расчете на одного человека) сократился на 10,5%, в то время как свежего мяса — увеличился на 2,5%. Рыбопродукция При этом квоты имеют тарифный характер (то есть существует первичная тарифная ставка и вто- ричная «запретительная» ставка). Кроме того, на отдельные виды товаров из 100 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ ПЭ № 4 2010 ПЭ № 4 2010 ряда стран требуется предварительное одобрение японского Министерства внешней торговли и промышленности. В частности, это правило распро- страняется на красную рыбу и продукцию из нее, поставляемую из Китая, Тайваня и КНДР, а также рыбу, выловленную за пределами японских тер- риториальных вод иностранными судами и переданную в море на японские суда для ее ввоза в Японию [28]. В текущем десятилетии в динамике производства рыбопродукции и особенно ее импорта наблюдался понижательный тренд (табл. 8). За пе- риод 2000—2008 гг. объем импорта рыбопродукции в Японии сократился с 15,7 млн долл. до 14,7 млн долл. При этом в последние несколько лет в стране наблюдается абсолютное сокращение объемов потребления некоторых ви- дов рыбы (в частности, норвежской семги, американской нерки и трески). Наличие данной тенденции обусловлено сокращением потребления рыбной продукции на внутреннем рынке вследствие появления в структуре пищево- го рациона населения страны большого ассортимента «легких в приготовле- нии» мясных продуктов1, а также повышения мировых цен на морепродукты, инициированного увеличением объемов потребления рыбы и морепродуктов в Китае, Европе и США. Таблица 8 Динамика производства, экспорта, импорта и потребления рыбопродукции в Японии, тыс. т Показатель 1995 2000 2005 2008 2008/ 2000, % Производство 6786,7 5817,6 5135,4 4981,1 85,6 рыболовство 5966,6 5054,8 4389,2 4248,7 84,1 аквакультура 820,1 762,8 746,2 732,4 96,0 Экспорт 235,5 221,5 470,1 517,9 233,8 Импорт 3494,2 3488,9 3336,1 2759,9 79,1 Видимое потребление* 10 045,4 9085,1 8001,4 7223,1 79,5 Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источник: составлено на основе данных [6; 7]. Динамика производства, экспорта, импорта и потребления рыбопродукции в Японии, тыс. т Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источник: составлено на основе данных [6; 7]. В настоящее время основными экспортерами водных биологических ре- сурсов на японский рынок являются КНР (17,1%), США (10,4%), Россия (8,7%), Таиланд (7,6%), Чили (7,1%), Вьетнам (5,2%), Индонезия (5,2%), Ре- спублика Корея (4,9%). К наиболее значимым товарным позициям японско- го импорта относятся креветки, тунец, лосось и форель, краб, угорь, треска. Они составляет около половины всего объема импорта. 101 Е. И. Деваева, Т. Е. Котова ПЭ № 4 2010 Российский экспорт продукции рыбного комплекса в 2008 г. оценивался в 1,28 млрд долл., что на 1,5% выше, чем в 2000 г. 1 По оценкам Корейского института экономики сельского хозяйства, по сравнению с 1976 г. в ежедневном рационе жителей Республики Корея мясных продуктов стало в 4 раза больше, молочных продуктов — в 10 раз. Рыбопродукция В структуре российских поставок рыбопродукции в Японию преобладают морепродукты. В 2008 г. за счет российских поставок формировалось свыше 17% общего объема япон- ского импорта ракообразных и моллюсков. В настоящее время России при- надлежат лидирующие позиции по поставкам мороженых и свежих крабов (453,8 млн долл.), нерки (113,8 млн долл.) и палтуса (37,0 млн долл.). В Республике Корея рыбное хозяйство также является важной отрас- лью экономики и социально значимым промыслом. Удельный вес рыбы в общем балансе белка животного происхождения в Республике Корея — один из самых высоких в мире: около 40%. Несмотря на то, что структура пита- ния претерпела за последнюю четверть века существенные изменения (в ней стало больше мясных и молочных продуктов1), потребление рыбы и море- продуктов в Республике Корея стабильно увеличивается. Так, если в 2000 г. среднедушевое потребление рыбы и морепродуктов составляло около 70 кг, то в 2008 г. этот показатель превысил 87 кг. Вместе с тем уровень самообеспе- ченности рыбой и морепродуктами Республики Корея в настоящее время не превышает 70%. К примеру, в 1980 г. уровень самообеспеченности достигал 138%, в 2000 г. — 94%. В текущем десятилетии в Республике Корея наблюдается рост объемов импорта рыбы и морепродуктов. При этом темпы роста импорта в значитель- ной степени опережали темпы роста экспорта, что привело к формированию отрицательного сальдо в торговле рыбопродукцией (табл. 9). Таблица 9 Динамика производства, экспорта, импорта и потребления рыбопродукции в Республике Корея, тыс. т Показатель 1995 2000 2005 2008 2008/ 2000, % Производство 2688,1 2118,4 2077,6 2417,7 114,1 рыболовство 2319,9 1825,0 1641,0 1943,9 106,5 аквакультура 368,2 293,4 436,6 473,8 161,5 Экспорт 435,1 527,8 405,4 576,7 109,3 Импорт 405,3 746,7 1265,1 1149,7 154,0 Видимое потребление* 2658,3 2337,3 2937,3 2990,7 128,0 Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источник: составлено на основе данных [6; 7]. Динамика производства, экспорта, импорта и потребления рыбопродукции в Республике Корея, тыс. т Примечание. * Видимое потребление = внутреннее производство + импорт — экспорт. Источник: составлено на основе данных [6; 7]. 102 ТОВАРНЫЕ РЫНКИ СЕВЕРО-ВОСТОЧНОЙ АЗИИ: ОРИЕНТИРЫ ДЛЯ ЭКСПОРТА ДВ РОССИИ ПЭ № 4 2010 В 2008 г. объем импорта составил 1,4 млрд долл., большая часть которо- го приходилась на Китай (35,8%), Россию (14,3%), США (9,1%) и Японию (4,5%). С целью защиты национальных производителей морепродуктов от импортируемой продукции (главным образом из Китая) правительство Ре- спублики Корея ужесточило таможенный режим, введя в 2008 г. 2 Для сравнения: в странах Юго-Восточной Азии к 2020 г. потребление рыбопродукции на душу населения в среднем составит 25,8 кг в год. Д д д р у ру щ р ф р д р ду ц д тарификации от 10 до 20%. 2 Для сравнения: в странах Юго-Восточной Азии к 2020 г. потребление рыбопродукции на душу населения в среднем составит 25,8 кг в год. До введения в действие регулирующих тарифов импорт этих видов продукции подлежал икации от 10 до 20%. ПЭ № 4 2010 стороны, с сохранением повышательной динамики цен на продукцию тра- диционного лова вследствие увеличения затрат на добычу рыбы и морепро- дуктов и истощения ресурсов Мирового океана, с другой — ростом дефицита продовольствия, особенно белкового происхождения. В результате к 2020 г. производство продукции аквакультуры возрастет почти на 80% относительно 2008 г., увеличившись до 70—75% в общем объеме производства продукции рыбного сектора. Пролонгация указанных выше тенденций до 2030 г. может создать для Дальнего Востока России определенные ограничения как в плане наращива- ния физических объемов поставок рыбы свежемороженой и морепродуктов в восточноазиатском направлении, так и увеличения в структуре экспорта доли рыбопродукции с более высокой добавленной стоимостью. СПИСОК ЛИТЕРАТУРЫ 1. Деваева Е. И., Котова Т. Е. Внешняя торговля Дальнего Востока России: совре- менное состояние и тенденции развития // Пространственная экономика. 2009. № 4. 1. Деваева Е. И., Котова Т. Е. Внешняя торговля Дальнего Востока России: совре- менное состояние и тенденции развития // Пространственная экономика. 2009. № 4. 1. Деваева Е. И., Котова Т. Е. Внешняя торговля Дальнего Востока России: совре- менное состояние и тенденции развития // Пространственная экономика. 2009. № 4. 2. Деваева Е. И., Котова Т. Е. Российское Дальневосточье и АТР: внешнеторго- вый аспект // Проблемы Дальнего Востока. 2007. № 6. 2. Деваева Е. И., Котова Т. Е. 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За период 2000—2008 гг. экспорт российской рыбы и морепродуктов в Республику Корея увеличился в 2,8 раза (для сравнения: экспорт рыбопро- дукции в Китай возрос в 2,4 раза, в Японию — в 1,2 раза), притом что цены на рыбопродукцию в среднем в 2—2,5 раза ниже, чем на японском, и в 1,2— 1,5 раза, чем на китайском рынке. Таким образом, предпринятые Японией меры по ужесточению требований к импорту морских биоресурсов привели к возрастанию роли Республики Корея как посредника в торговле рыбопро- дукцией. Наиболее весомы позиции России в южнокорейском импорте крабов, палтуса, камбалы, сельди, трески, печени, икры и молок. Доля российской продукции в импорте указанных видов рыбных товаров превышает 55%, а по свежим крабам и палтусу Россия практически полностью удовлетворяет импортные потребности Республики Корея. В перспективе, согласно прогнозам Министерства морского хозяйства и рыболовства Республики Корея, в стране ожидается дальнейшее увеличение потребления рыбы и морепродуктов, что обусловлено наблюдающимся в на- стоящее время в стране ростом интереса к здоровому питанию. В целом, согласно прогнозам FAO, при сохранении современных тен- денций, сложившихся на рынках рыбы и морепродуктов в странах СВА, в средне- и долгосрочной перспективе следует ожидать существенного роста потребления рыбы и морепродуктов, и главным образом в Китае [28]. Уже к 2020 г. потребление на душу населения в КНР достигнет 35,9 кг в год2 [15]. При этом важнейшим источником для удовлетворения возрастающего спроса на рыбу и морепродукты станет аквакультура. Это связано, с одной 103 Е. И. Деваева, Т. Е. Котова 16. Fishery and Aquaculture Statistics: 2006. Rome: FAO, 2008. 17. Global forest resources assessment 2010, country reports. China. Rome: FAO, 2005. 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https://openalex.org/W3095196981	https://escholarship.org/content/qt1vr006m6/qt1vr006m6.pdf?t=qmgomq	English	null	Cadherin-11 Is Required for Neural Crest Specification and Survival	Frontiers in physiology	2,020	cc-by	16,299	UC Davis UC Davis Previously Published Works Title Cadherin-11 Is Required for Neural Crest Specification and Survival Permalink https://escholarship.org/uc/item/1vr006m6 Authors Manohar, Subrajaa Camacho-Magallanes, Alberto Echeverria, Camilo et al. Publication Date 2020 DOI 10.3389/fphys.2020.563372 Peer reviewed UC Davis UC Davis Previously Published Works Title Cadherin-11 Is Required for Neural Crest Specification and Survival Permalink https://escholarship.org/uc/item/1vr006m6 Authors Manohar, Subrajaa Camacho-Magallanes, Alberto Echeverria, Camilo et al. Publication Date 2020 DOI 10.3389/fphys.2020.563372 Peer reviewed Cadherin-11 Is Required for Neural Crest Specification and Survival Subrajaa Manohar 1†, Alberto Camacho-Magallanes 1†, Camilo Echeverria Jr. 2 and Crystal D. Rogers2* 1 Department of Biology, School of Math and Science, California State University Northridge, Northridge, CA, United States, 2 Department of Anatomy, Physiology, and Cell Biology, UC Davis School of Veterinary Medicine, Davis, CA, United States Neural crest (NC) cells are multipotent embryonic cells that form melanocytes, craniofacial bone and cartilage, and the peripheral nervous system in vertebrates. NC cells express many cadherin proteins, which control their specification, epithelial to mesenchymal transition (EMT), migration, and mesenchymal to epithelial transition. Abnormal NC development leads to congenital defects including craniofacial clefts as well as NC-derived cancers. Here, we identify the role of the type II cadherin protein, Cadherin-11 (CDH11), in early chicken NC development. CDH11 is known to play a role in NC cell migration in amphibian embryos as well as cell survival, proliferation, and migration in cancer cells. It has also been linked to the complex neurocristopathy disorder, Elsahy-Waters Syndrome, in humans. In this study, we knocked down CDH11 translation at the onset of its expression in the NC domain during NC induction. Loss of CDH11 led to a reduction of bonafide NC cells in the dorsal neural tube combined with defects in cell survival and migration. Loss of CDH11 increased p53-mediated programmed-cell death, and blocking the p53 pathway rescued the NC phenotype. Our findings reveal an early requirement for CDH11 in NC development and demonstrated the complexity of the mechanisms that regulate NC development, where a single cell-cell adhesion protein simultaneous controls multiple essential cellular functions to ensure proper specification, survival, and transition to a migratory phase in the dorsal neural tube. Our findings may also increase our understanding of early cadherin-related NC developmental defects. INTRODUCTION a section of the journal Frontiers in Physiology Cadherin proteins are calcium dependent cell-cell adhesion molecules, which are essential for the development and maintenance of embryonic tissues (Giger and David, 2017; Taneyhill and Schiffmacher, 2017). Cadherins are single pass transmembrane proteins that contain a calcium- binding extracellular domain as well as a cytoplasmic domain which links with three catenin family proteins (α, β, and p120) and the actin cytoskeleton (Gul et al., 2017). Classical cadherins are divided into types I and II. Type I cadherins include epithelial cadherin (E-cadherin/ CDH1) and neural cadherin (N-cadherin/CDH2) among others, which have been implicated in both central nervous system (CNS) development in chick and zebrafish embryos (Dady et al., 2012; Miyamoto et al., 2015), and neural crest (NC) specification and the epithelial to mesenchymal transition (EMT) in frog, fish, and chicks (Piloto and Schilling, 2010; Rogers et al., 2013, 2018; Huang et al., 2016). Type II cadherins include Cadherin 7 (CDH7), Received: 18 May 2020 Accepted: 06 October 2020 Published: 30 October 2020 Edited by: Lisa Taneyhill, University of Maryland, College Park, United States Reviewed by: Dominique Alfandari, University of Massachusetts Amherst, United States Clemens Martin Franz, Kanazawa University, Japan Correspondence: Crystal D. Rogers crdrogers@ucdavis.edu †These authors have contributed equally to this work Reviewed by: Dominique Alfandari, University of Massachusetts Amherst, United States Clemens Martin Franz, Kanazawa University Japan Correspondence: Crystal D. Rogers crdrogers@ucdavis.edu Specialty section: This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology Specialty section: This article was submitted to Craniofacial Biology and Dental Research, Specialty section: This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology Received: 18 May 2020 Accepted: 06 October 2020 Published: 30 October 2020 Keywords: Cadherin-11, neural crest, specification, apoptosis, survival, p53, caspase Powered by the California Digital Library University of California eScholarship.org ORIGINAL RESEARCH published: 30 October 2020 doi: 10.3389/fphys.2020.563372 CDH11 Expression Starts in the Neural Tube Prior to NC Cell Formation Tube Prior to NC Cell Formation CDH11 function has been extensively examined during NC migration and EMT in amphibian embryos (Hadeball et al., 1998; Vallin et al., 1998; Borchers et al., 2001; Kashef et al., 2009; McCusker et al., 2009; Koehler et al., 2013; Abbruzzese et al., 2016), but less is known about its endogenous expression and role in amniotes, which encouraged us to begin by examining the spatiotemporal expression pattern of the CDH11 protein in the chicken embryo. First, protein lysate was collected at multiple developmental stages (HH4–6, HH8–10, and HH11–12) and western blot analysis was used to define the relevant stages. To test the antibody reactivity across species, we also used lysate from tailbud stage Ambystoma mexicanum (axolotl) whole embryos. Two antibodies against CDH11 were tested: a previously verified monoclonal mouse antibody against recombinant intracellular peptide of human CDH11 (Chalpe et al., 2010) and a rabbit polyclonal antibody directed against human CDH11 used previously in mouse tissue (Chang et al., 2017; Supplementary Figure S1A). The mouse antibody identified three bands (potentially different isoforms or posttranslationally modified versions of CDH11) between the sizes of 110–135 kD while the rabbit antibody identified only the mid-sized band and did not recognize axolotl CDH11. Further, the mouse antibody bound to an antigen in the non-neural ectoderm (Supplementary Figures S1B–E’, white arrow). Both antibodies show that CDH11 protein is expressed by stages HH4–6 through HH11–12 in chicken (Supplementary Figures S1A–M’). It is likely that the mouse antibody recognizes three versions of CDH11 (pro-, active full-length, cleaved) previously reported in Xenopus based on the absence of the smallest band from the early premigratory stages (McCusker et al., 2009). We also performed whole mount IHC using both antibodies, and although their expression profiles were similar in whole mount NC cells are a vertebrate-specific population of stem-like cells that form craniofacial bone, cartilage, pigment cells, and the peripheral and enteric nervous systems (Hutchins et al., 2018; Rogers and Nie, 2018). In avian embryos, NC cells begin as tightly adherent neuroepithelial cells in the dorsal neural tube. By going through an EMT, which is controlled by alterations in the expression of type I and II cadherin proteins (Taneyhill et al., 2007; Rogers et al., 2013; Scarpa et al., 2015), the NC cells detach from each other and the basal lamina and gain the ability to migrate. Citation: Cadherin 11 (Osteoblast-cadherin/CDH11), and Cadherin-6B (CDH6), which have been linked to CNS patterning, NC cell delamination, EMT, and migration during embryonic development (Taneyhill et al., 2007; Liu et al., 2008; Kashef et al., 2009; Schiffmacher et al., 2014, 2016). With varied timing and onset of protein expression, the regulation and function of cadherin proteins is clearly important for normal development of the CNS, NC cells, and NC derivatives. Here, we focus on identifying the role of Cadherin-11 (CDH11) during early avian embryogenesis. CDH11 has been identified as a Wnt signaling target and effector in developmental and disease systems (Hadeball et al., 1998; Chalpe et al., 2010; Satriyo et al., 2019). Originally defined as a mesenchymal marker with no expression in the undifferentiated neural tube in mouse embryos (Hoffmann and Balling, 1995), the transcript was subsequently reported in developing mouse neuroepithelia (Kimura et al., 1995, 1996), as well as migratory NC cells in chick and frog embryos (Vallin et al., 1998; Borchers et al., 2001; Chalpe et al., 2010). It has been identified as a major regulator of NC migration in Xenopus embryos (Vallin et al., 1998; Kashef et al., 2009; Abbruzzese et al., 2016; Langhe et al., 2016) and has been linked to tumor growth, cell survival, and EMT in disease models (Yoshioka et al., 2015; Piao et al., 2016; Row et al., 2016). Both reduced and increased levels of CDH11 are linked to patient survival and reduced metastasis in numerous cancers; however, its role is contrasting in different cancer cell types (Carmona et al., 2012; Lee et al., 2013). Specifically, high levels of CDH11 expression have been linked to poor prognosis in gastric cancer and triple-negative breast cancer (Chen et al., 2018; Satriyo et al., 2019), yet it maintains a pro-apoptotic tumor suppressor role in others (Marchong et al., 2010; Li et al., 2012). Although studies have linked CDH11 to NC migration its role in NC induction, specification, maintenance, or survival during premigratory stages has not been studied. Frontiers in Physiology \| www.frontiersin.org Citation: Manohar S, Camacho-Magallanes A, Echeverria C Jr and Rogers CD (2020) Cadherin-11 Is Required for Neural Crest Specification and Survival. Front. Physiol. 11:563372. doi: 10.3389/fphys.2020.563372 October 2020 \| Volume 11 \| Article 563372 1 Frontiers in Physiology \| www.frontiersin.org Cadherin-11 Required for NC Survival Manohar et al. (Abbruzzese et al., 2016; Schiffmacher et al., 2016; Rogers et al., 2018) to prevent developmental defects. Previous studies showed that perturbation of factors involved in this process can directly affect the formation and migratory ability of NC cells. Studies also identified links between cadherin proteins and NC cell migration and differentiation; however, there is little known about how type I or II cadherin proteins regulate premigratory NC development. Our study focuses on the role of CDH11 during the time point it first emerges in the NC domain in the dorsal neural tube. We thoroughly define the spatiotemporal localization of CDH11 in the neural plate and neural tube as well as in the pre- and post-migratory NC cells in the chick embryo during early stages Hamburger Hamilton (HH) stages 4–12. Loss of CDH11 expression reduces the premigratory NC population marked by PAX7, SOX9, SNAI2, and SOX10, and increases membrane-associated CDH1, F-actin, p53 and p53-mediated apoptosis in the presumptive NC regions. Our results indicate that the upregulation of CDH11 in the dorsal neural tube prior to NC migration is necessary for NC cell specification, survival, EMT, and migration. Cadherin 11 (Osteoblast-cadherin/CDH11), and Cadherin-6B (CDH6), which have been linked to CNS patterning, NC cell delamination, EMT, and migration during embryonic development (Taneyhill et al., 2007; Liu et al., 2008; Kashef et al., 2009; Schiffmacher et al., 2014, 2016). With varied timing and onset of protein expression, the regulation and function of cadherin proteins is clearly important for normal development of the CNS, NC cells, and NC derivatives. Cadherin 11 (Osteoblast-cadherin/CDH11), and Cadherin-6B (CDH6), which have been linked to CNS patterning, NC cell delamination, EMT, and migration during embryonic development (Taneyhill et al., 2007; Liu et al., 2008; Kashef et al., 2009; Schiffmacher et al., 2014, 2016). With varied timing and onset of protein expression, the regulation and function of cadherin proteins is clearly important for normal development of the CNS, NC cells, and NC derivatives. CDH11 Expression Starts in the Neural Tube Prior to NC Cell Formation (Q-T’) At late HH9 (9 SS) expression is maintained in the NC cells undergoing EMT and migrating out of the neural tube. (U–X’) At HH11 (15 SS), CDH11 expression is weaker in the neural tube and is maintained in the migratory NC cells marked by PAX7. Dashed boxes indicate zoom regions depicted by grayscale images. Scale bars as indicated in first row. CDH11 Expression Starts in the Neural Tube Prior to NC Cell Formation Abnormal NC development can cause congenital defects known as neurocristopathies, which include cleft palate, craniofacial abnormalities, albinism, and defects in the enteric and peripheral nervous systems among others (Reissmann and Ludwig, 2013; Lopez et al., 2018). Bi-allelic mutations in CDH11 have specifically been linked to Elsahy- Waters syndrome, which is a combination of abnormal craniofacial developmental morphologies including those likely induced by neurocristopathies (Harms et al., 2018). The processes of NC specification and EMT are tightly regulated at multiple levels by signaling molecules (Bhattacharya et al., 2018), epigenetic modifiers (Hu et al., 2012), transcription factors (Simoes-Costa et al., 2015), and adhesion molecules October 2020 \| Volume 11 \| Article 563372 2 Manohar et al. Cadherin-11 Required for NC Survival (Supplementary Figures S1B–M), in section, the mouse antibody also bound to an epitope localized in non-neural ectoderm (Supplementary Figures S1C’–E’, arrows). Due to its specificity, we chose to use the rabbit antibody for the rest of our experiments. We also verified specificity of the antibody by performing CDH11 loss and gain of function experiments and visualizing reduction or exogenous expression of the protein in whole mount (Supplementary Figures S2A–D,H–J, S3) and transverse sections (Supplementary Figures S2E–G,K–M, S3). (Supplementary Figures S1B–M), in section, the mouse antibody also bound to an epitope localized in non-neural ectoderm (Supplementary Figures S1C’–E’, arrows). Due to its specificity, we chose to use the rabbit antibody for the rest of our experiments. We also verified specificity of the antibody by performing CDH11 loss and gain of function experiments and visualizing reduction or exogenous expression of the protein in whole mount (Supplementary Figures S2A–D,H–J, S3) and transverse sections (Supplementary Figures S2E–G,K–M, S3). y To characterize the spatiotemporal localization of CDH11 in early avian development, we performed IHC using anti-CDH11 in conjunction with previously characterized markers of NC cells (PAX7 and HNK1; Del Barrio and Nieto, 2004; Basch et al., 2006). The results identify CDH11 expression in the neural plate of 1 somite stage (SS) embryos at HH7 (Figures 1A–D,A’–D’, Supplementary Figures S1F–M’), but is expressed at much lower levels in the neural plate border, which expresses PAX7 and CDH1 as NC cells are being induced (Figures 1B–D,B’–D’). As the neural tube begins to fuse at 5 SS (HH8). CDH11 expression is maintained throughout the neural tube, co-localizing with dorsal cells expressing CDH1 (Figures 1E–H,E’–H’). CDH11 Expression Starts in the Neural Tube Prior to NC Cell Formation CDH11 expression starts to emerge in a subset of PAX7-positive cells in the dorsal neural tube (Figures 1I–L,I’–L’, Supplementary Figures S1H–M). At 7 SS (HH9), as NC cells begin to delaminate and undergo EMT, CDH11 is upregulated in the most proximal PAX7-positive cells (Figures 1M–P,M’–P’). As HNK1 expression begins in the early migrating NC cells, the leading cells co-express CDH11 (Figures 1O,P,O’,P’). In 9 SS (late HH9) embryos, as NC cells begin to migrate away from the midline, and all migratory cells are positive for both CDH11 and PAX7 (Figures 1Q-T,Q’–T’), while the most lateral cells express HNK1 (Figures 1S,T). Focusing on CDH11-postive cells at 7 SS (Figure 1M’) and 9 SS (Figure 1Q’) shows that CDH11 appears membrane-localized as NC cells collectively migrate out of the neural tube. At 15 SS (HH11), CDH11 remains in the neural tube and the migratory NC cells, co-localizing with PAX7 (Figures 1U–X,U’–X’); however, the cellular localization in later migratory NC appears more punctate (Figures 1Q’–T’). Our results support previous studies in frog by demonstrating the endogenous expression of CDH11 in migrating chick NC cells (McCusker et al., 2009; Abbruzzese et al., 2016; Mathavan et al., 2017). These data confirm that CDH11 is expressed during NC cell EMT and migration, but introduce novel expression in epithelial premigratory NC cells suggesting that CDH11 may play an earlier role in NC development. FIGURE 1 \| CDH11 expression in NC cells starts during specification stages. (A–X’) Immunohistochemistry (IHC) using antibodies against CDH11 (yellow), PAX7 (pink) to mark NC cells and neural plate border, CDH1 (green) to mark cell-cell junctions and epithelial tissues, and HNK1 (blue) to mark migratory NC cells as well as parts of the endoderm, mesoderm, and notochord, or stained with DAPI (blue) to mark nuclei. At (A–D’) HH7[1 somite stage (SS)], when NC are induced at the neural plate border, CDH11 is expressed in the neural plate/ tube, but is only expressed in a subset of border cells with PAX7. Examples of cells positive for both PAX7 and CDH11 are marked with pink arrows. (E-H’, I-L’) At HH8 (5 SS), CDH11 co-localizes with CDH1 in the developing neural tube. Black arrow marks cell in premigratory NC region positive for CDH11. (M–P’) At HH9 (7 SS) is strongly upregulated in the premigratory NC cells marked by PAX7 and early migrating NC cells marked by HNK1. Frontiers in Physiology \| www.frontiersin.org CDH11 Is Necessary for NC Cell Population Maintenance During Specification In contrast, at 5 SS (HH8), when CDH11 expression emerges in the dorsal neural tube, the number of PAX7-positive NC cells was reduced by 40% (Figures 2C–D’, n = 19, p = 0.0005). At 10 SS (HH10), FIGURE 2 \| CDH11 is required for NC cell population specification. Embryos were injected at HH4 and collected at multiple stages to analyze NC progenitor marker, PAX7. (A-F’) IHC for PAX7 on embryos injected with CDH11MO and collected between 3 SS and 10 SS. (G–H’) IHC for Pax7 in embryo injected with ContMO and collected at 6 SS. (I,J) Actual PAX7+ cell counts of uninjected and morpholino-injected sides. (K) Corrected total cell fluorescence of PAX7 expression in section images injected with either CDH11MO or ContMO. (A) Whole mount IHC for PAX7 in HH8- (3 SS) embryo with (B) overlay with CDH11MO (green). (A’,B’) Transverse section of (A,B) with PAX7-positive NC cells circled. Mean number of cells at HH8- is 21.43 on uninjected and 21.14 on CDH11MO-injected side, p = 0.94, n = 14. (C) Whole mount IHC for PAX7 in HH8 embryo with (C) overlay with CDH11MO (green). (C’,D’) Transverse section of (C,D) with PAX7-positive NC cells boxed. Mean number of cells at HH8 is 49.53 on uninjected and 29.89 on CDH11MO-injected side, p = 0.0005, n = 19. (E) Whole mount IHC for PAX7 in HH10 embryo with (F) overlay with CDH11MO (green). (F’) Transverse section of (F) with PAX7-positive NC cells. Mean number of cells at HH10 is 56.00 on uninjected and 36.57 on CDH11MO-injected side, p = 0.01, n = 7. (G) Whole mount IHC for PAX7 in HH8 embryo with (H) overlay with ContMO (green). (G’,H’) Transverse sections of (G,H) with PAX7-positive NC cells circled. Mean number of cells on uninjected side is 35.8 and on ContMO-injected side is 34.4, p = 0.74, n = 14. Dashed boxes were drawn around NC cell population from uninjected side and are mirrored on injected sides to demonstrate changes in the NC cell population density. At 5 SS the NC cell population is less dense in the CDH11MO-side compared to uninjected when compared to 3 SS and ContMO-injected embryos. CDH11 Is Necessary for NC Cell Population Maintenance During Specification (K,L) Fluorescence intensity calculated using NIH ImageJ (see methods) from HH5 (n = 8, p = 0.57), HH8 (N = 11, p = 0.11), HH9 (n = 8, p = 0.17), HH10 (n = 7, p = 0.46), and ContMO HH8–9 (n = 12, p = 0.34). Scale bars are as marked (100 μm for whole mount and 50 μm for sections). Anterior to top in all whole mount images, dorsal to top in all sections. Loss of CDH11 reduces the PAX7-positive NC cell population after induction (I, HH5-3SS) and at a point between specification and determination (5 SS). FIGURE 2 \| CDH11 is required for NC cell population specification. Embryos were injected at HH4 and collected at multiple stages to analyze NC progenitor marker, PAX7. (A-F’) IHC for PAX7 on embryos injected with CDH11MO and collected between 3 SS and 10 SS. (G–H’) IHC for Pax7 in embryo injected with ContMO and collected at 6 SS. (I,J) Actual PAX7+ cell counts of uninjected and morpholino-injected sides. (K) Corrected total cell fluorescence of PAX7 expression in section images injected with either CDH11MO or ContMO. (A) Whole mount IHC for PAX7 in HH8- (3 SS) embryo with (B) overlay with CDH11MO (green). (A’,B’) Transverse section of (A,B) with PAX7-positive NC cells circled. Mean number of cells at HH8- is 21.43 on uninjected and 21.14 on CDH11MO-injected side, p = 0.94, n = 14. (C) Whole mount IHC for PAX7 in HH8 embryo with (C) overlay with CDH11MO (green). (C’,D’) Transverse section of (C,D) with PAX7-positive NC cells boxed. Mean number of cells at HH8 is 49.53 on uninjected and 29.89 on CDH11MO-injected side, p = 0.0005, n = 19. (E) Whole mount IHC for PAX7 in HH10 embryo with (F) overlay with CDH11MO (green). (F’) Transverse section of (F) with PAX7-positive NC cells. Mean number of cells at HH10 is 56.00 on uninjected and 36.57 on CDH11MO-injected side, p = 0.01, n = 7. (G) Whole mount IHC for PAX7 in HH8 embryo with (H) overlay with ContMO (green). (G’,H’) Transverse sections of (G,H) with PAX7-positive NC cells circled. Mean number of cells on uninjected side is 35.8 and on ContMO-injected side is 34.4, p = 0.74, n = 14. Dashed boxes were drawn around NC cell population from uninjected side and are mirrored on injected sides to demonstrate changes in the NC cell population density. CDH11 Is Necessary for NC Cell Population Maintenance During Specification i Next, to understand the stage at which CDH11 is necessary for NC cell development, and to determine whether CDH11 was required for induction, specification, or maintenance of the NC population, in addition to its role in migration, a time-course experiment was performed. We used a translation- blocking CDH11MO, which effectively reduced CDH11 fluorescence intensity on the injected side of the embryo compared to the uninjected side by approximately 50% (Supplementary Figures S2A–G, S3). CDH11 expression was inhibited at gastrula stage (HH4), prior to the expression of the neural plate border marker, PAX7. After injection, embryos were collected at stages HH5 and HH7 and IHC for PAX7 was performed. We next counted the number of PAX7-positive October 2020 \| Volume 11 \| Article 563372 Frontiers in Physiology \| www.frontiersin.org 3 Cadherin-11 Required for NC Survival Manohar et al. there continued to be 35% less PAX7-positive cells on the CDH11MO-injected side, suggesting that the NC cells did not recover prior to migration (Figures 2E–F’,I, n = 7, p = 0.01). Embryos injected with a non-specific control morpholino (ContMO), did not exhibit significant differences in the number of PAX7-positive cells between the injected and uninjected sides (Figures 2G–H’, n = 14, p = 0.74). We additionally assessed the changes in fluorescence intensity at each stage after CDH11 knockdown and although the fluorescence was cells and measured fluorescence intensity on the morpholino- injected side compared to the uninjected side. As expected, at NC induction and early specification stages prior to the onset of CDH11 expression upregulation (HH5- HH8-), the number of PAX7-positive NC progenitors in the neural plate border was unchanged (Figures 2A–B’,I, n = 14, p = 0.94). In contrast, at 5 SS (HH8), when CDH11 expression emerges in the dorsal neural tube, the number of PAX7-positive NC cells was reduced by 40% (Figures 2C–D’, n = 19, p = 0.0005). At 10 SS (HH10), cells and measured fluorescence intensity on the morpholino- injected side compared to the uninjected side. As expected, at NC induction and early specification stages prior to the onset of CDH11 expression upregulation (HH5- HH8-), the number of PAX7-positive NC progenitors in the neural plate border was unchanged (Figures 2A–B’,I, n = 14, p = 0.94). Frontiers in Physiology \| www.frontiersin.org CDH11 Is Necessary for NC Cell Population Maintenance During Specification Mean number of SOX10+ cells is 20.35 on uninjected and 11.87 on CDH11MO-injected side, p = 0.01, n = 18. (J–M’) Overlay from the same embryo to demonstrate reduction in NC progenitors (PAX7) and definitive NC cells (SOX9, SOX10) after CDH11 MO-injection (n = 8/9 embryos with reduced cells). (N) IHC for SOX2 in transverse section from HH8 embryo with (O) overlay with CDH11MO (green). (P) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of SOX2+ cells is 80.20 on uninjected and 79.50 on CDH11MO-injected side, p = 0.90, n = 5. All graphs show mean (indicated on graph) and median (line within graph). Scale bar for (A,B,D,E,G,H,N,O) indicated in (A) and scale bar for (J-M’) indicated in (J). Reducing CDH11 significantly reduces the entire population of premigratory NC cells without affecting the SOX2-positive neural tube progenitors. Loss of CDH11 Reduces Expression of NC Specifiers SNAI2, SOX9, and SOX10 (D) IHC for SNAI2 in transverse section from HH9 embryo with (E) overlay with CDH11MO (green). (F) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of SNAI2+ cells is 22.75 on uninjected and 13.75 on CDH11MO-injected side, p = 0.001, n = 16. (G) IHC for SOX10 in transverse section from HH8 embryo with (H) overlay with CDH11MO (green). (I) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of SOX10+ cells is 20.35 on uninjected and 11.87 on CDH11MO-injected side, p = 0.01, n = 18. (J–M’) Overlay from the same embryo to demonstrate reduction in NC progenitors (PAX7) and definitive NC cells (SOX9, SOX10) after CDH11 MO-injection (n = 8/9 embryos with reduced cells). (N) IHC for SOX2 in transverse section from HH8 embryo with (O) overlay with CDH11MO (green). (P) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of SOX2+ cells is 80.20 on uninjected and 79.50 on CDH11MO-injected side, p = 0.90, n = 5. All graphs show mean (indicated on graph) and median (line within graph). Scale bar for (A,B,D,E,G,H,N,O) indicated in (A) and scale bar for (J-M’) indicated in (J). Reducing CDH11 significantly reduces the entire population of premigratory NC cells without affecting the SOX2-positive neural tube progenitors. The loss of CDH11 thus reduces the amount of progenitors and definitive NC cells prior to NC cell migration. However, each NC specifier protein drives specific programs with regards to NC cell development. SNAI2 inhibits Cdh6B and Cdh1 expression to drive cell migration (Taneyhill et al., 2007; Tien et al., 2015) and it is also linked to the inhibition of apoptotic activity in NC cells (Tribulo et al., 2004), while the SOXE proteins, SOX9 and SOX10 are linked with the progression of NC cell migration (Cheung and Briscoe, 2003). To determine the mechanisms downstream of CDH11 knockdown that led to a reduction in the NC population, we next analyzed the impact on cell death and proliferation. Loss of CDH11 Reduces Expression of NC Specifiers SNAI2, SOX9, and SOX10 Speci e s S , SO 9, a d SO 0 As reported by multiple groups, in the NC gene regulatory network (GRN) the factors in the neural plate border (i.e., PAX7, PAX3) drive the expression of bonafide NC markers (SNAI2, SOX9, SOX10, etc.) as neurulation proceeds (Basch et al., 2006; Plouhinec et al., 2014; Williams et al., 2019). These factors are then responsible for altering the expression of specific cadherin proteins and allowing for NC cell EMT and migration (Taneyhill et al., 2007; Huang et al., 2016; Taneyhill and Schiffmacher, 2017). To determine if loss of CDH11 universally reduced the NC cell population by reducing definitive NC cells, embryos were unilaterally injected with CDH11MO, electroporated at HH4, and IHC was used to detect bonafide NC cell markers (SOX9, SNAI2, SOX10) at HH8–9 (5 SS to 7 SS). Loss of CDH11 significantly reduced SOX9-postive NC cells by 41% (Figures 3A–C, n = 11, p = 0.04). SNAI2-positive cells were reduced by 34% (Figures 3D–F, n = 16, p = 0.001) and SOX10-positive NC cells were reduced by 41.7% (Figures 3G–I, n = 19, p = 0.01). We next knocked down CDH11 and performed IHC for PAX7, SOX9, and SOX10 in the same embryos to confirm that the loss of CDH11 was affecting both NC cell progenitors and the premigratory bonafide NC population. We identified that both cell populations were reduced in the absence of CDH11 (Figures 3J–M’, n = 8/9). Finally, we wanted to determine if the CDH11-knockdown phenotype was NC-specific or if loss of CDH11 affected all ectodermal derivatives. Therefore, we analyzed the expression of SOX2, a neural tube progenitor marker, which was unaffected in the CDH11MO-innjected side compared to the UI-side (Figures 3N–P, n = 5, p = 0.9). FIGURE 3 \| Loss of CDH11 reduces definitive NC cells. To determine if loss of CDH11 affected neural progenitors and definitive NC cells in addition to NC progenitors, embryos were injected with CDH11MO or ContMO and electroporated, and IHC was performed for definitive NC cells (SOX9, SNAI2, SOX10) and neural progenitors (SOX2). (A) IHC for SOX9 in transverse section from HH8 embryo with (B) overlay with CDH11MO (green). (C) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of SOX9+ cells is 20.34 on uninjected and 11.85 on CDH11MO- injected side, p = 0.04, n = 11. CDH11 Is Necessary for NC Cell Population Maintenance During Specification At 5 SS the NC cell population is less dense in the CDH11MO-side compared to uninjected when compared to 3 SS and ContMO-injected embryos. (K,L) Fluorescence intensity calculated using NIH ImageJ (see methods) from HH5 (n = 8, p = 0.57), HH8 (N = 11, p = 0.11), HH9 (n = 8, p = 0.17), HH10 (n = 7, p = 0.46), and ContMO HH8–9 (n = 12, p = 0.34). Scale bars are as marked (100 μm for whole mount and 50 μm for sections). Anterior to top in all whole mount images, dorsal to top in all sections. Loss of CDH11 reduces the PAX7-positive NC cell population after induction (I, HH5-3SS) and at a point between specification and determination (5 SS). October 2020 \| Volume 11 \| Article 563372 4 Frontiers in Physiology \| www.frontiersin.org Cadherin-11 Required for NC Survival Manohar et al. reduced in the injected vs. uninjected sides, due to variability in the NC population responses to CDH11MO, the changes in fluorescence were not significant (Figure 2K, p > 0.05). Taken together, loss of CDH11 significantly reduced PAX7+ NC cells after induction during the stages at which NC specifier genes and proteins are normally upregulated suggesting a role for CDH11 specifically in NC specification or maintenance in preparation for EMT, prior to its role in NC migration. FIGURE 3 \| Loss of CDH11 reduces definitive NC cells. To determine if loss of CDH11 affected neural progenitors and definitive NC cells in addition to NC progenitors, embryos were injected with CDH11MO or ContMO and electroporated, and IHC was performed for definitive NC cells (SOX9, SNAI2, SOX10) and neural progenitors (SOX2). (A) IHC for SOX9 in transverse section from HH8 embryo with (B) overlay with CDH11MO (green). (C) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of SOX9+ cells is 20.34 on uninjected and 11.85 on CDH11MO- injected side, p = 0.04, n = 11. (D) IHC for SNAI2 in transverse section from HH9 embryo with (E) overlay with CDH11MO (green). (F) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of SNAI2+ cells is 22.75 on uninjected and 13.75 on CDH11MO-injected side, p = 0.001, n = 16. (G) IHC for SOX10 in transverse section from HH8 embryo with (H) overlay with CDH11MO (green). (I) Graph showing difference between uninjected and CDH11MO-injected sides. CDH11 Is Required for NC Cell Survival q The reduction in NC cells after CDH11 knockdown could be caused by two cellular responses. To determine if this phenotype was a result of increased cell death (or reduced October 2020 \| Volume 11 \| Article 563372 Frontiers in Physiology \| www.frontiersin.org 5 Cadherin-11 Required for NC Survival Manohar et al. FIGURE 4 \| Loss of CDH11 increases cell death. To determine the cause of reduced NC cell population after CDH11 knockdown, IHC was performed for (A-C) p53, (D,D’,G,G’,I,I’,L,L’) activated caspase 3 (Casp3) to mark apoptotic cells, (E,E’,G,G’,J,J’,L,L’) PAX7, or (N–U) phosphorylated histone H3 (PH3) to mark mitotic cells. (A,B) IHC for p53 in transverse section. (C) Graph showing difference between fluorescence intensity on uninjected and CDH11MO- njected sides (n = 17, p = 0.02). (D) IHC for Casp3 or (E) PAX7 in whole mount or for (D’) Casp3 or (E’) PAX7 in transverse section, (F) CDH11MO in whole mount and (G,G’) are overlays. (H) Graph showing increased number of Casp3-positive cells on CDH11MO side. Mean number Casp3+ apoptotic cells is 17.90 on uninjected and 31.86 on CDH11MO-injected side, n = 14, p = 0.04. (I) IHC for Casp3 or (J) PAX7 in whole mount or for (I’) Casp3 or (J’) PAX7 in transverse section, (K) ContMO in whole mount and (L,L’) are overlays. (M) Graph showing difference between uninjected and ContMO-injected sides. Mean number of Casp3+ apoptotic bodies is 9.36 on uninjected and 9.36 on ContMO-injected side, p = 0.97, n = 14. (N) IHC for PH3 in whole mount and (O) overlay with CDH11MO. (P) Transverse section from HH8 embryo injected with CDH11MO on right side (green). (Q) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of PH3+ cells is 6.20 on uninjected and 5.20 on CDH11MO-injected side, p = 0.50, n = 20. (R) IHC or PH3 in whole mount embryo injected with (S) ContMO. (T) Transverse section. (U) Graph showing difference between uninjected and ContMO- njected sides. Mean number of PH3+ cells is 5.88 on uninjected and 4.75 on ContMO-injected side, p = 0.54, n = 8. Loss of CDH11 increases cell death on njected side. All graphs show mean (indicated on graph) and median (line within graph). Scale bar whole mount images indicated in (A) and for sections in (C). Asterisk indicates injected side in sections. CDH11 Is Required for NC Cell Survival Mean number Casp3+ apoptotic cells is 17.90 on uninjected and 31.86 on CDH11MO-injected side, n = 14, p = 0.04. (I) IHC for Casp3 or (J) PAX7 in whole mount or for (I’) Casp3 or (J’) PAX7 in transverse section, (K) ContMO in whole mount and (L,L’) are overlays. (M) Graph showing difference between uninjected and ContMO-injected sides. Mean number of Casp3+ apoptotic bodies is 9.36 on uninjected and 9.36 on ContMO-injected side, p = 0.97, n = 14. (N) IHC for PH3 in whole mount and (O) overlay with CDH11MO. (P) Transverse section from HH8 embryo injected with CDH11MO on right side (green). (Q) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of PH3+ cells is 6.20 on uninjected and 5.20 on CDH11MO-injected side, p = 0.50, n = 20. (R) IHC for PH3 in whole mount embryo injected with (S) ContMO. (T) Transverse section. (U) Graph showing difference between uninjected and ContMO- injected sides. Mean number of PH3+ cells is 5.88 on uninjected and 4.75 on ContMO-injected side, p = 0.54, n = 8. Loss of CDH11 increases cell death on injected side. All graphs show mean (indicated on graph) and median (line within graph). Scale bar whole mount images indicated in (A) and for sections in (C). Asterisk indicates injected side in sections. Previous results in Xenopus embryos linked loss of CDH11 to increased Wnt-dependent cell cycling and suggested that loss of CDH11 was positively correlated with NC cell proliferation (Koehler et al., 2013). To determine if NC cell proliferation was altered after CDH11 knockdown at the premigratory stage, we injected and electorporated CDH11MO or ContMO unilaterally at HH4 and performed IHC for phosphorylated histone H3 (PH3), a marker of mitotic cells (Figures 4N–U). We determined that neither CDH11MO (Figures 4N–Q, n = 20, p = 0.57) nor ContMO (Figures 4R–U, n = 8, p = 0.54) significantly changed the number of PH3-positive cells suggesting that there is no consistent role for CDH11 in NC proliferation. These data demonstrate a requirement for CDH11 in NC cell survival. CDH11 Is Required for NC Cell Survival cell survival) or of a reduction NC cell proliferation (or reduced population growth), we unilaterally injected chicken embryos with CDH11MO or ContMO, electroporated at HH4, and performed IHC for markers of cell death and cell proliferation. To determine the requirement for CDH11 in NC cell survival, IHC was performed for tumor protein p53 (p53) as previous work in chicken NC cells demonstrated that p53 plays a role in NC cell EMT and that excess p53 reduces the premigratory NC cell population (Rinon et al., 2011). The fluorescence intensity of p53 expression was measured in the dorsal region of the neural tube, and in the absence of CDH11, p53 expression was increased in the CDH11MO-injected side of the neural tube compared to the uninjected side (Figures 4A–C, n = 17, p = 0.02, Supplementary Figures S4A–D). We next analyzed expression of the p53-mediated apoptosis effector protein, activated Caspase-3 (Casp3) together with PAX7 (Zou et al., 1999). After CDH11 knockdown, the total number of Casp3- positive cells was counted in the injected and uninjected sides of each embryo. Expression of Casp3 was significantly increased by 71.5% in CDH11MO-injected sides compared with the uninjected side (Figures 4D,D’,G,H, n = 14, p = 0.04), concurrent with a reduction in PAX7-positive cells (Figures 4E,E’,G,G’). However, embryos injected with ContMO showed no significant difference in Casp3 or PAX7 between the injected versus uninjected sides (Figures 4I-M, n = 14, p = 0.97). We also performed terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis to further confirm the presence of apoptotic cells in the neural tube after CDH11 knockdown. We identified an increase in fluorescence intensity of the TUNEL staining in the injected side of CDH11MO-injected neural tubes compared to the uninjected side (Supplementary Figures S4E–H,J, n = 12, p = 0.02) while ContMO-injected embryos had no significant difference in TUNEL staining between the injected and uninjected sides (Supplementary Figure S4I, n = 5, p = 0.53). Loss of CDH11 is thus correlated with an increase in cell death and CDH11 may be necessary for NC cell survival. FIGURE 4 \| Loss of CDH11 increases cell death. To determine the cause of reduced NC cell population after CDH11 knockdown, IHC was performed for (A-C) p53, (D,D’,G,G’,I,I’,L,L’) activated caspase 3 (Casp3) to mark apoptotic cells, (E,E’,G,G’,J,J’,L,L’) PAX7, or (N–U) phosphorylated histone H3 (PH3) to mark mitotic cells. (A,B) IHC for p53 in transverse section. CDH11 Is Required for NC Cell Survival (C) Graph showing difference between fluorescence intensity on uninjected and CDH11MO- injected sides (n = 17, p = 0.02). (D) IHC for Casp3 or (E) PAX7 in whole mount or for (D’) Casp3 or (E’) PAX7 in transverse section, (F) CDH11MO in whole mount and (G,G’) are overlays. (H) Graph showing increased number of Casp3-positive cells on CDH11MO side. Mean number Casp3+ apoptotic cells is 17.90 on uninjected and 31.86 on CDH11MO-injected side, n = 14, p = 0.04. (I) IHC for Casp3 or (J) PAX7 in whole mount or for (I’) Casp3 or (J’) PAX7 in transverse section, (K) ContMO in whole mount and (L,L’) are overlays. (M) Graph showing difference between uninjected and ContMO-injected sides. Mean number of Casp3+ apoptotic bodies is 9.36 on uninjected and 9.36 on ContMO-injected side, p = 0.97, n = 14. (N) IHC for PH3 in whole mount and (O) overlay with CDH11MO. (P) Transverse section from HH8 embryo injected with CDH11MO on right side (green). (Q) Graph showing difference between uninjected and CDH11MO-injected sides. Mean number of PH3+ cells is 6.20 on uninjected and 5.20 on CDH11MO-injected side, p = 0.50, n = 20. (R) IHC for PH3 in whole mount embryo injected with (S) ContMO. (T) Transverse section. (U) Graph showing difference between uninjected and ContMO- injected sides. Mean number of PH3+ cells is 5.88 on uninjected and 4.75 on ContMO-injected side, p = 0.54, n = 8. Loss of CDH11 increases cell death on injected side. All graphs show mean (indicated on graph) and median (line within graph). Scale bar whole mount images indicated in (A) and for sections in (C). Asterisk indicates injected side in sections. FIGURE 4 \| Loss of CDH11 increases cell death. To determine the cause of reduced NC cell population after CDH11 knockdown, IHC was performed for (A-C) p53, (D,D’,G,G’,I,I’,L,L’) activated caspase 3 (Casp3) to mark apoptotic cells, (E,E’,G,G’,J,J’,L,L’) PAX7, or (N–U) phosphorylated histone H3 (PH3) to mark mitotic cells. (A,B) IHC for p53 in transverse section. (C) Graph showing difference between fluorescence intensity on uninjected and CDH11MO- injected sides (n = 17, p = 0.02). (D) IHC for Casp3 or (E) PAX7 in whole mount or for (D’) Casp3 or (E’) PAX7 in transverse section, (F) CDH11MO in whole mount and (G,G’) are overlays. (H) Graph showing increased number of Casp3-positive cells on CDH11MO side. Blocking p53-Mediated Apoptosis Rescues NC Cellsi Mean number of PAX7+ cells is 47.77 on uninjected and 57.23 on CDH11MO + CDH11-GFP-injected side, p = 0.15, n = 13. Mean number of PAX7+ cells is 35.06 on uninjected and 39.94 on CDH11MO + p53MO-injected side, p = 0.48, n = 16. (L) Graph showing difference in Casp3 expression between uninjected and injected sides. Mean number of Casp3+ cells is 17.90 on uninjected and 31.86 on CDH11MO- injected side, p = 0.04, n = 14. Mean number of Casp3 + cells is 40.20 on uninjected and 36.80 on CDH11MO + CDH11-GFP-injected side, p = 0.71, n = 10. Mean number of Casp3 + cells is 16.50 on uninjected and 14.17 on CDH11MO + p53MO-injected side, p = 0.68, n = 12. All graphs show mean (indicated on graph) and median (line within graph). Phenotypes were rescued by co-injection with full length CDH11 as well as by blocking p53 translation suggesting that the NC phenotype is due to cell death after loss of CDH11. Scale bars for (E,F) are as marked in (E) and (G-J) are marked in (G). FIGURE 5 \| Blocking p53-mediated apoptosis rescues the NC fate. To determine if the NC and Casp3 phenotypes resulted from p53-mediated apoptosis, embryos were injected with multiple combinations of treatments to attempt to rescue the phenotype. (A–D) Whole mount IHC for PAX7 in HH8- HH9 embryos after (A) CDH11MO, (B) CDH11MO + CDH11-GFP, (C) p53MO, or (D) CDH11MO + p53MO. Inset shows treatment injection (green). (E) IHC for PAX7 in transverse section from HH9 embryo with (F) overlay with CDH11MO + CDH11-GFP (green). (G) IHC for PAX7 in transverse section from HH9 embryo with (H) overlay with Blocking p53-Mediated Apoptosis Rescues NC Cellsi Previous studies have identified both SNAI2 and SOX9 as factors that prevent apoptosis in NC cells in Xenopus and chick embryos, respectively (Tribulo et al., 2004; Cheung et al., 2005). Further, increased p53 expression has been linked to reduction October 2020 \| Volume 11 \| Article 563372 Frontiers in Physiology \| www.frontiersin.org 6 Manohar et al. Cadherin-11 Required for NC Survival in SNAI2 protein expression and increased craniofacial defects in chick and mouse (Rinon et al., 2011). Due to the reduction in the expression of the NC specifier proteins and increased in p53 and Casp3 expression after CDH11 knockdown, we hypothesized that blocking the p53-mediated apoptotic pathway would rescue the phenotype caused by reduction of CDH11. To confirm that the NC phenotype was specific to changes in CDH11 expression, we first compared embryos injected with CDH11MO alone to those co-injected with CDH11MO and full length CDH11-GFP rescue construct and performed IHC for PAX7 expression to examine NC development. As expected, whereas loss of CDH11 reduced PAX7-expressing cells at stages after HH8 significantly (Figures 5A,K, n = 18, p = 0.02), PAX7 expression was rescued by co-injection of CDH11-GFP (Figures 5B,E,F,K, n = 13, p = 0.15). We next co-injected CDH11MO with a p53 translation-blocking morpholino (p53MO) to rescue the loss of PAX7. Injection of p53MO alone had little effect on PAX7 expression (Figure 5C), but interestingly, co-injection of CDH11MO with p53MO was able to partially rescue the PAX7-positive cell population (Figures 5D,G,H,K, n = 16, p = 0.48). To confirm that the renewed PAX7 expression was caused by a reduction in CDH11- p53-mediated cell death, we verified the rescued phenotype by analyzing Casp3 expression in CDH11MO and p53MO co-injected embryos (Figures 5I,J,L, n = 14, p = 0.04), injection of p53MO alone did not significantly change levels of Casp3 in embryos (Figure 5L, n = 10, p = 0.71). However, blocking p53 was able to partially rescue the increased Casp3 expression (Figures 5I,J,L, n = 12, p = 0.68). These data demonstrate that loss of CDH11 reduces NC cells due to increased p53-mediated cell death, and that blocking p53-mediated apoptosis can rescue the loss of NC cells. FIGURE 5 \| Blocking p53-mediated apoptosis rescues the NC fate. To determine if the NC and Casp3 phenotypes resulted from p53-mediated apoptosis, embryos were injected with multiple combinations of treatments to attempt to rescue the phenotype. Blocking p53-Mediated Apoptosis Rescues NC Cellsi (I) IHC for Casp3 in transverse section from HH8 embryo with (J) overlay with CDH11MO + p53MO (green). (K) Graph showing difference in PAX7 expression between uninjected and injected sides. Mean number of PAX7+ cells is 42.8 on uninjected and 28.6 on CDH11MO- injected side, p = 0.02, n = 18. Mean number of PAX7+ cells is 47.77 on uninjected and 57.23 on CDH11MO + CDH11-GFP-injected side, p = 0.15, n = 13. Mean number of PAX7+ cells is 35.06 on uninjected and 39.94 on CDH11MO + p53MO-injected side, p = 0.48, n = 16. (L) Graph showing difference in Casp3 expression between uninjected and injected sides. Mean number of Casp3+ cells is 17.90 on uninjected and 31.86 on CDH11MO- injected side, p = 0.04, n = 14. Mean number of Casp3 + cells is 40.20 on uninjected and 36.80 on CDH11MO + CDH11-GFP-injected side, p = 0.71, n = 10. Mean number of Casp3 + cells is 16.50 on uninjected and 14.17 on CDH11MO + p53MO-injected side, p = 0.68, n = 12. All graphs show mean (indicated on graph) and median (line within graph). Phenotypes were rescued by co-injection with full length CDH11 as well as by blocking p53 translation suggesting that the NC phenotype is due to cell death after loss of CDH11. Scale bars for (E,F) are as marked in (E) and (G-J) are marked in (G). FIGURE 5 \| Blocking p53-mediated apoptosis rescues the NC fate. To determine if the NC and Casp3 phenotypes resulted from p53-mediated apoptosis, embryos were injected with multiple combinations of treatments to attempt to rescue the phenotype. (A–D) Whole mount IHC for PAX7 in HH8- HH9 embryos after (A) CDH11MO, (B) CDH11MO + CDH11-GFP, (C) p53MO, or (D) CDH11MO + p53MO. Inset shows treatment injection (green). (E) IHC for PAX7 in transverse section from HH9 embryo with (F) overlay with CDH11MO + CDH11-GFP (green). (G) IHC for PAX7 in transverse section from HH9 embryo with (H) overlay with CDH11MO + p53MO (green). (I) IHC for Casp3 in transverse section from HH8 embryo with (J) overlay with CDH11MO + p53MO (green). (K) Graph showing difference in PAX7 expression between uninjected and injected sides. Mean number of PAX7+ cells is 42.8 on uninjected and 28.6 on CDH11MO- injected side, p = 0.02, n = 18. Blocking p53-Mediated Apoptosis Rescues NC Cellsi (A–D) Whole mount IHC for PAX7 in HH8- HH9 embryos after (A) CDH11MO, (B) CDH11MO + CDH11-GFP, (C) p53MO, or (D) CDH11MO + p53MO. Inset shows treatment injection (green). (E) IHC for PAX7 in transverse section from HH9 embryo with (F) overlay with CDH11MO + CDH11-GFP (green). (G) IHC for PAX7 in transverse section from HH9 embryo with (H) overlay with CDH11MO + p53MO (green). (I) IHC for Casp3 in transverse section from HH8 embryo with (J) overlay with CDH11MO + p53MO (green). (K) Graph showing difference in PAX7 expression between uninjected and injected sides. Mean number of PAX7+ cells is 42.8 on uninjected and 28.6 on CDH11MO- injected side, p = 0.02, n = 18. Mean number of PAX7+ cells is 47.77 on uninjected and 57.23 on CDH11MO + CDH11-GFP-injected side, p = 0.15, n = 13. Mean number of PAX7+ cells is 35.06 on uninjected and 39.94 on CDH11MO + p53MO-injected side, p = 0.48, n = 16. (L) Graph showing difference in Casp3 expression between uninjected and injected sides. Mean number of Casp3+ cells is 17.90 on uninjected and 31.86 on CDH11MO- injected side, p = 0.04, n = 14. Mean number of Casp3 + cells is 40.20 on uninjected and 36.80 on CDH11MO + CDH11-GFP-injected side, p = 0.71, n = 10. Mean number of Casp3 + cells is 16.50 on uninjected and 14.17 on CDH11MO + p53MO-injected side, p = 0.68, n = 12. All graphs show mean (indicated on graph) and median (line within graph). Phenotypes were rescued by co-injection with full length CDH11 as well as by blocking p53 translation suggesting that the NC phenotype is due to cell death after loss of CDH11. Scale bars for (E,F) are as marked in (E) and (G-J) are marked in (G). FIGURE 5 \| Blocking p53-mediated apoptosis rescues the NC fate. To determine if the NC and Casp3 phenotypes resulted from p53-mediated apoptosis, embryos were injected with multiple combinations of treatments to attempt to rescue the phenotype. (A–D) Whole mount IHC for PAX7 in HH8- HH9 embryos after (A) CDH11MO, (B) CDH11MO + CDH11-GFP, (C) p53MO, or (D) CDH11MO + p53MO. Inset shows treatment injection (green). (E) IHC for PAX7 in transverse section from HH9 embryo with (F) overlay with CDH11MO + CDH11-GFP (green). (G) IHC for PAX7 in transverse section from HH9 embryo with (H) overlay with CDH11MO + p53MO (green). CDH11 Is Required for Normal NC Cell EMT, Morphology, and Migration Overall, loss of CDH11 significantly reduces NC cell population, affects their morphology, and reduces cell migration as a result. All graphs show mean (indicated on graph) and median (line within graph). Scale bars for (A-D) are as marked in (A), (A’,B’) are marked in (A’), (E,G) are marked in (E) (F,H) are marked in (F) and (F’–H”) are marked in (F’) FIGURE 6 \| CDH11 knockdown affects cell morphology and NC cell migration. To determine if the NC determination and cell death phenotype affects cell morphology in vivo (A–D’) embryos were injected unilaterally with CDH11MO and electroporated at HH4, and IHC was performed for (A–B’) CDH1 to mark epithelial cells and (C–D’) SOX9 to mark definitive NC cells. Dashed boxes in (A-D) indicate location of zoom in from (A’–D’).To determine if the CDH11 knockdown phenotype affects cell morphology and migration ex vivo (E–H”) neural tube explants were dissected from HH8 embryos, cultured on fibronectin coated slides, and stained for filamentous actin (F-actin). (A) IHC for CDH1 in transverse section from 10 SS embryo with (B) overlay with CDH11MO (green). (A’) Zoom in of dashed box from (A) showing increased CDH1 expression in dorsal neural tube in CDH11MO-injected versus uninjected side. (B’) Overlay with CDH11MO. (C) IHC for SOX9 in transverse section from the same 10 SS embryo from (A) with (D) overlay with CDH11MO (green) demonstrating reduced migration on CDH11-injected side. (C’,D’) Zoom in of dashed box from (C,D). (E-F”) Staining for F-actin in explant from embryo unilaterally injected with CDH11MO and electroporated at HH4, at (E) 20X and (F) 40X magnification. (F’) Zoom in of single follower cell from CDH11MO-injected explant. (F”) Zoom in of single leading cell from CDH11MO- injected explant. Both cells are significantly closer to epithelial explant and smaller than uninjected cells. (G–H”) Staining for F-actin in explant from uninjected side at (G) 20X and (H) 40X magnification. (H’) Zoom in of grouped follower cells from uninjected explant. (H”) Zoom in of single leading cell from uninjected explant. (I) Graph showing in vivo difference in CDH1 between uninjected and CDH11MO-injected sides. Corrected mean total cell fluorescence of CDH1 in the dorsal neural tube is 1645.5 on uninjected and 2202.9 on CDH11MO-injected side, p = 0.02, n = 11. (J) Graph showing difference in migration in vivo from midline of PAX7 and SOX9-positive cells between uninjected and CDH11MO-injected sides. CDH11 Is Required for Normal NC Cell EMT, Morphology, and Migration , p gy, g Finally, since cell adhesion molecules such as cadherins play a critical role in morphological changes during NC EMT and migration, we investigated how the NC cell features were affected by the early loss of CDH11 in the premigratory cells in vivo. To this end, we analyzed changes in the expression of E-cadherin/CDH1, a membrane-bound type I cadherin protein that has been linked to NC cell specification (Rogers et al., 2018) and to NC cell EMT and migration in both frog and chick embryos (Rogers et al., 2013; Huang et al., 2016). CDH11MO was unilaterally injected and electroporated at HH4, and cell morphology, CDH1 fluorescence intensity, and cell migration distance were measured in HH10 (10 SS) embryos. Loss of CDH1 enhanced CDH1 protein fluorescence in NC cells on the injected side (Figures 6A–B’, n = 11, p = 0.02), which resulted in defective migration while cells from the uninjected side had already migrated. We separately measured the distance from the midline that PAX7 and SOX9-positive cells traveled in CDH11MO-injected cells compared to uninjected sides because their expression differs in premigratory NC cell populations. CDH11MO-injected SOX9-positive cells migrated 31% less than the uninjected side (Figures 6C–D’,J, n = 17, p = 0.026) and PAX7-positive cells migrated 43.8% less than the uninjected side (Figure 6J, n = 19, p = 0.029). Our results suggest that CDH11 is necessary for NC specification, survival, and EMT. As a result of the early phenotype, cell morphology and migration remain affected at migratory stages. f We also performed NC explant assays to better assess the morphology and migratory ability of the cells lacking CDH11 ex vivo to determine if the NC migration defects in embryos October 2020 \| Volume 11 \| Article 563372 Frontiers in Physiology \| www.frontiersin.org 7 Cadherin-11 Required for NC Survival Manohar et al. FIGURE 6 \| CDH11 knockdown affects cell morphology and NC cell migration. To determine if the NC determination and cell death phenotype affects cell morphology in vivo (A–D’) embryos were injected unilaterally with CDH11MO and electroporated at HH4, and IHC was performed for (A–B’) CDH1 to mark epithelial cells and (C–D’) SOX9 to mark definitive NC cells. CDH11 Is Required for Normal NC Cell EMT, Morphology, and Migration Dashed boxes in (A-D) indicate location of zoom in from (A’–D’).To determine if the CDH11 knockdown phenotype affects cell morphology and migration ex vivo (E–H”) neural tube explants were dissected from HH8 embryos, cultured on fibronectin coated slides, and stained for filamentous actin (F-actin). (A) IHC for CDH1 in transverse section from 10 SS embryo with (B) overlay with CDH11MO (green). (A’) Zoom in of dashed box from (A) showing increased CDH1 expression in dorsal neural tube in CDH11MO-injected versus uninjected side. (B’) Overlay with CDH11MO. (C) IHC for SOX9 in transverse section from the same 10 SS embryo from (A) with (D) overlay with CDH11MO (green) demonstrating reduced migration on CDH11-injected side. (C’,D’) Zoom in of dashed box from (C,D). (E-F”) Staining for F-actin in explant from embryo unilaterally injected with CDH11MO and electroporated at HH4, at (E) 20X and (F) 40X magnification. (F’) Zoom in of single follower cell from CDH11MO-injected explant. (F”) Zoom in of single leading cell from CDH11MO- injected explant. Both cells are significantly closer to epithelial explant and smaller than uninjected cells. (G–H”) Staining for F-actin in explant from uninjected side at (G) 20X and (H) 40X magnification. (H’) Zoom in of grouped follower cells from uninjected explant. (H”) Zoom in of single leading cell from uninjected explant. (I) Graph showing in vivo difference in CDH1 between uninjected and CDH11MO-injected sides. Corrected mean total cell fluorescence of CDH1 in the dorsal neural tube is 1645.5 on uninjected and 2202.9 on CDH11MO-injected side, p = 0.02, n = 11. (J) Graph showing difference in migration in vivo from midline of PAX7 and SOX9-positive cells between uninjected and CDH11MO-injected sides. Average distance migrated away from midline by PAX7+ cells is 136.8 μm on uninjected and 76.93 μm on CDH11MO-injected side, p = 0.006, n = 11 cells. Average distance migrated away from midline by SOX9+ cells is 166.87 μm on uninjected and 115.22 μm on CDH11MO-injected side, p = 0.002, n = 19. (K) Graph showing average distance migrated ex vivo by cells from explant is 73.7 μm from uninjected explant and 53.6 μm from CDH11MO-injected explant, p = 0.02, n = 17 cells. (L) Graph showing average cell length is 36.8 μm in uninjected explants and 19.0 μm in CDH11MO-injected explants, p = 5.6E-08, n = 23 cells. CDH11 Is Required for Normal NC Cell EMT, Morphology, and Migration Average distance migrated away from midline by PAX7+ cells is 136.8 μm on uninjected and 76.93 μm on CDH11MO-injected side, p = 0.006, n = 11 cells. Average distance migrated away from midline by SOX9+ cells is 166.87 μm on uninjected and 115.22 μm on CDH11MO-injected side, p = 0.002, n = 19. (K) Graph showing average distance migrated ex vivo by cells from explant is 73.7 μm from uninjected explant and 53.6 μm from CDH11MO-injected explant, p = 0.02, n = 17 cells. (L) Graph showing average cell length is 36.8 μm in uninjected explants and 19.0 μm in CDH11MO-injected explants, p = 5.6E-08, n = 23 cells. Overall, loss of CDH11 significantly reduces NC cell population, affects their morphology, and reduces cell migration as a result. All graphs show mean (indicated on graph) and median (line within graph). Scale bars for (A-D) are as marked in (A), (A’,B’) are marked in (A’), (E,G) are marked in (E), (F,H) are marked in (F), and (F’–H”) are marked in (F’). strongly adherent to the epithelial explant (compare Figures 6E,F’–G,H’). In addition to exhibiting migration defects, cells lacking CDH11 were significantly smaller more rounded and lacked filopodia similar to Xenopus NC cells lacking CDH11 (Kashef et al., 2009; compare Figures 6F’,F”-H’,H”, n = 23 cells, p = 5.6E-08). are due to intrinsic or extrinsic properties. Embryos were electroporated with CDH11MO at HH4 and neural tube explants were dissected from the embryos at HH8 (3 SS–6 SS), cultured for 8 h, and then stained for filamentous actin (F-actin). Explants were repeated in triplicate and both leading edge and follower cells were measured (distance and size) individually. Cells lacking CDH11 migrated away from the explant 27.3% less than uninjected cells (Figures 6E–F”,K, n = 17, p = 0.02). The average migration distance from the epithelial explant by CDH11MO-injected cells was 53.6 μm while uninjected cells demonstrated a normal migratory ability, formed both lamellipodia and filopodia, and migrated approximately 73.7 μm from the explant (Figures 6G–H”,K). Very few cells were physically able to detach from the collective group in CDH11MO-injected explants, and most remained strongly adherent to the epithelial explant (compare Figures 6E,F’–G,H’). In addition to exhibiting migration defects, cells lacking CDH11 were significantly smaller more rounded and lacked filopodia similar to Xenopus NC cells lacking CDH11 (Kashef et al., 2009; compare Figures 6F’,F”-H’,H”, n = 23 cells, p = 5.6E-08). Frontiers in Physiology \| www.frontiersin.org DISCUSSION Defining the specific roles of cell adhesion proteins in early development is necessary as abnormal expression of these proteins is linked to cellular anomalies and congenital defects. With dynamic expression profiles, differential downstream signaling; and implications in cell survival, specification, and Defining the specific roles of cell adhesion proteins in early development is necessary as abnormal expression of these proteins is linked to cellular anomalies and congenital defects. With dynamic expression profiles, differential downstream signaling; and implications in cell survival, specification, and October 2020 \| Volume 11 \| Article 563372 8 Cadherin-11 Required for NC Survival Manohar et al. migration, cadherin proteins are involved in multiple a of embryonic development. Here, we examined the locali and function of CDH11 protein in early avian NC cells. C is expressed in the neural tube prior to NC cell format turned on in the NC cells at the premigratory stages, maintained in migratory NC cells. Loss of CDH11 re the number of specified cells (SNAI2, SOX9, SOX10-po that normally proceed to cell migration and reduced pr that are crucial for NC cell survival (SNAI2, SOX9). Furthe loss of CDH11 increased levels of CDH1 in premigrato cells, which failed to migrate normally. In contrast to pr studies, loss of CDH11 had little effect on cell prolife but increased p53 expression and p53-mediated cell de the neural tube, which was either a result of losing and SOX9 or caused the reduction in those proteins. Inh of p53 in cells deficient for CDH11 partially rescued th of NC cells and the increase in Casp3 (Figure 7A). Pr studies in frog embryos (Borchers et al., 2001; Kashef 2009; Langhe et al., 2016) have reported that CDH11-de cells exhibit migratory and morphological defects. Our shed light on the CDH11-mediated events in NC pr migration and allows us to posit that at least some migratory defects may be secondarily caused by defects specification and cell survival. Critical Timing of CDH11 Function NC cells are a dynamic population of cells con by multiple levels of secreted morphogens, transcr factors, epigenetic modifiers, and cell-cell adhesion mo A FIGURE 7 \| Summary diagram of CDH11-knockdown phenotype. (A) (CDH11-). DISCUSSION Normal cells undergo EMT, exit the neural tube, migrate colle lamellipodial and filopodial projections form as the cells navigate through mediated apoptosis due to either (1) inability to complete EMT/migration SOX10 positive cells are all significantly reduced in the absence of CDH (Martik and Bronner, 2017). To understand the potential role of CDH11 in early development, we characterized the spatiotemporal expression and localization of the protein in vivo in stages earlier than those previously identified (Chalpe et al., 2010). Our data indicate that CDH11 protein is expressed in the neuroepithelium during neurulation and is specifically upregulated above its neuroepithelial levels in the bonafide NC cells (SOX9+, SNAI2+) prior to EMT (Figure 1). The bulk of previous work studying CDH11 function in NC cells focused on its necessity for normal NC cell migration including its post-translational processing (McCusker et al., 2009; Abbruzzese et al., 2016), the formation of focal adhesions, protrusive activity, and extracellular matrix dynamics (Kashef et al., 2009; Langhe et al., 2016; Row et al., 2016). Here, we focused on understanding the pre-migratory role of CDH11 in NC cell development. CDH11 is expressed at very low levels in the neural plate border cells during NC induction, and our results suggest that it is not necessary for NC induction (3 SS and earlier), but rather it is necessary for NC cell specification and survival (5 SS and later; Figure 2). In support of our results, previous studies in Xenopus embryos showed that both exogenous CDH11 and dominant negative CDH11 expression reduced the population of undifferentiated NC cells and inhibited NC cell migration without affecting neural plate specification (Borchers et al., 2001). Interestingly, the same study reported that NC cells gained neural cell identities in the presence of dominant negative CDH11, in line with our finding that CDH11 may be necessary for specification during premigratory stages. We believe that our results fill a gap in the previously reported phenotype. Loss of CDH11 reduces migration, cadherin proteins are involved in multiple aspects of embryonic development. Here, we examined the localization and function of CDH11 protein in early avian NC cells. CDH11 is expressed in the neural tube prior to NC cell formation, is turned on in the NC cells at the premigratory stages, and is maintained in migratory NC cells. Critical Timing of CDH11 Function NC cells are a dynamic population of cells controlled by multiple levels of secreted morphogens, transcription factors, epigenetic modifiers, and cell-cell adhesion molecules A B FIGURE 7 \| Summary diagram of CDH11-knockdown phenotype. (A) Image depicting NC cell development in normal NC cells (CDH11+) and cells lacking CDH11 (CDH11-). Normal cells undergo EMT, exit the neural tube, migrate collectively, and then progressively mesenchymalize as development proceeds. Normal lamellipodial and filopodial projections form as the cells navigate through the extracellular matrix. In the absence of CDH11, NC cells are induced, but undergo p53- mediated apoptosis due to either (1) inability to complete EMT/migration or (2) altered intracellular signaling in the absence of CDH11. PAX7, SOX9, SNAI2, and SOX10 positive cells are all significantly reduced in the absence of CDH11 while p53 and Casp3 are upregulated. (B) Simplified NC GRN identifying NC specifiers and multiple putative inputs into the CDH11 upstream regulatory region as identified by ATAC-seq performed on NC cells (Williams et al., 2019). Little is known about the downstream targets and effectors of most cadherin proteins in the NC GRN with the exception of CDH6B (Schiffmacher et al., 2016) and CDH11 in migratory NC cells (Kashef et al., 2009; Koehler et al., 2013), and therefore identifying their targets in premigratory NC cells is essential. Direct binding relationships are indicated with solid lines while putative regulatory relationships are indicated by dashed lines. Letters indicate species in which experiments were performed: x = Xenopus, c = chicken, m = mouse. GRN information sourced from Rogers and Nie (2018). A B B FIGURE 7 \| Summary diagram of CDH11-knockdown phenotype. (A) Image depicting NC cell development in normal NC cells (CDH11+) and cells lacking CDH11 (CDH11-). Normal cells undergo EMT, exit the neural tube, migrate collectively, and then progressively mesenchymalize as development proceeds. Normal lamellipodial and filopodial projections form as the cells navigate through the extracellular matrix. In the absence of CDH11, NC cells are induced, but undergo p53- mediated apoptosis due to either (1) inability to complete EMT/migration or (2) altered intracellular signaling in the absence of CDH11. PAX7, SOX9, SNAI2, and SOX10 positive cells are all significantly reduced in the absence of CDH11 while p53 and Casp3 are upregulated. (B) Simplified NC GRN identifying NC specifiers and multiple putative inputs into the CDH11 upstream regulatory region as identified by ATAC-seq performed on NC cells (Williams et al., 2019). DISCUSSION Loss of CDH11 reduced the number of specified cells (SNAI2, SOX9, SOX10-positive) that normally proceed to cell migration and reduced proteins that are crucial for NC cell survival (SNAI2, SOX9). Furthermore, loss of CDH11 increased levels of CDH1 in premigratory NC cells, which failed to migrate normally. In contrast to previous studies, loss of CDH11 had little effect on cell proliferation but increased p53 expression and p53-mediated cell death in the neural tube, which was either a result of losing SNAI2 and SOX9 or caused the reduction in those proteins. Inhibition of p53 in cells deficient for CDH11 partially rescued the loss of NC cells and the increase in Casp3 (Figure 7A). Previous studies in frog embryos (Borchers et al., 2001; Kashef et al., 2009; Langhe et al., 2016) have reported that CDH11-deficient cells exhibit migratory and morphological defects. Our results shed light on the CDH11-mediated events in NC prior to migration and allows us to posit that at least some of the migratory defects may be secondarily caused by defects in cell specification and cell survival. Frontiers in Physiology \| www.frontiersin.org Critical Timing of CDH11 Function Little is known about the downstream targets and effectors of most cadherin proteins in the NC GRN with the exception of CDH6B (Schiffmacher et al., 2016) and CDH11 in migratory NC cells (Kashef et al., 2009; Koehler et al., 2013), and therefore identifying their targets in premigratory NC cells is essential. Direct binding relationships are indicated with solid lines while putative regulatory relationships are indicated by dashed lines. Letters indicate species in which experiments were performed: x = Xenopus, c = chicken, m = mouse. GRN information sourced from Rogers and Nie (2018). October 2020 \| Volume 11 \| Article 563372 Frontiers in Physiology \| www.frontiersin.org 9 Cadherin-11 Required for NC Survival Manohar et al. the definitive NC cell populations (Figures 2, 3) because without CDH11, these cells undergo p53-mediated cell death (Figures 4, 5). The timing of cell death occurs after induction but prior to NC cell EMT and migration. Our data suggest one of two possibilities; intracellular signaling downstream of CDH11 is necessary for the maintenance and survival of the premigratory NC cells, or that the cells require the presence of CDH11 for normal cell-cell adhesion-related EMT preparatory steps prior to migration, and without it they die. Future experiments are designed to test those hypotheses. Although CDH11 and other cadherins currently reside at the end of the NC GRN and are thought to function solely as regulators of NC EMT and migration downstream of NC specifiers, recent work that characterized all open enhancers in premigratory NC cells indeed identified putative binding sites in the CDH11 enhancer for SNAI2 and SOX9 (Figure 7B; Williams et al., 2019). Their enhancer analyses suggest that SNAI2 and SOX9 may drive CDH11 expression prior to EMT while FOXD3 and SOX2 may repress it. Future work will determine which of these factors functions upstream of CDH11 in the NC cells and whether the effects of CDH11 knockdown on cell specification and survival are direct (SOX9/ SNAI2➔CDH11➔cell specification/survival) or indirect through changes in NC specifiers (CDH11➔SOX9/SNAI2➔cell specification/survival). to Xenopus embryos (Supplementary Figure S1A), likely creating an extracellular fragment that would interact with the full length CDH11 to drive NC cell migration (McCusker et al., 2009). In our study, the NC cells have activated NC specifier proteins (SOX9, SNAI2, SOX10) and attempt to migrate, but cannot due to abnormal levels of CDH1 that prevent proper mesenchymalization (Figure 6; Rogers et al., 2013). Critical Timing of CDH11 Function Cells lacking CDH11 have increased F-actin cabling (Figure 6), which has previously been linked to the execution phase of cell death and is required for the formation of apoptotic bodies in embryonic carcinoma cells (Neradil et al., 2005). Additionally, actin-induced activation of the Ras signaling pathway has been previously linked to apoptosis (Gourlay and Ayscough, 2006). In contrast, overexpression of CDH11 may function to activate cell death via a Wnt-or Rho-dependent mechanism as previously described (Li et al., 2012; Row et al., 2016), and future experiments are designed to identify CDH11-specific apoptotic mechanisms. CDH11 and p53 Mediated Apoptosis CDH11 and p53 Mediated Apoptosis Our initial investigation into the role of CDH11 during early embryogenesis stemmed from its expression in developing NC cells and the varied accounts of its function in cancer cells. Previous studies in embryos demonstrated that CDH11 is necessary for NC migration (Vallin et al., 1998; Kashef et al., 2009; Abbruzzese et al., 2016; Langhe et al., 2016), but very few studies dissected its function in formation or survival in vivo (Borchers et al., 2001). Additionally, the role of CDH11 in cancer cells is variable. In murine retinoblastoma, CDH11 functions as a tumor suppressor, and overexpression of the gene in mouse models resulted in increased cell death (Marchong et al., 2010). This study relates to our data demonstrating that changes in CDH11 induces cell death or reduces NC cell survival; however, the mechanisms driving cell death downstream of gain and loss of CDH11 are likely unique in each situation. Both SLUG/SNAI2 (Tribulo et al., 2004) and SOX9 (Cheung et al., 2005) have demonstrated anti-apoptotic activity in NC cells, and their presence in the neural folds is correlated with reduced cell death. The previous studies paired with our data showing that loss of CDH11 reduces SOX9 and SNAI2 expression while concurrently increasing p53 expression (Figures 3, 5), suggests a link between CDH11 and the p53-mediated apoptotic pathway. However, rather than driving cell death indirectly, it is possibly based on its requirement for CDH11 in NC migration. The loss of CDH11, whether through changes in cell-cell adhesion or intracellular signaling, drives cell death. Loss of CDH11 in the premigratory cells creates a defect in the mechanisms driving NC delamination and EMT. Western blot analysis using protein lysates from progressives stages of chicken embryos confirmed that chicken CDH11 undergoes processing and cleavage similar Frontiers in Physiology \| www.frontiersin.org Implications and Considerations for Disease Studies Contrasting conclusions about the role of CDH11 in the disease state may be clarified if its function is assessed in the context of epithelial vs. mesenchymal cells. Loss of CDH11 prior to NC cell migration prevents the cells from leaving the neural tube efficiently, thereby activating the p53-mediated apoptotic pathway. The lack of specific expression in NC progenitors and generalized neural tube expression until just prior to the stage of migration suggests that CDH11 may play another role in the developing neuroepithelium. These data support a dual role for CDH11 during development. The upregulated expression of CDH11 in premigratory NC cells at 5 SS coincides with the wild type expression of SNAI2, SOX9, and SOX10, three proteins that are necessary for NC cell migration. It also coincides with the stage at which N-cadherin is reduced in the dorsal neural tube (Rogers et al., 2018). We believe that the functional type II cadherin complex is required for the completion of EMT and that loss of CDH11 leads to an increased tension on the cytoskeletal elements of the NC cells as evidenced by the increased CDH1 expression and F-actin localization causing activation of the p53-mediated apoptotic pathway. This hypothesis is supported by previous work demonstrating tightly controlled cadherin proteins in the NC EMT process (Coles et al., 2007; Rogers et al., 2013; Schiffmacher et al., 2014, 2016; Scarpa et al., 2015). In essence, the cells are programmed to migrate, but because they are unable, they are directed to die. As altering CDH11 affects cell survival, CDH11 may be functioning as it does in cancer cells, as pro-apoptotic stemness modulator that functions via the WNT and Rho pathways. Future experiments will focus on understanding the specific mechanisms downstream of CDH11 that regulate the NC cell population. Possible Mechanisms Downstream of CDH11i In Xenopus, CDH11 controls filopodia and lamellipodia formation by binding to the guanine nucleotide exchange factor (GEF) October 2020 \| Volume 11 \| Article 563372 10 Cadherin-11 Required for NC Survival Manohar et al. proteins. Overexpression of cdc42, Rac1, and RhoA in frog embryos lacking CDH11, rescues cranial NC cell migration (Kashef et al., 2009), and the GEF proteins specifically regulate NC cell protrusion and migration likely mediated by Rac1 (Kratzer et al., 2020). Therefore, loss of CDH11 may cause alterations in intracellular signaling and cytoskeletal rearrangements specifically linked to altered Rac/Rho signaling downstream of CDH11. proteins. Overexpression of cdc42, Rac1, and RhoA in frog embryos lacking CDH11, rescues cranial NC cell migration (Kashef et al., 2009), and the GEF proteins specifically regulate NC cell protrusion and migration likely mediated by Rac1 (Kratzer et al., 2020). Therefore, loss of CDH11 may cause alterations in intracellular signaling and cytoskeletal rearrangements specifically linked to altered Rac/Rho signaling downstream of CDH11. in 50 ms at 100 ms intervals. Injections of the morpholinos (0.5 mM-1 mM) were paired with 0.5–1.5 mg/ml of carrier plasmid DNA (Voiculescu et al., 2008) to enhance cell uptake of treatment. Injections were performed by air pressure using a glass micropipette targeted to the presumptive neural plate and neural plate border region. DNA plasmids pCAGGS- CDH11-IRES-GFP1, Sirius-H2B-C-10 (injected as marker for CDH11MO) were a gift from Michael Davidson to Addgene (Addgene plasmid # 552262; RRID:Addgene_55226) and were introduced in a similar manner to morpholinos described above. HH stage 4–5 electroporations were conducted on whole chick embryo explants placed ventral side up on filter paper rings. CDH11 also interacts with the proteoglycan, Syndecan-4, which maintains cell-substrate adhesion during cell migration. Loss of Syndecan-4 increased Rac activity, and inhibition of Rac1 by Syndecan-4 regulated the migration of NC cells (Matthews et al., 2008; Langhe et al., 2016). However, the non-canonical Wnt Planer Cell Polarity (PCP) Pathway promotes RhoA activity, which is necessary for NC cell migration, and inhibition of RhoA increased in Rac activation which can induce cell death (Matthews et al., 2008). It is possible that if the NC phenotype caused by loss of CDH11 is not directly related to cell-cell adhesion specific migration defects, rather, CDH11 may be a novel regulator that functions between Rac1 and RhoA, and loss of CDH11 may activate Rac1, preventing the cells from migrating and inducing apoptosis. Immunohistochemistry Immunohistochemistry (IHC) was performed as described previously (Strobl-Mazzulla and Bronner, 2012; Rogers et al., 2013). Briefly, for IHC, chicken embryos were fixed on filter paper in 4% paraformaldehyde (PFA) in phosphate buffer for 15–25 min at room temperature. After fixation, embryos were washed in 1X TBS (500 mM Tris-HCl, pH 7.4, 1.5 M NaCl, and 10 mM CaCl2) containing 0.1% Triton X-100 (TBST+ Ca2+). Short fixation times and TBST+ Ca2+ were used to enhance the IHC for cadherin proteins specifically. IHC using longer fixation or PBS + Triton without Ca2+ resulted in much lower antigen signal. For blocking, embryos were incubated in TBST+ Ca2+ and 10% donkey serum for 1 h at room temperature. Primary antibodies were diluted in blocking solution and incubated with embryos for 3 h at room temperature or for 24–48 h at 4°C. After incubation with primary antibodies, whole embryos were washed in TBST + Ca2+, incubated with AlexaFluor secondary antibodies diluted in blocking buffer (1,500) for 3 h at room temperature or 12–24 h at 4°C. They were then washed in TBST+ Ca2+, and post-fixed in 4% PFA for 30 min−1 h at room temperature. Antibodies used in the study (Table 1): Rabbit α-Cadherin-11 (Cell Signaling Technologies, #4442), Mouse α-Cadherin-11 (Invitrogen, #5B2H5), Mouse α-E-cadherin (BD Transduction Laboratories, 61081), Rabbit α-Active Caspase-3 (R and D Systems, #AF835), Mouse α-PAX7 (DSHB), Rabbit α-SOX9 (EMD Millipore, #ab5535), Rabbit α-SOX2 (Abcam, #ab97959), Mouse α-SOX10 (Proteintech, #66786-1-Ig), Rabbit α-SLUG/SNAI2 (Cell Signaling Technology, #9585S), Mouse α-p53 (Millipore, CBL404), and Rabbit α-Phospho histone H3 (R7D Systems, #ab5176). After IHC all embryos were imaged in both whole mount and transverse section (after cryosectioning) using a Zeiss Imager M2 with Apotome capability and Zen optical processing software. Loss of CDH11 causes p53-mediated cell death and reduces the number of bonafide NC cells in addition to causing morphological and migratory defects both in vivo and ex vivo. Our analyses add new information to previous discoveries, demonstrating that CDH11 is not solely required for migration, but plays an important role prior to NC cell emigration from the neural tube. Possible Mechanisms Downstream of CDH11i Future studies will continue to investigate the mechanisms that cause a reduction in the NC cell population in CDH11-deficient cells, but it is clear from our studies and others that CDH11 plays a complex and important role in NC cell formation and survival prior to its role in migration. Microinjection and Electroporationl Translation blocking antisense fluorescein or biotin-labeled morpholinos to CDH11 (CDH11MO; 5'-TATTTTGTAGGCA CAGGAGTATCCA-3'), p53 (5'-CAATGGTTCCATCTCCTCC GCCATG-3') and a non-specific control morpholino (ContMO; 5'-CCTCTTACCTCAGTTACAATTTATA-3') were microinjected into the right side of a Hamburger-Hamilton stage 4–5 chicken embryo and platinum electrodes were placed vertically across the embryos and electroporated with five pulses of 6.3–6.8 V Chicken Embryos y Fertilized chicken eggs were obtained from local sources (Sunstate Ranch, CA and the UC Davis Hopkins Avian Facility) and incubated at 37°C to the desired stages according to the criteria of Hamburger and Hamilton (HH). Use and experiments on embryos was approved by the California State University Northridge IACUC protocol: 1516-012a, c and the UC Davis IACUC protocol #21448. 1VectorBuilder.com 2http://n2t.net/addgene:55226 1VectorBuilder.com 2http://n2t.net/addgene:55226 Cell Counts and Statistical Analysis y All experiments were repeated three to four times. Cell counts and fluorescence intensity represented in box plots were either performed manually in Adobe Photoshop or were performed using NIH ImageJ. Cell counts were averaged from one to three sections per embryo. Mean, median, and standard deviation were calculated using Microsoft Excel across all biological replicates. The value of p was calculated in Microsoft excel using a Student’s t-Test with a 2-tailed distribution with unequal variance between samples for stringency. p-values under 0.05 are considered statistically significant. All cell counts are availables in Supplementary Tables 1–7. 7.4 with 150 mM NaCl plus 1.0% NP-40 and EDTA-free protease inhibitor (Roche cOmplete, #11697498001). SDS page was run on precast 8–12% bis-tris gel (Invitrogen, #NP0321BOX) for 3 h at 60 V; gel was transferred to nitrocellulose using the Invitrogen iBlot2 Dry Blotting System. Nitrocellulose membranes were washed in TBST+ Ca2+, blocked and incubated with primary antibody in TBST+ Ca2+ with 5.0% milk or 5.0% BSA, were then incubated in (5%) milk protein in TBST+ Ca2+ with secondary antibodies, and visualized using Prometheus ProSignal Femto ECL Reagent (#: 20-302B) and exposed to Prometheus ProSignal ECL Blotting Film, 5 × 7 in. (#: 30-507 L). ETHICS STATEMENT The animal study was reviewed and approved by California State University IACUC UC Davis IACUC. The animal study was reviewed and approved by California State University IACUC UC Davis IACUC. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. Imaging and Fluorescence Quantification Fluorescence images were taken using Zeiss ImagerM2 with Apotome.2 and Zen software (Karl Zeiss). Fluorescence was quantified using NIH ImageJ by averaging the relative intensity of 1–6 images per embryo. Specifically, when multiple section images were available, intensity would be measured individually and then averaged over one individual. N’s represent unique individuals. Background was subtracted uniformly across the images using the background subtraction function in NIH ImageJ with a rolling-ball radius of 50.00 pixels before quantitation (Hutchins and Szaro, 2013). For Casp3, fluorescence was compared between half neural tubes while all other analyses compared fluorescence in the dorsal half of the neural tubes on the injected and uninjected sides. Half embryos injected with CDH11MO or ContMO were compared to the uninjected or control side. Western Blot Embryo lysate was isolated from 10 to 20 manually dissected chicken embryos from stages HH4–6, HH8–10, and HH11–12 or tailbud stage axolotl embryos for Western blot analysis. Lysate was isolated using lysis buffer: 50 mM Tris-HCL pH October 2020 \| Volume 11 \| Article 563372 Frontiers in Physiology \| www.frontiersin.org 11 Cadherin-11 Required for NC Survival Manohar et al. TABLE 1 \| Antibodies used in study. Antibody Dilution SOURCE IDENTIFIER Mouse monoclonal anti-CDH11 (CDH11/Cadherin- OB) IgG1 1:250 IHC/1:10,000 WB Invitrogen 5B2H5 Rabbit polyclonal anti-CDH11 (CDH11/OB Cadherin) IgG 1:200 IHC/1:10,000 WB Cell Signaling Technologies 4442 Mouse anti-Pax7 IgG1 1:5 DSHB PAX7 Mouse anti-HNK1 IgM 1:5 DSHB 3H5 Mouse anti-CDH1 IgG2a 1:500 BD Biosciences 610181 Rabbit anti-SOX2 IgG 1:250 Abcam ab97959 Rabbit anti-SOX9 1:500 EMD Millipore ab5535 Rabbit anti-SLUG (SNAI2) 1:250 Cell Signaling Technology 9585S Mouse anti-SOX10 IgG2a 1:500 Proteintech 66786-1-Ig Rabbit anti- Phospho histone H3 1:500 Abcam ab5176 Mouse anti-p53 1:250 Millipore CBL404 Rabbit anti-Active Caspase-3 1:500 R and D Systems AF835 Ex vivo Neural Tube Explants For explant assays, embryos were electroporated with 0.75 mM CDH11MO plus Sirius carrier DNA on the right side of the embryo at HH4. Embryos were cultured until HH8 as described previously (Sauka-Spengler and Barembaum, 2008). At HH8, the neural tubes were dissected out of the embryo in Ringer’s solution and subsequently placed in 8-well chamber slides (Millicell EZ SLIDE 8-well glass, sterile, # PEZGS0816) that were coated with 100 μg/ml fibronectin. The explants were cultured in DMEM with 10% FBS, 2 mM l-glutamine, and 100 units of penicillin with 0.1 mg/ml streptomycin at 37°C with 0.5% CO2 for 8 h. After incubation, explants were fixed using 4% PFA, washed in TBST + Ca2+, and incubated with Phalloidin stain. Cytoskeletal stain was: Invitrogen Molecular Probes Alexa Fluor 568 Phalloidin (#A12380). TUNEL Assay For TUNEL assay, the Click-iT Plus TUNEL Alexa Fluor 488 and 647 were used. Protocol was as the manufacturer suggests. Briefly, embryos were fixed for 15 min in 4% PFA in phosphate buffer, washed in TBST + Ca2+, washed with deionized H2O, incubated with TdT reaction mix at 37°C, washed and incubated with Click-iT reaction for 30–60 min at room temperature. REFERENCES Gul, I. 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A., and Bronner-Fraser, M. (2007). A critical role for Cadherin6B in regulating avian neural crest emigration. Dev. Biol. 312, 533–544. doi: 10.1016/j.ydbio.2007.09.056 Dady, A., Blavet, C., and Duband, J. L. (2012). Timing and kinetics of E- to N-cadherin switch during neurulation in the avian embryo. Dev. Dyn. 241, 1333–1349. doi: 10.1002/dvdy.23813 Koehler, A., Schlupf, J., Schneider, M., Kraft, B., Winter, C., and Kashef, J. (2013). Loss of Xenopus cadherin-11 leads to increased Wnt/beta-catenin signaling and up-regulation of target genes c-myc and cyclin D1 in neural crest. Dev. Biol. 383, 132–145. doi: 10.1016/j.ydbio.2013.08.007 Del Barrio, M. G., and Nieto, M. A. (2004). Relative expression of Slug, RhoB, and HNK-1 in the cranial neural crest of the early chicken embryo. Dev. Dyn. ACKNOWLEDGMENTS SM, and CR. Funding Acquisition: CR (funds were provided either by NIH R15 HD092170-01 from the NICHD, CSUN startup funding, and UC Davis startup funding). Methodology: AC-M, SM, and CR. Supervision: CR. Writing: CR (wrote manuscript and performed editing), SM (wrote manuscript and performed editing), and AC-M (performed editing). All authors contributed to the article and approved the submitted version. SM, and CR. Funding Acquisition: CR (funds were provided either by NIH R15 HD092170-01 from the NICHD, CSUN startup funding, and UC Davis startup funding). Methodology: AC-M, SM, and CR. Supervision: CR. Writing: CR (wrote manuscript and performed editing), SM (wrote manuscript and performed editing), and AC-M (performed editing). All authors contributed to the article and approved the submitted version. We thank our colleagues from the Rogers Lab at California State University Northridge in the Department of Biology and those at UC Davis in the Department of Anatomy, Physiology, and Cell Biology, who provided insight and expertise that greatly assisted the research. FUNDING The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys.2020.563372/ full#supplementary-material This research was supported in part by a National Institute of Health, NICHD grant to CR (R15HD092170-01) and startup funds from UC Davis. AUTHOR CONTRIBUTIONS Conceptualization: CR and AC-M. Data Curation: AC-M (functional experiments and imaging), SM (characterization, functional experiments, and imaging), CR (functional experiments, data analysis, and imaging), and CE (functional experiments and imaging). Formal Analysis (cell counts, statistics): AC-M, October 2020 \| Volume 11 \| Article 563372 Frontiers in Physiology \| www.frontiersin.org 12 Cadherin-11 Required for NC Survival Manohar et al. REFERENCES Loss of Tbx3 in murine neural crest reduces enteric glia and causes cleft palate, but does not influence heart development or bowel transit. Dev. 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Pleiotropic effects of coat colour- associated mutations in humans, mice and other mammals. Semin. Cell Dev. Biol. 24, 576–586. doi: 10.1016/j.semcdb.2013.03.014 Yoshioka, R., Kita, Y., Nagahira, A., Manno, A., Makita, N., Tomita, U., et al. (2015). Quantitative analysis of cadherin-11 and beta-catenin signalling during proliferation of rheumatoid arthritis-derived synovial fibroblast cells. J. Pharm. Pharmacol. 67, 1075–1082. doi: 10.1111/jphp.12410 Rinon, A., Molchadsky, A., Nathan, E., Yovel, G., Rotter, V., Sarig, R., et al. (2011). p53 coordinates cranial neural crest cell growth and epithelial- mesenchymal transition/delamination processes. Development 138, 1827–1838. REFERENCES doi: 10.1242/dev.053645 Zou, H., Li, Y., Liu, X., and Wang, X. (1999). An APAF-1.Cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem. 274, 11549–11556. doi: 10.1074/jbc.274.17.11549 Rogers, C. D., and Nie, S. (2018). Specifying neural crest cells: from chromatin to morphogens and factors in between. Wiley Interdiscip. Rev. Dev. Biol. 7:e322. doi: 10.1002/wdev.322 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. Rogers, C. D., Saxena, A., and Bronner, M. E. (2013). Sip1 mediates an E-cadherin-to-N-cadherin switch during cranial neural crest EMT. J. Cell Biol. 203, 835–847. doi: 10.1083/jcb.201305050 Copyright © 2020 Manohar, Camacho-Magallanes, Echeverria and Rogers. 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. Rogers, C. D., Sorrells, L. K., and Bronner, M. E. (2018). A catenin-dependent balance between N-cadherin and E-cadherin controls neuroectodermal cell fate choices. Mech. Dev. 152, 44–56. doi: 10.1016/j.mod.2018.07.003 Row, S., Liu, Y., Alimperti, S., Agarwal, S. K., and Andreadis, S. T. (2016). Cadherin-11 is a novel regulator of extracellular matrix synthesis and tissue mechanics. J. Cell Sci. 129, 2950–2961. doi: 10.1242/jcs.183772 October 2020 \| Volume 11 \| Article 563372 Frontiers in Physiology \| www.frontiersin.org 14
https://openalex.org/W3001806592	https://krjournal.com/index.php/krj/article/download/76/389	English	null	ON TOTALLY SUPRA N-CONTINUOUS FUNCTION AND TOTALLY SUPRA N-CLOSED MAP	Kongunadu research journal	2,015	cc-by	2,779	1. INTRODUCTION Definition 2.3 (Mashhour et al., 1983) Let (X, τ) be a topological space and µ be a supra topology on X. We call µ a supra topology associated with τ , if τ ⊆ µ. In 1983, Mashhour et al. (1983) introduced the notion of supra topological spaces and studied, continuous functions and s* -continuous functions. Jamal M.Mustafa (2012) introduced and studies a class of functions called totally supra b-continuous and slightly supra b-continuous functions in supra topological spaces. Definition 2.4 Let (X, µ) be a supra topological space. A set A of X is called (i) supra semi- open set (Levine, 1991), if A ⊆ clµ(intµ(A)). (i) supra semi- open set (Levine, 1991), if A ⊆ clµ(intµ(A)). (ii) supra α -open set (Devi et al., 2008), if A ⊆ intµ(clµ (intµ (A))). In this paper, we introduce the concept of totally supra N-continuous function and totally supra N-closed map and investigated the relationship of these functions with other functions in supra topological spaces. (iii) supra Ω closed set (Noiri and Sayed, 2005), if sclµ(A) ⊆ intµ (U ),whenever A ⊆ U , U is supra open set. (iii) supra Ω closed set (Noiri and Sayed, 2005), if sclµ(A) ⊆ intµ (U ),whenever A ⊆ U , U is supra open set. Definition 2.1(Mashhour et al.,1983) A subfamily µ of X is said to be supra topology on X if The complement of above supra closed set is supra open and vice versa. The complement of above supra closed set is supra open and vice versa. i) X ,   Ai   i  j  Ai   ii) If then . The pair (X,µ) is called supra topological space. i) X ,   Ai   i  j  Ai   ii) If then . The pair (X,µ) is called supra topological space. Vidyarani, L. and M. Vigneshwaran* Department of Mathematics, Kongunadu Arts and Science College(Autonomous), Coimbatore-641029, Tamilnadu, India. E.mail: vignesh.mat@gmail.com TRACT Vidyarani, L. and M. Vigneshwaran Department of Mathematics, Kongunadu Arts and Science College(Autonomous), Coimbatore-641029, Tamilnadu, India. E.mail: vignesh.mat@gmail.com A map f:(X, τ ) → (Y, σ) is said to be (i) supra N-continuous (Vidyarani and Vigneshwaran, 2013a), if f-1(V) is supra N- closed in (X, τ) for every supra closed set V of (Y, σ). The element of µ are called supra open sets in (X, µ) and the complement of supra The element of µ are called supra open sets in (X, µ) and the complement of supra open set is called supra closed sets and it is denoted by µc. (ii) Perfectly supra N-continuous (Vidyarani and Vigneshwaran, 2013a), if f-1(V) is supra clopen in (X, τ) for every supra N-closed set V of (Y, σ). (ii) Perfectly supra N-continuous (Vidyarani and Vigneshwaran, 2013a), if f-1(V) is supra clopen in (X, τ) for every supra N-closed set V of (Y, σ). open set is called supra closed sets and it is denoted by µc. open set is called supra closed sets and it is denoted by µc. ABSTRACT In this paper, we introduce the concept of totally supra N-continuous function and totally supra N-closed map and investigated the relationship of these functions with other functions. Mathematics subject Classification: 54C05, 54C10. DOI:10.26524/krj65 DOI:10.26524/krj65 Kong. Res. J. 2(1) : 47-49, 2015 Kongunadu Arts and Science College, Coimbatore. Kong. Res. J. 2(1) : 47-49, 2015 Kongunadu Arts and Science College, Coimbatore. Kong. Res. J. 2(1) : 47-49, 2015 Kongunadu Arts and Science College, Coimbatore. Kong. Res. J. 2(1) : 47-49, 2015 ISSN 2349-2694 2. PRELIMINARIES (iv) supra N-closed set (Vidyarani and Vigneshwaran, 2013), if Ωclµ (A) ⊆ U , whenever A ⊆ U , U is supra α open set. Definition 2.1(Mashhour et al.,1983) Definition 2.5 A map f:(X, τ ) → (Y, σ) is said to be 3. TOTALLY SUPRA N-CONTINUOUS FUNCTIONS Definition 3.1 A map f:(X, τ ) → (Y, σ) is called totally supra continuous function if the inverse image of every supra open set in (Y, σ) is supra clopen in (X, τ ). Proof Let f:(X, τ ) → (Y, σ) be a totally supra continuous function. Let V be supra open set in (Y, σ). Since f is totally supra continuous function, then f -1(V) is supra clopen in (X, τ ). Implies f -1(V) is supra N-clopen in (X, τ ). Hence f -1 (V) is supra N-open in (X, τ ). Therefore f is supra N-continuous function. Definition 3.2 A map f:(X, τ ) → (Y, σ) is called totally supra N-continuous function if the inverse image of every supra open set in (Y, σ) is supra N- clopen in (X, τ ). The converse of the above theorem need not be true. It is shown by the following example. Theorem 3.3 Every strongly supra N-continuous function is totally supra N-continuous function. Example 3.8 Let X=Y={a, b, c} and τ = {X, φ, {a},{b},{a,b},{b, c}} , σ = {Y, φ, {a},{b,c} }. N-closed set in (X, τ ) are {X, φ, {a},{b},{a,b},{b, c}}. N-closed set in (Y, σ) are {Y, φ, {a},{b},{c},{a,b},{b, c},{a,c}}. f:(X, τ ) → (Y, σ) be the function defined by f(a)=c, f(b)=b, f(c)=a. Here f is supra N-continuous but not totally supra continuous, since V={b,c} is supra open in (Y, σ) but f -1({b,c}) = {a,b} is supra open but not supra closed set in (X, τ ). Proof Let f:(X, τ ) → (Y, σ) be a strongly supra N- continuous function. Let V be supra open set in (Y, σ). Then V is supra N-open set in (Y, σ), since every supra open set is supra N-open set. Since f is strongly supra N-continuous function f -1(V) is both supra open and supra closed in (X, τ ). Implies f -1(V) is supra N-clopen in (X, τ ). Therefore f is totally supra N-continuous function. Theorem 3.9 Every totally supra continuous function is totally supra N-continuous function. The converse of the above theorem need not be true. It is shown by the following example. The converse of the above theorem need not be true. It is shown by the following example. Proof Let f:(X, τ ) → (Y, σ) be a totally supra continuous function. 3. TOTALLY SUPRA N-CONTINUOUS FUNCTIONS Let V be supra open set in (Y, σ). Since f is totally supra continuous function, then f -1(V) is supra clopen in (X, τ ). Implies f -1(V) is supra N-clopen in (X, τ ). Therefore f is totally supra N- continuous function. Example 3.4 Let X=Y={a, b, c} and τ = {X, φ, {a},{b},{a,b},{b, c}} , σ = {Y, φ, {a} }. N-closed set in (X, τ ) are {X, φ, {a},{b},{a,b},{b, c}}. N-closed set in (Y, σ) are {Y, φ, {a},{b},{c},{a,b},{b, c},{a,c}}. f:(X, τ ) → (Y, σ) be the function defined by f(a)=a, f(b)=c, f(c)=b. Here f is totally supra N-continuous but not strongly supra N-continuous, since V={a,c} is supra N-closed in (Y, σ) but f -1({a,c}) = {a,b} is supra open but not supra closed set in (X, τ ). The converse of the above theorem need not be true. It is shown by the following example. The converse of the above theorem need not be true. It is shown by the following example. Example 3.10 Let X=Y={a, b, c} and τ = {X, φ, {a}} , σ = {Y, φ, {a},{b},{a,b},{b,c} }. N-closed set in (X, τ) are {X, φ, {a},{b},{c},{a,b},{b, c},{a,c}}. N-closed set in (Y, σ) are {Y, φ, {a},{b},{b, c},{a,c}}. f:(X, τ ) → (Y, σ) be the function defined by f(a)=b, f(b)=a, f(c)=c. Here f is totally supra N-continuous but not totally supra continuous, since V={a,b} is supra open in (Y, σ) but f -1({a,b}) = {a,b} is not supra clopen set in (X, τ ). Theorem 3.5 Every totally supra N-continuous function is supra N-continuous function. Theorem 3.5 Every totally supra N-continuous function is supra N-continuous function. Proof Let f:(X, τ ) → (Y, σ) be a totally supra N- continuous function. Let V be supra open set in (Y, σ). Since f is totally supra N-continuous function, then f -1(V) is supra N-clopen in (X, τ ). Implies f - 1(V) is supra N-open in (X, τ ). Therefore f is supra N- continuous function. Theorem 3.11 If f:(X, τ ) → (Y, σ) is totally supra N- continuous and g: (Y, σ) →(Z,η) is supra continuous then gοf: (X, τ ) →(Z,η) is totally supra N-continuous. Theorem 3.11 If f:(X, τ ) → (Y, σ) is totally supra N- continuous and g: (Y, σ) →(Z,η) is supra continuous then gοf: (X, τ ) →(Z,η) is totally supra N-continuous. Definition 2.2 (Mashhour et al., 1983) (iii) strongly supra N-continuous (Vidyarani and Vigneshwaran, 2013a), if f-1(V) is supra closed in (X, τ) for every supra N-closed set V of (Y, σ). (iii) strongly supra N-continuous (Vidyarani and Vigneshwaran, 2013a), if f-1(V) is supra closed in (X, τ) for every supra N-closed set V of (Y, σ). The supra closure of a set A is denoted by clµ (A), and is defined as supra cl(A) = ∩ {B : B is supra closed and A ⊆ B}. The supra interior of a set A is denoted by intµ(A), and is defined as supraint(A) = ∪ {B : B is supra open and A ⊇ B}. (iv) supra N-closed map (Vidyarani and Vigneshwaran, 2013b), if f(V) is supra N- closed in (Y, σ) for every supra closed set V of (X, τ ). 47 totally supra N-continuous, since V={a} is supra open in (Y, σ) but f -1({a}) = {c} is supra N-closed but not supra N-open set in (X, τ ). (v) strongly supra N-closed map (Vidyarani and Vigneshwaran, 2013b), if f(V) is supra N- closed in (Y, σ) for every supra N-closed set V of (X, τ ). Theorem 3.7 Every totally supra continuous function is supra N-continuous function. 4. TOTALLY SUPRA N-CLOSED MAP Definition 4.1 A map f:(X, τ) → (Y, σ) is said to be totally supra N-closed map, if f(V) is supra clopen in (Y, σ) for every supra N-closed set V of (X, τ ). Proof Let V be supra N-closed set in X, then f(V) is supra clopen in Y, since f is totally supra N-closed map. Implies f(V) is supra closed in Y. Then f(V) is supra N-closed in Y, since every supra closed set is supra N-closed set. Since g is totally supra N-closed map g(f(V)) is supra clopen in Z. Hence gof is totally supra N-closed map. Theorem 4.2 Every totally supra N-closed map is supra N-closed map. Proof Let f:(X, τ ) → (Y, σ) be a totally supra N- closed map. Let V be supra closed set in (X, τ ), then V is supra N-closed set in (X, τ ), since every supra closed set is supra N-closed set. Since f is totally supra N-closed map, then f(V) is supra clopen in (Y, σ). Implies f(V) is supra closed in (Y, σ). Therefore f(V) is supra N-closed in (Y, σ). Therefore f is supra N-closed map. 3. TOTALLY SUPRA N-CONTINUOUS FUNCTIONS Here f is strongly supra N-closed map but not totally supra N- closed map, since V={b} is supra N-closed in (X, τ ) but f ({b}) = {c} is supra open but not supra closed set in (Y, σ). Proof Let V be supra open set in Z. Since g is totally supra N-continuous, then g-1(V) is supra N-closed and supra N-open set in Y. Since f is perfectly supra N-continuous, then f-1g-1(V) is supra clopen in X. Implies f-1 g-1(V) is supra N-clopen in X. Hence gοf is totally supra N-continuous. ) but f ({b}) = {c} is supra open but not supra closed set in (Y, σ). Theorem 4.6 If f:(X, τ) → (Y, σ) is totally supra N- closed map and g: (Y, σ) →(Z,η) is totally supra N- closed map then gοf: (X, τ ) →(Z,η) is totally supra N- closed map. 3. TOTALLY SUPRA N-CONTINUOUS FUNCTIONS The converse of the above theorem need not be true. It is shown by the following example. Proof Let V be supra open set in Z. Since g is supra continuous, then g-1(V) is supra open set in Y. Since f is totally supra N-continuous, then f-1 (g-1(V)) is supra N-clopen in X. Hence gοf is totally supra N- continuous. Example 3.6 Let X=Y={a, b, c} and τ = {X, φ, {a},{b},{a,b},{b, c}} , σ = {Y, φ, {a},{b,c} }. N-closed set in (X, τ ) are {X, φ, {a},{b},{a,b},{b, c}}. N-closed set in (Y, σ) are {Y, φ, {a},{b},{c},{a,b},{b, c},{a,c}}. f:(X, τ ) → (Y, σ) be the function defined by f(a)=c, f(b)=b, f(c)=a. Here f is supra N-continuous but not Example 3.6 Let X=Y={a, b, c} and τ = {X, φ, {a},{b},{a,b},{b, c}} , σ = {Y, φ, {a},{b,c} }. N-closed set in (X, τ ) are {X, φ, {a},{b},{a,b},{b, c}}. N-closed set in (Y, σ) are {Y, φ, {a},{b},{c},{a,b},{b, c},{a,c}}. f:(X, τ ) → (Y, σ) be the function defined by f(a)=c, f(b)=b, f(c)=a. Here f is supra N-continuous but not 48 Theorem 3.12 If f:(X, τ ) → (Y, σ) is perfectly supra N-continuous and g: (Y, σ) →(Z,η) is totally supra N- continuous then gοf: (X, τ ) →(Z,η) is totally supra N- continuous. Theorem 3.12 If f:(X, τ ) → (Y, σ) is perfectly supra N-continuous and g: (Y, σ) →(Z,η) is totally supra N- continuous then gοf: (X, τ ) →(Z,η) is totally supra N- continuous. Example 4.5 Let X=Y={a, b, c} and τ = {X, φ,{a},{a,b}} , σ = {Y, φ, {a},{c},{a,c} }. N-closed set in (X, τ ) are {X, φ, {b},{c},{a,c},{b, c}}. N-closed set in (Y, σ) are {Y, φ, {a},{b},{c},{a,b},{b,c}}. f:(X, τ ) → (Y, σ) be the function defined by f(a)=a, f(b)=c, f(c)=b. Here f is strongly supra N-closed map but not totally supra N- closed map, since V={b} is supra N-closed in (X, τ ) but f ({b}) = {c} is supra open but not supra closed set in (Y, σ). Example 4.5 Let X=Y={a, b, c} and τ = {X, φ,{a},{a,b}} , σ = {Y, φ, {a},{c},{a,c} }. N-closed set in (X, τ ) are {X, φ, {b},{c},{a,c},{b, c}}. N-closed set in (Y, σ) are {Y, φ, {a},{b},{c},{a,b},{b,c}}. f:(X, τ ) → (Y, σ) be the function defined by f(a)=a, f(b)=c, f(c)=b. REFERENCES Devi, R., S. Sampathkumar and M. Caldas, (2008). On supra α open sets and sα-continuous maps, Gen. Math. 16(2): 77-84. Jamal M. Mustafa, (2012). Totally supra b-continuous and slightly supra b-continuous functions, stud. Univ. Babes. Bolyai Math. 57(1): 135-144. The converse of the above theorem need not be true. It is shown by the following example. The converse of the above theorem need not be true. It is shown by the following example. Example 4.3 Let X=Y={a, b, c} and τ = {X, φ, {a,b}} , σ = {Y, φ, {a,b},{b,c} }. N-closed set in (X, τ ) are {X, φ, {c},{a,c},{b, c}}. N-closed set in (Y, σ) are {Y, φ, {a},{c},{a,c}}. f:(X, τ ) → (Y, σ) be the function defined by f(a)=c, f(b)=b, f(c)=a. Here f is supra N- closed map but not totally supra N-closed map, since V={b,c} is supra N-closed in (X, τ ) but f ({b,c}) = {a,b} is supra open but not supra closed set in (Y, σ). Levine, N., (1991). Semi-open sets and Semi- continuity in topological spaces, Amer. Math. 12: 5-13. Mashhour, A.S., A.A. Allam, F.S. Mahmoud and F.H. Khedr, (1983). On supra topological spaces, Indian J. Pure and Appl.Math.14(A): 502-510. Noiri, T. and O.R. Sayed, (2005). On Ω closed sets and Ωs closed sets in topological spaces, Acta Math., 4:307-318. Theorem 4.4 Every totally supra N-closed map is strongly supra N-closed map. Vidyarani, L. and M. Vigneshwaran, (2013). N- Homeomorphism and N-Homeomorphism in supra topological spaces. Int. J. Math. Stat. Inv. 1(2): 79-83. Proof Let f:(X, τ ) → (Y, σ) be a totally supra N- closed map. Let V be supra N-closed set in (X, τ ). Since f is totally supra N-closed map, then f(V) is supra clopen in (Y, σ). Implies f(V) is supra closed in (Y, σ). Therefore f(V) is supra N-closed in (Y, σ). Therefore f is strongly supra N-closed map. Vidyarani, L. and M. Vigneshwaran, (2013a). Some forms of N-closed maps in supra Topological spaces. IOSR J. Math. 6(4): 13-17. Vidyarani, L. and M. Vigneshwaran, (2013b). On supra N-closed and sN-closed sets in supra Topological spaces. Int. J. Math. Arch. 4(2): 255- 259. The converse of the above theorem need not be true. It is shown by the following example. 49
https://openalex.org/W2972030945	https://discovery.dundee.ac.uk/ws/files/34582169/Screw_pile_LM_MA_AG_MC_R1.pdf	English	null	Finite element modelling of the uplift behaviour of screw piles in sand	null	2,019	public-domain	2,740	University of Dundee G-PFEM Numerical Assessment of Screw Pile Undrained Capacity Monforte, L.; Arroyo, Marcos; Gens, Antonio; Ciantia, Matteo Published in: ISSPEA 2019: DOI: 10.20933/100001123 Publication date: 2019 Document Version Peer reviewed version Link to publication in Discovery Research Portal Citation for published version (APA): Monforte, L., Arroyo, M., Gens, A., & Ciantia, M. (2019). G-PFEM Numerical Assessment of Screw Pile Undrained Capacity. In C. Davidson, M. J. Brown, J. A. Knappett, A. J. Brennan, C. Augarde, W. Coombs, L. Wang, D. Richards, D. White, & A. Blake (Eds.), ISSPEA 2019: 1st International Symposium on Screw Piles for Energy Applications (pp. 95-100). University of Dundee. https://doi.org/10.20933/100001123 Published in: ISSPEA 2019: DOI: 10.20933/100001123 Publication date: 2019 Document Version Peer reviewed version Link to publication in Discovery Research Portal Citation for published version (APA): Monforte, L., Arroyo, M., Gens, A., & Ciantia, M. (2019). G-PFEM Numerical Assessment of Screw Pile Undrained Capacity. In C. Davidson, M. J. Brown, J. A. Knappett, A. J. Brennan, C. Augarde, W. Coombs, L. Wang, D. Richards, D. White, & A. Blake (Eds.), ISSPEA 2019: 1st International Symposium on Screw Piles for Energy Applications (pp. 95-100). University of Dundee. https://doi.org/10.20933/100001123 Citation for published version (APA): Monforte, L., Arroyo, M., Gens, A., & Ciantia, M. (2019). G-PFEM Numerical Assessment of Screw Pile Undrained Capacity. In C. Davidson, M. J. Brown, J. A. Knappett, A. J. Brennan, C. Augarde, W. Coombs, L. Wang, D. Richards, D. White, & A. Blake (Eds.), ISSPEA 2019: 1st International Symposium on Screw Piles for Energy Applications (pp. 95-100). University of Dundee. https://doi.org/10.20933/100001123 General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Citation for published version (APA): Monforte, L., Arroyo, M., Gens, A., & Ciantia, M. (2019). G-PFEM Numerical Assessment of Screw Pile Undrained Capacity. In C. Davidson, M. J. Brown, J. A. Knappett, A. J. Brennan, C. Augarde, W. Coombs, L. Wang, D. Richards, D. White, & A. Blake (Eds.), ISSPEA 2019: 1st International Symposium on Screw Piles for Energy Applications (pp. 95-100). University of Dundee. https://doi.org/10.20933/100001123 L Monforte, M Arroyo, A Gens and M O Ciantia G-PFEM NUMERICAL ASSESSMENT OF SCREW PILE UNDRAINED CAPACITY Proceedings of the International Symposium on Screw Piles for Energy Applications, Dundee, Scotland,27 – 28 May 2019. G-PFEM NUMERICAL ASSESSMENT OF SCREW PILE UNDRAINED CAPACITY L MONFORTE, M ARROYO and A GENS Universitat Politécnica de Catalunya (UPC), Barcelona (Spain) - Departmen of Civil and Environmental Engineering lluis.monforte@upc.edu L MONFORTE, M ARROYO and A GENS Universitat Politécnica de Catalunya (UPC), Barcelona (Spain) - Department of Civil and Environmental Engineering lluis monforte@upc edu M O CIANTIA School of Science and Engineering, University of Dundee SUMMARY: This contribution describes a preliminary parametric analysis of the factors affecting the pull-out resistance of screw-piles in undrained conditions. The numerical simulations rely on the Particle Finite Element method, a method known for its capabilities to tackle large deformations and rapid changing boundaries at large strains. A total stress analysis –assuming a quasi-incompressible elastic model along with a Tresca plastic model- is used to simulate the clayey soil behavior. Contact constraints are imposed to the solution with a penalty approach. As a first step, a two-dimensional geometry is used and the pile resistance to pullout and penetratoin is assessed. General rights i h d Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 INTRODUCTION The fast growth of off-shore developments poses new challenges and increases the demand for cost-effective and reliable foundation solutions. Screw piles (or helical piles) have been proposed as a potential innovative alternative foundation in offshore environments. This kind of foundation is capable supporting large uplift loads. Examples of its application include the support of pipelines and the foundations for transmission towers1,2. The optimization of the geometry of the screw pile has been historically addressed by experimental means and also through the use of quite simplified analytical solutions. Although much insight is gained form such analyses, a number of basic features of the problem are left aside. Consequently, nowadays, numerical models are predominant. The Finite Element method (FEM) allows to accurately simulate the non-linear behavior of clayey soils using well- honed tools of continuum mechanics (field equations, constitutive material descriptions). However, advanced variants of FEM are required in order to avoid mesh tangling and distortion when the interaction between various deformable or rigid bodies is included. One such advanced variant is the Particle Finite Element Method (PFEM), which is here employed to simulate the screw piles in clayey soil. The work is structured as follows: first, the numerical method is outlined; after presenting a validation analysis (the simulation of the insertion of a pile) some preliminary results are highlighted; finally, some conclusions are drawn. Monforte, Arroyo, Gens and Ciantia Model set-up All the numerical simulations reported in this work share the same constitutive parameters. The soil is assumed to obey a Tresca yield criterion and a quasi-incompressible elastic model, with a Young modulus E = 2980 kPa, Poisson’s ratio ν = 0.49 and undrained shear strength Su = 10 kPa, which results in a rigidity index of Ir = G/ Su =100. The pile is considered completely rigid; this hypothesis is approximate enough due to the high ratio between the pile Young’s modulus and that of the soil. The contact constraints are imposed in the solution using a Penalty Method. This includes an inbuilt tension cut-off, since no tensions are allowed at the soil-structure interface. The tangential part of the contact is modelled employing the so-called elastic-plastic analogy and, as customary in total stress analyses, the maximum allowable contact tangential stress is taken as a fraction α of the undrained shear strength of the soil. At the beginning of the simulations, the pile is wished-in-place; thus, installation effects are not considered here. The soil initial state is characterized by a total pressure equal to p = 200 kPa and null deviatoric stresses q = 0 (K0 = 1). Therefore, at the top boundary a load of 200 kPa is applied. Both displacement components are restricted at the bottom boundary, whereas only the horizontal component is imposed at the vertical boundaries. NUMERICAL MODEL The Particle Finite Element Method is characterized by a particle discretization of the domain: every time step a finite element mesh – whose nodes are the particles – is constructed using a Delaunay’s tessellation and the solution is evaluated using this mesh with well-shaped, low- order elements. The continuum is modeled using an Updated Lagrangian formulation3,4. Additionally, h-adaptive routines are employed to obtain a better discretization of the domain. New particles are introduced in areas where large plastic dissipation is generated. These zones must be refined because the number of particles may become too low to obtain an accurate solution. On the contrary, due to high shear deformations, particles may locally concentrate in the same region of the domain. To overcome the difficulties that may follow from that, particles that are closer than a characteristic distance are removed. Numerical simulations have been carried out by means of the numerical code G-PFEM5, specially developed for the analysis of large strain contact problems in geomechanics. The code is able to accurately simulate the interaction between fluid-saturated porous media and rigid structures using low-order elements for efficiency. Techniques to alleviate volumetric locking are required: in this work, a mixed stabilized formulation6. The code is capable of handling coupled problems within quasi-static7 or fully dynamic settings8. However, in this preliminary study only a relatively simple axisymmetric undrained total-stress model is employed. This approach is reasonable, as the loading of piles in clayey soils occurs at a relatively high velocity compared with the hydraulic properties of clay and undrained conditions prevail. Validation analysis: penetration of a simple pile To showcase the possibilities of the method, results of the penetration of a pile are first presented. Two different cases are presented, one assuming a completely smooth interface and the other with an adhesion equal to half the undrained strength or α = 0.5. q g Figure 1 presents the main result of interest, namely the bearing capacity factor, Nc, d fi d q g Figure 1 presents the main result of interest, namely the bearing capacity factor, Nc, defined as: 𝑁𝑐 = 𝑞𝑡𝑖𝑝 – 𝜎𝑣0 𝑆𝑢 𝑁𝑐 = 𝑞𝑡𝑖𝑝 – 𝜎𝑣0 𝑆𝑢 𝑢 where 𝑞𝑡𝑖𝑝 is the total tip force dified by the projected area of the pile whereas 𝜎𝑣0 stands for the initial total vertical stress. It is clear that after a normalized penetration of one radii, both G-PFEM NUMERICAL ASSESSMENT OF SCREW PILE UNDRAINED CAPACITY cases reach a stationary state. Little influence of the contact roughness is visible in the results: for the smooth case, the mean bearing capacity factor is 8.97 whereas a value of 9.33 is obtained for the rough case. The obtained end bearing capacity factor assuming a smooth interface, 𝑁𝑐= 8.97, is in very close agreement with the traditional value proposed by Skempton9, 𝑁𝑐= 9. Figure 2 illustrates for the smooth case how the failure mechanism accompanies the pile, by plotting incremental plastic shear strain at several penetration depths (ranging from 1 to 5 radii). Figure { SEQ Figure \* ARABIC }: Closed-ended pile. Normalized penetration curve for the smooth and rough cases. Figure { SEQ Figure \* ARABIC }: Closed-ended pile. Normalized penetration curve for the smooth and rough cases. Figure { SEQ Figure \* ARABIC }: Closed-ended pile. Smooth interface. Incremental plastic shear strain. Figure { SEQ Figure \* ARABIC }: Closed-ended pile. Smooth interface. Incremental plastic shear strain. Monforte, Arroyo, Gens and Ciantia RESULTS AND DISCUSSION Once the accuracy of the developed numerical framework has been demonstrated, screw piles may be confidently simulated. Two different geometries are assessed with one and two helices (see Figure 3). In both cases a pull-out vertical displacement equal to 0.5 times the radius of the pile is imposed at the head of the pile. Figure 4 reports the load-displacement curves, where the pile displacement has been normalized by the radius of the pile whereas the load is normalized by the projected area of the pile. At zero displacement a pressure of 200 kPa has to be supplied to neutralize the effect of the initial stress state. For the single helix case, the pile rapidly reaches a steady state with a limit resistance of around 240 kPa. Meanwhile, for two helices, the limit resistance is around 450 kPa with slower resistance mobilization. Three different bearing capacity factors are also presented: for the tip of the pile (introduced previously), and for each individual helix, (defined as the total vertical force acting on their surfaces divided by the projected area and normalized by Su). Bearing capacity at the tip is negative; in contrast to the analysis of pile penetration the soil fails in an inverse mode. In the pile that only has one helix, the bearing capacity factor of the helix is close to 12. In contrast, in the case with two helices this capacity factor is of around 9 9. Figure 3: Total vertical stress [in kPa] (top) and incremental plastic shear strain (bottom) for cases with one (left) and two helices (right) Figure 3: Total vertical stress [in kPa] (top) and incremental plastic shear strain (bottom) for cases with one (left) and two helices (right) Figure 3: Total vertical stress [in kPa] (top) and incremental plastic shear strain (bottom) for cases with one (left) and two Figure 3: Total vertical stress [in kPa] (top) and incremental plastic shear strain (bottom) for cases with one (left) and two helices (right) G-PFEM NUMERICAL ASSESSMENT OF SCREW PILE UNDRAINED CAPACITY Figure 4: Evolution of the pile resistance, tip bearing capacity and bearing capacity factor of each screw in terms of the normalized uplift. Results for the geometry with two plates are depicted in blue. Figure 4: Evolution of the pile resistance, tip bearing capacity and bearing capacity factor of each screw in terms of the normalized uplift. RESULTS AND DISCUSSION Results for the geometry with two plates are depicted in blue. Figure 5: Push-pull sequence. Evolution of the pile resistance, tip bearing capacity and bearing capacity factors. Results for two plates are depicted in blue. Results during the penetration phase are plotted with a discontinuous line Figure 5: Push-pull sequence. Evolution of the pile resistance, tip bearing capacity and bearing capacity fact two plates are depicted in blue. Results during the penetration phase are plotted with a discontinuous line In both cases (Figure 3) the vertical stress (negative in compression) is reduced below the tip of the pile, whereas it is in high compression above the upper helix. Interestingly, no large variations in the stress field appear in the region between the two helices. Also, due to the moderately high initial stress (200 kPa), all the soil remains in contact with the structure during pull-out. Figure 3 also reports incremental plastic shear strain, to give an indication of the failure In both cases (Figure 3) the vertical stress (negative in compression) is reduced below the tip of the pile, whereas it is in high compression above the upper helix. Interestingly, no large variations in the stress field appear in the region between the two helices. Also, due to the moderately high initial stress (200 kPa), all the soil remains in contact with the structure during pull-out. Figure 3 also reports incremental plastic shear strain, to give an indication of the failure Monforte, Arroyo, Gens and Ciantia mechanism. With two helices, a cylinder-shape failure mechanism is observed between both helical plates, since they are relatively close to each other (2 R). For a single helix, a flow- around mechanism is obtained. Finally, another set of simulations was run in a first approximation to installation effects. This time, first, the pile is penetrated 0.5 R and then pulled out by the same amount. Figure 5 presents load-displacement curves, where the penetration phase is depicted with discontinuous lines and the subsequent pulling test with a continuous line. No large discrepancies from the previous result are observed for the pulling phase. ACKNOWLEDGEMENT This research was partly funded by the Spanish MEC through grant BIA2017-84752-R This research was partly funded by the Spanish MEC through grant BIA2017-84752-R CONCLUSION This contribution has outlined preliminary results of the numerical simulation of screw pile pull-out by means of the Particle Finite Element method. In particular, the effect of the number of helical plates has been assessed. Details of the total resistance and also the failure mechanism have been shown. The developed numerical scheme appears to be a promising tool for the simulation of tool-soil interaction. REFERENCES 1.Wang, D., Merifield, R. S: and Gaudin, C. Uplift behavior of helical anchors in clay. Canadian Geotechnical Journal, 50: 575-584 (2013). 2 Al-Baghdadi, T. Screw piles as offshore foundations. Numerical and physical modelling. PhD Thesis (University of Dundee) (2018) 3 Oñate, E., Idelsohn, S.R., Del Pin, F. & Aubry, R. The Particle Finite Element Method – an overview. International Journal of Computational Methods 1(2) 267-307 (2004). 4. Rodriguez, J.M., Carbonell, J.M., Cante, J.C. & Oliver, J. The Particle Finite Element Method (PFEM) in thermo-mechanical problems. Int. J. for Numerical Methods in Engineering, 107(9): 733-785 (2016). 5. Monforte, L., Arroyo, M., Carbonell, J.M. & Gens, A. Numerical simulation of undrained insertion problems in geotechnical engineering with the particle finite element method (PFEM). Computers and Geotechnics. 82, 144.156 (2017). 6. Monforte, L., Carbonell, J.M., Arroyo, M. & Gens, A. Performance of mixed formulations for the particle finite element method in soil mechanics problems. Computational Particle Mechanics. 4(3), 269-284 (2017). 7. Monforte, L., Arroyo, M., Carbonell, J. M., & Gens, A. (2018). Coupled effective stress analysis of insertion problems in geotechnics with the Particle Finite Element Method. Computers and Geotechnics, 101, 114-129. 8. Monforte, L., Navas, P., Carbonell, J. M., Arroyo, M., & Gens, A. (2019). Low‐ order stabilized finite element for the full Biot formulation in soil mechanics at finite strain. Int. J. for Numerical and Analytical Methods in Geomechanics, 43(7), 1488-1515. y 9. Skempton, A. W. The Bearing capacity of clays. Building Research Congress, London, The Institution of Civil Engineering, 180-189 (1951).
https://openalex.org/W4246788463	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0196523&type=printable	English	null	Correction: Distance, accessibility and costs. Decision-making during childbirth in rural Sierra Leone: A qualitative study	PloS one	2,018	cc-by	224	CORRECTION Reference 1. Treacy L, Bolkan HA, Sagbakken M (2018) Distance, accessibility and costs. Decision-making during childbirth in rural Sierra Leone: A qualitative study. PLoS ONE 13(2): e0188280. https://doi.org/10. 1371/journal.pone.0188280 PMID: 29462152 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Treacy L, Bolkan HA, Sagbakken M (2018) Correction: Distance, accessibility and costs. Decision-making during childbirth in rural Sierra Leone: A qualitative study. PLoS ONE 13(4): e0196523. https://doi.org/10.1371/journal. pone.0196523 Correction: Distance, accessibility and costs. Decision-making during childbirth in rural Sierra Leone: A qualitative study Laura Treacy, Håkon A. Bolkan, Mette Sagbakken The affiliation for the first and third author is incorrect. Laura Treacy is affiliated with Nad- heim Kirkens Bymisjon, Norbygata 45, 0190 Oslo, Norway. Mette Sagbakken is affiliated with Department of Nursing and Health Promotion, Faculty of Health Sciences, OsloMet—Oslo Metropolitan University, Postboks 4 St. Olavs plass 0130, Oslo, Norway. 1. Treacy L, Bolkan HA, Sagbakken M (2018) Distance, accessibility and costs. Decision-making during childbirth in rural Sierra Leone: A qualitative study. PLoS ONE 13(2): e0188280. https://doi.org/10. 1371/journal.pone.0188280 PMID: 29462152 Published: April 23, 2018 Copyright: © 2018 Treacy 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. 1 / 1 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196523 April 23, 2018
https://openalex.org/W2952599565	https://europepmc.org/articles/pmc6603296?pdf=render	English	null	IRT scoring procedures for TIMSS data	MethodsX	2,019	cc-by	4,331	Gregory Camillia,, John A. Dosseyb Gregory Camillia,, John A. Dosseyb a Rutgers University, United States b Illinois State University, United States a Rutgers University, United States b Illinois State University, United States Contents lists available at ScienceDirect Contents lists available at ScienceDirect MethodsX 6 (2019) 1506–1511 MethodsX 6 (2019) 1506–1511 A B S T R A C T In large-scale international assessment programs, results for mathematics proﬁciency are typically reported for jurisdictions such as provinces or countries. An overall score is provided along with subscores based on content subdomains deﬁned in the test speciﬁcations. In this paper, an alternative method for obtaining empirical subscores is described, where the empirical subscores are based on an exploratory item response theory (IRT) factor solution. This alternative scoring is intended to augment rather than to replace traditional scoring procedures. The IRT scoring method is applied to the mathematics achievement data from the Trends in International Mathematics and Science Study (TIMSS). A brief overview of the method is given, and additional material is given for validation of the empirical subscores. The ultimate goal of scoring is to provide diagnostic feedback in the form of naturally occurring item clustering. This provides useful information in addition to traditional subscores based on test speciﬁcations. As shown by Camilli and Dossey (2019), the achievement ranks of countries may change depending on which empirical subscore of mathematics is considered. Traditional subscores are highly correlated and tend to provide similar rank orders. The methods takes advantage of the TIMSS sampling design, speciﬁcally pairs of jackknife zones, to aggregate categorical to higher-order sampling units for IRT factor analysis. The methods takes advantage of the TIMSS sampling design, speciﬁcally pairs of jackknife zones, to aggregate categorical to higher-order sampling units for IRT factor analysis. Once factor scores are estimated for sampling units and interpreted, they are aggregated to the jurisdiction level (countries, states, provinces) using sampling weights. The procedure for obtaining standard errors of jurisdictional level scores combines cross-sampling-unit variance and Monte Carlo sampling variation. Once factor scores are estimated for sampling units and interpreted, they are aggregated to the jurisdiction level (countries, states, provinces) using sampling weights. The procedure for obtaining standard errors of jurisdictional level scores combines cross-sampling-unit variance and Monte Carlo sampling variation. Full technical details of the IRT factoring procedures are given in Camilli and Fox (2015). Fox (2010) provides additional background for Bayesian item response modeling techniques. The estimation algorithm is based on stochastic approximation expectation-maximization (SAEM). Full technical details of the IRT factoring procedures are given in Camilli and Fox (2015). Fox (2010) provides additional background for Bayesian item response modeling techniques. The estimation algorithm is based on stochastic approximation expectation-maximization (SAEM). © 2019 The Authors. A R T I C L E I N F O Method name: Empirical subscore estimation Keywords: Multidimensional scoring, Item response theory, IRT, Exploratory factor analysis, Mathematics achievement, Empirical subscores, International assessment, Diagnostic information, TIMSS Article history: Received 30 April 2019; Accepted 18 June 2019; Available online 21 June 2019 https://doi.org/10.1016/j.mex.2019.06.015 2215-0161/© 2019 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/). * Corresponding author. Introduction In large-scale international assessments, results for mathematics achievement are typically reported for jurisdictions such as provinces or countries. An overall score and content subscore scores are provided, but the most conspicuous feature of reporting is a set of ranks based on total scores. In this paper, a method for interpreting factor structures and deriving associated empirical factor scores is described relative to data from the Trends in International Mathematics and Science Study (TIMSS. A brief overview of the method is given below, and additional details for subscore estimation are given in Camilli and Fox [1]. Fox [2] provides a thorough background for Bayesian item response modeling techniques. In the companion paper [3] to the present article, a discussion of the TIMSS framework for mathematics assessment is provided and substantive results are broken down by participating countries. Speciﬁcations Table Mathematics Estimating multidimensional subscores for international comparisons in mathematics achievement Empirical subscore estimation Camilli, G. & Dossey, J. A. (2019). Obtaining multidimensional national proﬁles for TIMSS 2007 and 2011 mathematics. Journal of Mathematical Behavior. https://doi.org/10.1016/j. jmathb.2019.02.001. Camilli, G. & Fox, J.-P. (2015). An aggregate IRT procedure for exploratory factor analysis. Journal of Educational and Behavioral Statistics, 40, 377-401. https://doi.org/10.3102/ 1076998615589185. Gregory Camilli Subject area: More speciﬁc subject area: Mathematics Estimating multidimensional subscores for international comparisons in mathematics achievement Subject area: More speciﬁc subject area: Method name: Name and reference of original method: Method name: Name and reference of original method: Empirical subscore estimation Camilli, G. & Dossey, J. A. (2019). Obtaining multidimensional national proﬁles for TIMSS 2007 and 2011 mathematics. Journal of Mathematical Behavior. https://doi.org/10.1016/j. jmathb.2019.02.001. Camilli, G. & Fox, J.-P. (2015). An aggregate IRT procedure for exploratory factor analysis. Journal of Educational and Behavioral Statistics, 40, 377-401. https://doi.org/10.3102/ 1076998615589185. Traditional and empirical subscores A single score on an assessment and its associated rank encourage policy makers to view student achievement in terms of a “horserace” among countries or states. Describing performance in terms of subscores relating to different kinds of mathematics skills would at ﬁrst appear to encourage more strategic use of test information. For this purpose, framework- based subscores for TIMSS can be obtained and examined with International Data Explorer made available by the National Center for Education Statistics at https://nces.ed.gov/surveys/ international/ide/. Alternatively, subscores can be devised empirically using exploratory factor analysis. A B S T R A C T Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). A R T I C L E I N F O Method name: Empirical subscore estimation Keywords: Multidimensional scoring, Item response theory, IRT, Exploratory factor analysis, Mathematics achievement, Empirical subscores, International assessment, Diagnostic information, TIMSS Article history: Received 30 April 2019; Accepted 18 June 2019; Available online 21 June 2019 * Corresponding author. E-mail addresses: greg.camilli@gse.rutgers.edu (G. Camilli), jdossey44@gmail.com (J.A. Dossey). https://doi.org/10.1016/j.mex.2019.06.015 2215-0161/© 2019 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/). G. Camilli, J.A. Dossey / MethodsX 6 (2019) 1506–1511 G. Camilli, J.A. Dossey / MethodsX 6 (2019) 1506–1511 1507 Speciﬁcations Table Subject area: Mathematics More speciﬁc subject area: Estimating multidimensional subscores for international comparisons in mathematics achievement Method name: Empirical subscore estimation Name and reference of original method: Camilli, G. & Dossey, J. A. (2019). Obtaining multidimensional national proﬁles for TIMSS 2007 and 2011 mathematics. Journal of Mathematical Behavior. https://doi.org/10.1016/j. jmathb.2019.02.001. Camilli, G. & Fox, J.-P. (2015). An aggregate IRT procedure for exploratory factor analysis. Journal of Educational and Behavioral Statistics, 40, 377-401. https://doi.org/10.3102/ 1076998615589185. Resource availability: Gregory Camilli Factor selection and interpretation A three-factor solution was obtained at fourth grade for TIMSS 2007 and 2011 after transforming the initial solution to simple structure using the oblimin rotation. In both grades, the factor structure was observed to be nearly the same for 2007 and 2011. Choice of the number of factors was driven by three factors. First, eigenvalues are a byproduct of the aggregate model method, and these serve as a rough guide when employed in scree plots. This procedure usually indicated a factor structure of 2–5 factors. For example, from Table 4 of Camilli and Fox [1], the ﬁrst ﬁve eigenvalues are given for Grade 4 TIMSS data in 2007.Inordertheseare147.14,10.89,7.83,2.88,and2.63.Thescreeplotindicatedabreakbetweenthe3-and 4-factor solutions, though factors 4 and 5 showed moderate stability between 2007 and 2011. Next, substantive interpretations of the factors loadings were developed. Only factors with clear substantive interpretations were considered. Third, we conducted reliability analyses (reported below), and choose factors with cross-cycle (2007, 2011) correlations of .89 or higher. Finally, factor scores uik for each country i for each JKZ k on each rotated factor were generated. Aggregate factor analysis Aggregate factor analysis The data were then analyzed with the aggregate model of Camilli and Fox [1]. The goal of exploratory factor analysis at the higher-order level is to discover clusters of items that perform similarly across countries. The clusters are identiﬁed by expert interpretation of a loading matrix resulting from an exploratory factor analysis, and then scores are derived that correspond the factor structure. In short, the clusters are the “factors” of factor analysis, and the empirical subscores are factor scores. Factor selection and interpretation Data Data were obtained from the TIMSS 2007 mathematics for Grade 4 [4]. In TIMSS 2007, there were 59 jurisdictions with about 150 schools per jurisdiction and over 200 items matrix sampled over students (who each typically take about 25 items). It is convenient to refer to the higher-order units as countries, but TIMSS participants may include jurisdictions such as countries, states, or provinces within a country. With this usage in mind, the number of countries differs by both grade and topic (science or mathematics). For Grade 4, for example, there are 41 jurisdictions, 177 items, and 194,000 students. Students receive a subset of items (matrix sampling), but each item is paired with all other items within a country so that items can be analyzed simultaneously. Matrix sampling is a common design for large-sale assessments that do not report scores at the student level. The method described in this paper and Camilli and Fox [1] works well for TIMSS data, but has not yet been extended to other related methods of sampling. 1508 G. Camilli, J.A. Dossey / MethodsX 6 (2019) 1506–1511 A large number of items were not presented to individuals at the school level due to item matrix sampling procedures. Thus, school is not a practical unit for analysis. To address this sparseness issue, jackknife zones (JKZ) were used as units, where JKZs are formed by sorting schools on both explicit and implicit background variables and then pairing adjacent schools [5]. This process can be thought of as creating pairs of schools. This resulted in approximately 3000 level 2 units (or pairs) at each year-grade combination. The data set for analysis then consisted of a set of three variables for each jackknife unit within a country for each item: the total sample size for the given item, the weighted number of correct responses, and the weighted number of incorrect responses. Because individuals’ responses to an item within a sampling unit (jackknife zone) are aggregated, this can substantially reduce both the size of the data set and processing time. This modeling approach is a natural extension of the work by Mislevy [6,7] and Mislevy and Bock [8]. Factor interpretation There are many items in TIMSS, and an exploratory analysis yields m*Q factor loadings for m items and Q factors. For this reason, a method was constructed for interpreting factors based on two different statistics. First, the 10 highest loadings were considered in developing factor descriptions, which were determined by examining both the actual items and item information provided on the TIMSS website. Second, to describe factor resolution the full range of loadings were examined to determine whether the majority were positive for any given factor. As an illustration, consider TIMSS 2011 at grade 4 for 178 total items. The following tallies were used to evaluate the factor structure: the number of positive loadings, the number of loadings with magnitude <.1, and the number of negative loadings <.1. For factor 1, these tallies were 176, 4, and 1. One moderately-sized negative loading was observed (this item involved fractions and decimals), with 75 positive loadings higher in magnitude. For factor 2, the tallies were 130, 77, and 18. One moderate negative loading was observed, while 20 positive loadings higher in magnitude were observed. For factor 3, the tallies were 127, 80, and 13. One moderate negative loading was observed, with 15 positive loadings higher in magnitude. Similar results were obtained for other year/grade combinations. Choosing the largest negative loading to evaluate the number of larger positive loadings tends to understate factor resolution. If the second highest negative loading was chosen, the number of G. Camilli, J.A. Dossey / MethodsX 6 (2019) 1506–1511 1509 positive higher loadings higher in magnitude roughly doubles for factors 2 and 3. We also used this procedure to conﬁrm the number of factors chosen; factors not chosen (factors 4 and 5) performed worse on these tally statistics. Note also, that items used to interpret factors always had substantially higher loadings than the highest negative loading. A substantive interpretation of each factor is provided below. p For 2007 Grade 4 TIMSS data, items not sharing the fundamental theme suggested by the factor name tended not to occur for factors 2 and 3, with the caveat that as the loadings approached zero (all items have loadings because this is EFA), there was less consistency with the overall theme. For factor 1 (having to do with visual aspects of problem-solving) some items did at ﬁrst appear inconsistent. One such item concerned whole numbers: “Maria has 6 red boxes. Factor Descriptions Grades 4 and 8 Factor Descriptions Grades 4 and 8 Below, the factors are brieﬂy described. Full interpretations as well as sample items loading on the factors are given in Camilli and Dossey [3]. Factor 1_4. Factor 1 involves items requiring numerical reasoning with both charts and symbols, graphical reasoning related to data display, interpreting geometric shapes, and locating objects on a map. This factor can be thought of as reasoning with visual scaffolds, and is shortened to “Graphical Reasoning.” Factor 2_4. Factor 2 involves whole numbers and number sentences. These items all involve solving for an unknown rather than simple counting. This subscore is labeled “Solving for Unknowns.” Factor 3_4. Factor 3 is clearly focused on fractions and decimals from a variety of perspec factor is labeled “Fractions and Decimals.” Factor 1_8. Factor 1 predominantly involves items requiring applications of numerical skills in contextual settings. This factor is labeled simply as “General Numerical.” Factor 2_8. Factor 2 involves solving mathematical problems, but here, the focus is clearly on algebraic reasoning involving rates, ratios, and proportions. For shortened reference, this factor is labeled “Algebra and Ratios.” Factor interpretation Each red box has 4 pencils inside. She also has 3 blue boxes. Each blue box has 4 pencils inside. How many pencils does Maria have altogether?” Another whole number question asked “How much do the apples weigh in grams? This item included a drawing of a scale with apples on the weight tray and an analog read out. Thus, both items upon further examination were determined to reﬂect the overall factor theme due to their strong visual elements. The factors are described in more detail below. More information on factor interpretation can be obtained by contacting the ﬁrst author, including the TIMSS items with the highest loadings of the three mathematics factors for 2007 Grade 4. sb2 qi ¼ P kwik uqik uqi 2 =P kwik: This is the weighted between sampling zone variance. sw2 qi ¼ P kwiks2 qik=P kwik; where sw2 qi is the estimate of MCMC error associated with the empirical subscore obtained through Gibbs sampling. Given ni of sampling zones within country i (about 75), approximate the standard error as: SE uqi ¼ sb2 qi þ sw2 qi =ni 1=2 . Given ni of sampling zones within country i (about 75), approximate the standard error as: SE uqi ¼ sb2 qi þ sw2 qi =ni 1=2 . For TIMSS ofﬁcial score reports, the SEs of country means is obtained by a jackknife replication procedure. For the present analysis, SEs were approximated by a simpler method. However, the correlation between the approximate SEs for the ﬁrst factor and the jackknife SEs for the overall TIMSS score were r = .85 and r = .75 at Grades 4 and with outliers (Dubai and Qatar) removed. Reliability of empirical subscores Subscore value-added Subscore value-added As shown in Table 1, the intercorrelations among empirical subscores in ui were low to moderate (.15–58), and higher in Grade 8 than in Grade 4. Thus, factor-based subscores provide more distinct information for understanding national-level achievement. In contrast, intercorrelations among TIMSS domain scores are much higher at r > .95. To determine whether subscores provide practically distinct information, Feinberg and Wainer [10] suggested the VAR or values-added ratio based on the PRMSE of Haberman [11]. Sinharay et al. [12] Table 1 Observed correlations between empirical subscores. Correlation Pair 2007 Grade 4 2011 Grade 4 2007 Grade 8 2011 Grade 8 u1 u2 .31 (.34) .45 (.45) .60 (.56) .63 (.52) u1 u3 .58 (.61) .48 (.42) u2 u3 .20 (36) .15 (.02) Note: At Grade 4, u1 = General Factor, u2 = Solving for Unknowns, u3 = Fractions and Decimals; at Grade 8, u1 = General Factor, u2 = Algebra and Ratios. Spearman rank-order correlations in parentheses. Reliability of empirical subscores Obtaining parallel forms reliability is problematic in the case of TIMSS, because there are not parallel forms in the classical sense, but rather multiple spiraled forms containing matrix-sampled items. In reports of testing outcomes, reliability coefﬁcients for TIMSS scores are not given (e.g., [9]). However, to understand the reliability of the empirical subscores, two approaches were taken: simulation and test-retest. The simulation approach consisted of four steps: (1) estimate item parameters and level 2 scores with the aggregate model and designate these as generating parameters, (2) simulate individual-level item responses based on generating values, (3) carry out a new analysis of the simulated data, and (4) correlate generating level 2 scores (u) with those estimated from the simulated data. This procedure was carried out by Camilli and Fox [1]. They found correlations of .95, .90, and .89 for the ﬁrst three factors in 2007 Grade 4 Data. On the other hand, correlations for the fourth and ﬁfth factors were notable lower at .54 and .43. As noted below, the decision for choosing the number of factors involved this reliability as one of three criteria. Because item response data were generated according with an individual MIRT model that is consistent with the MIRT model used for analysis, these reliabilities may be inﬂated. Actual data may vary from MIRT assumptions. For an alternative to the simulation approach, a test-retest reliability was carried out for the three 2007 and 2011 subscores at Grade 4. Cross-year correlations of .96, .95, and .89 were found for country-level empirical subscores. Similar results were found at Grade 8 for 2007 and 2011 with reliabilities for the ﬁrst two factors at .96 and .93 (there was no indication of additional factors at Grade 8). These latter coefﬁcients may be deﬂated due to national changes in instruction or curriculum across the four-year interval. Computing country level factor scores and standard errors The aggregate MIRT (multidimensional IRT) model was proposed by Camilli and Fox [1] for addressing computational issues with large numbers of items and groups. They showed how factor scores uik could be generated for jackknife zone k of country i within Gibbs estimation cycles. Note we refer to u as a vector (of length Q) of empirical subscores. The new methodology reported here is how this information is aggregated to the country level in three steps: Compute the aggregate sampling weights of each i; k unit by wik ¼ P jwijk, where i, j, and k subscript countries, individual examinees within country, and sampling zones within countries, respectively. Compute the aggregate sampling weights of each i; k unit by wik ¼ P jwijk, where i, j, and k subscript countries, individual examinees within country, and sampling zones within countries, respectively. Compute the average country level score by ui ¼ P kwikuik=P kwik aggregating across sampling zones. The vector u holds q ¼ 1; 2; :::; Q empirical subscores obtained from the aggregate factor analysis. Compute the average country level score by ui ¼ P kwikuik=P kwik aggregating across sampling zones. The vector u holds q ¼ 1; 2; :::; Q empirical subscores obtained from the aggregate factor analysis. Approximate the standard error (SE) for each individual subscore uqi within country i by 510 G. Camilli, J.A. Dossey / MethodsX 6 (2019) 1506–1511 G. Camilli, J.A. Dossey / MethodsX 6 (2019) 1506–1511 1510 sb2 qi ¼ P kwik uqik uqi 2 =P kwik: This is the weighted between sampling zone varianc Table 1 Feinberg and Jurich [13] recommend a minimum value of VAR = 1.1 for subscores to be reported. The VARs for the TIMSS empirical subscores exceed this criterion, and this conclusion is unlikely to be altered with plausible changes in the equivalence assumptions above. The VARs were higher at Grade 4 than Grade 8. To determine whether subscores provide practically information above and beyond the total scale score, Feinberg and Jurich [10] suggested values-added index based on the work of Haberman [11]. We computed these value-added indices for each empirical subscore, and found they all exceeded the threshold recommended by Feinberg and Jurich [13]. The value-added indices were higher at Grade 4 than Grade 8. The reliability results above demonstrate that the empirical subscores are reliable enough to provide diagnostic information, while the value-added analysis indicates the subscores are distinct enough to provide non-overlapping diagnostic information. In short, the empirical subscores were found to be both distinct and informative based on a number of psychometric criteria. Acknowledgments This article have been funded by National Science Foundation, Education and Human Resources, Grant/Award Number: 1433760. Table 1 Note: At Grade 4, u1 = General Factor, u2 = Solving for Unknowns, u3 = Fractions and Decimals; at Grade 8, u1 = General Factor, u2 = Algebra and Ratios. Spearman rank-order correlations in parentheses. G. Camilli, J.A. Dossey / MethodsX 6 (2019) 1506–1511 1511 G. Camilli, J.A. Dossey / MethodsX 6 (2019) 1506–1511 1511 provided the formula provided the formula provided the formula VAR ¼ r1 r2 3r4 ; ð1Þ where r1 is subscore reliability, r3 is the disattenuated correlation of the subscore and total score, and r4 is total score reliability. To apply this formula to TIMSS empirical country-level subscores, several equivalences must be assumed. First, from Table 1 of Camilli and Dossey [3], r4 = .96–.99 can be obtained from the ﬁrst row, assuming the ﬁrst factor is synonymous with the overall score. As noted above, subscore reliabilities can be obtained as r1 = .89–.95. Finally, disattenuated subscore-total correlations can be obtained from rows 2 and 3 of Table 5 of Camilli and Dossey [3]. For illustration, empirical subscore at 2007 Grade 8 can be examined. In this case, r1 = .96, r3 ¼ :77= ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ :96 :97 p ¼ :80, and r4 = .97, so VAR = 1.54. Feinberg and Jurich [13] recommend a minimum value of VAR = 1.1 for subscores to be reported. The VARs for the TIMSS empirical subscores exceed this criterion, and this conclusion is unlikely to be altered with plausible changes in the equivalence assumptions above. The VARs were higher at Grade 4 than Grade 8. where r1 is subscore reliability, r3 is the disattenuated correlation of the subscore and total score, and r4 is total score reliability. To apply this formula to TIMSS empirical country-level subscores, several equivalences must be assumed. First, from Table 1 of Camilli and Dossey [3], r4 = .96–.99 can be obtained from the ﬁrst row, assuming the ﬁrst factor is synonymous with the overall score. As noted above, subscore reliabilities can be obtained as r1 = .89–.95. Finally, disattenuated subscore-total correlations can be obtained from rows 2 and 3 of Table 5 of Camilli and Dossey [3]. For illustration, empirical subscore at 2007 Grade 8 can be examined. In this case, r1 = .96, r3 ¼ :77= ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ :96 :97 p ¼ :80, and r4 = .97, so VAR = 1.54. References [1] G. Camilli, J.-P. Fox, An aggregate IRT procedure for exploratory factor analysis, J. Educ. Behav. Stat. 40 (2015) 377–401. [2] J.-P. Fox, Bayesian Item Response Modeling: Theory and Applications, Springer, New York, 2010. [3] G. Camilli, J.A. Dossey, Multidimensional national proﬁles for T (2019), doi:http://dx.doi.org/10.1016/j.jmathb.2019.02.001. g [5] P. Foy, Estimating standard errors for the TIMSS and PIRLS 2011 achievement scales, in: M.O. Martin, I.V.S. Mullis (Eds.), Methods and Procedures in TIMSS and PIRLS 2011, TIMSS & PIRLS International Study Center, Boston College, Boston, MA, 2012, pp. 1–7. Retrieved 4-18-2018 from http://timssandpirls.bc.edu/methods/pdf/TP11_Reviewing_Achievement.pdf. [5] P. Foy, Estimating standard errors for the TIMSS and PIRLS 2011 achievement scales, in: M.O. Martin, I.V.S. Mullis (Eds.), Methods and Procedures in TIMSS and PIRLS 2011, TIMSS & PIRLS International Study Center, Boston College, Boston, MA, 2012, pp. 1–7. Retrieved 4-18-2018 from http://timssandpirls.bc.edu/methods/pdf/TP11_Reviewing_Achievement.pdf. pp p // p / /p / g [6] R.J. Mislevy, Item response models for grouped data, J. Educ. Stat. 83 (1983) 271–288. [6] R.J. Mislevy, Item response models for grouped data, J. Educ. Stat. 83 (1983) 271–288. [7] R.J. Mislevy, Estimation of latent group effects, J. Am. Stat. Assoc. 80 (1986) 993–997. [7] R.J. Mislevy, Estimation of latent group effects, J. Am. Stat. Assoc. 80 (1986) 993 997. [8] R.J. Mislevy, R.D. Bock, A hierarchical item-response model for educational testing, in: R.D. Bock (Ed.), Multilevel Analysis of Educational Data, Academic Press, San Diego, CA, 1989, pp. 57–74. [8] R.J. Mislevy, R.D. Bock, A hierarchical item-response model for educational testing, in: R.D. Bock (Ed.), Multilevel Analysis of Educational Data, Academic Press, San Diego, CA, 1989, pp. 57–74. is, M.O. Martin, P. Foy, A. Arora, TIMSS 2011 International Results in Mathematics, TIMSS & PIRLS International ter, Boston, 2012. y [10] R.A. Feinberg, H. Wainer, A simple equation to predict a subscore’s value, Educ. Meas. Issues Pract. 33 (3) (2014) 55–56. [10] R.A. Feinberg, H. Wainer, A simple equation to predict a subscore’s value, Educ. Meas. Issues Pract. 33 (3) (2014) 55–56. [11] S.J. Haberman, When can subscores have value? J. Educ. Behav. Stat. 33 (2008) 204–229. [12] S. Sinharay, S.J. Haberman, K. Boughton, Too simple to be useful: a comment on Feinberg and Wainer (2014), Educ. Meas. Issues Pract. 34 (2015) 6–8.
https://openalex.org/W4376135781	https://figshare.com/articles/journal_contribution/Supplementary_Table_S2_from_A_Phase_I_II_Trial_of_HER2_Vaccine_Primed_Autologous_T-Cell_Infusions_in_Patients_with_Treatment_Refractory_HER2_Overexpressing_Breast_Cancer/22802179/1/files/40534810.pdf	English	null	Supplementary Table S1 from A Phase I/II Trial of HER2 Vaccine–Primed Autologous T-Cell Infusions in Patients with Treatment Refractory HER2–Overexpressing Breast Cancer	null	2,023	cc-by	964	1 Supplemental Table S2. Detailed adverse events Gr.1 Gr.2 Gr.3 Gr.4 Gr.1 Gr.2 Gr.3 Gr.4 Allergy/Immunology Allergic rhinitis (including sneezing, nasal stuffiness, 2 1 postnasal drip) Allergic reaction/hypersensitivity (including drug 1 1 fever) Auditory/Ear Tinnitus 1 Blood/Bone Marrow Leukopenia 2 2 8 3 2 Lymphopenia 1 1 1 1 6 9 5 2 Hgb low 3 1 7 7 Platelets low 2 2 ANC/AGC low 2 4 3 1 Other MCV high 1 MCH high 1 2 RDW-CV high 1 Eosinophils high 1 RBC high 3 Unclassified cells – blasts present 1 MCHC low 2 HCT low 2 1 4 4 RBC low 1 6 3 Immature granulocytes high 1 Cardiac Arrhythmia Cardiac Arrhythmia - Other - 1 Cardiac General Hypotension 1 Hypertension 1 Other Tachycardia 1 Tachypnea 1 Constitutional Symptoms Insomnia 1 Fever (in the absence of neutropenia, where 5 neutropenia is defined as ANC <1.0 x 10e9/L) Rigors/chills 2 4 4 Fatigue (asthenia, lethargy, malaise) 2 1 8 6 Weight loss 2 Sweating (diaphoresis) 1 Unrelated Related 1 Supplemental Table S2. Detailed adverse events Gr.1 Gr.2 Gr.3 Gr.4 Gr.1 Gr.2 Gr.3 Gr.4 Allergy/Immunology Allergic rhinitis (including sneezing, nasal stuffiness, 2 1 postnasal drip) Allergic reaction/hypersensitivity (including drug 1 1 fever) Auditory/Ear Tinnitus 1 Blood/Bone Marrow Leukopenia 2 2 8 3 2 Lymphopenia 1 1 1 1 6 9 5 2 Hgb low 3 1 7 7 Platelets low 2 2 ANC/AGC low 2 4 3 1 Other MCV high 1 MCH high 1 2 RDW-CV high 1 Eosinophils high 1 RBC high 3 Unclassified cells – blasts present 1 MCHC low 2 HCT low 2 1 4 4 RBC low 1 6 3 Immature granulocytes high 1 Cardiac Arrhythmia Cardiac Arrhythmia - Other - 1 Cardiac General Hypotension 1 Hypertension 1 Other Tachycardia 1 Tachypnea 1 Constitutional Symptoms Insomnia 1 Fever (in the absence of neutropenia, where 5 neutropenia is defined as ANC <1.0 x 10e9/L) Rigors/chills 2 4 4 Fatigue (asthenia, lethargy, malaise) 2 1 8 6 Weight loss 2 Sweating (diaphoresis) 1 Unrelated Related 1 Supplemental Table S2. Detailed adverse events (continue-) 2 Supplemental Table S2. Detailed adverse events (continue-) Gr.1 Gr.2 Gr.3 Gr.4 Gr.1 Gr.2 Gr.3 Gr.4 Dermatology/Skin Dry skin 1 Injection site reaction/extravasation changes 1 43 9 Hyperpigmentation 1 2 Pruritus/itching 1 1 Rash/desquamation 1 1 3 Rash: acne/acneiform 4 Hair loss/alopecia (scalp or body) 1 Hyperpigmentation - vaccination site 1 Dermatology/Skin - Other (Specify,-) 1 Endocrine Hot flashes/flushes 2 1 Gastrointestinal Diarrhea 6 1 2 Nausea 4 10 2 1 Vomiting 2 1 2 1 Anorexia 2 5 1 Constipation 6 1 2 Dehydration 1 Infection Other UTI 1 4 HSV 4 Pulmonary/Upper Respiratory – NOS 1 2 Pulmonary/Upper Respiratory – sinus 1 Varicella 1 Lymphatics Edema:limb 2 2 1 Edema:head and neck 1 Other Axilla swelling 1 Metabolic/Laboratory Calcium, serum-low (hypocalcemia) 1 4 Potassium, serum-low (hypokalemia) 2 5 Sodium, serum-low (hyponatremia) 2 13 2 Creatinine high 1 Alkaline phosphatase high 1 2 ALT, SGPT high 1 2 Albumin, serum-low (hypoalbuminemia) 1 11 2 AST, SGOT high 1 5 1 Unrelated Related 2 3 Supplemental Table S2. 1 Supplemental Table S2. Detailed adverse events Gr.1 Gr.2 Gr.3 Gr.4 Gr.1 Gr.2 Gr.3 Gr.4 Allergy/Immunology Allergic rhinitis (including sneezing, nasal stuffiness, 2 1 postnasal drip) Allergic reaction/hypersensitivity (including drug 1 1 fever) Auditory/Ear Tinnitus 1 Blood/Bone Marrow Leukopenia 2 2 8 3 2 Lymphopenia 1 1 1 1 6 9 5 2 Hgb low 3 1 7 7 Platelets low 2 2 ANC/AGC low 2 4 3 1 Other MCV high 1 MCH high 1 2 RDW-CV high 1 Eosinophils high 1 RBC high 3 Unclassified cells – blasts present 1 MCHC low 2 HCT low 2 1 4 4 RBC low 1 6 3 Immature granulocytes high 1 Cardiac Arrhythmia Cardiac Arrhythmia - Other - 1 Cardiac General Hypotension 1 Hypertension 1 Other Tachycardia 1 Tachypnea 1 Constitutional Symptoms Insomnia 1 Fever (in the absence of neutropenia, where 5 neutropenia is defined as ANC <1.0 x 10e9/L) Rigors/chills 2 4 4 Fatigue (asthenia, lethargy, malaise) 2 1 8 6 Weight loss 2 Sweating (diaphoresis) 1 Unrelated Related Detailed adverse events (continue-) Gr.1 Gr.2 Gr.3 Gr.4 Gr.1 Gr.2 Gr.3 Gr.4 Metabolic/Laboratory (continued) Other Carbon dioxide high 1 Protein high 1 BUN low 6 Chloride high 2 GFR low 3 Protein low 3 Chloride low 2 Carbon dioxide low 1 Neurology Dizziness 1 Neuropathy: sensory 1 Other Tingling lips, chest during leukapheresis 1 Pain Arthralgia 4 1 1 1 Myalgia 6 2 4 Pain 1 2 1 2 Abdomen NOS 3 1 Back 4 1 1 Bone 1 3 6 5 Extremity-limb 1 Head/headache 5 2 1 14 1 2 Neuralgia/peripheral nerve 1 Chest wall 1 Pulmonary/Upper Respiratory Cough 9 2 1 Other pharyngitis/rhinitis 1 Renal/Genitourinary Urinary frequency/urgency 1 Other Bacteria present 4 4 protein 1 Ketones present 5 1 Leukocyte esterase present 2 8 WBC present 2 9 Mucus present 3 8 Protein present 1 4 Unrelated Related Supplemental Table S2. Detailed adverse events (continue-) Metabolic/Laboratory (continued) Bone 1 3 6 5 Extremity-limb 1 Head/headache 5 2 1 14 1 2 Neuralgia/peripheral nerve 1 Chest wall 1 Pulmonary/Upper Respiratory Cough 9 2 1 Other pharyngitis/rhinitis 1 Renal/Genitourinary Urinary frequency/urgency 1 Other Bacteria present 4 4 protein 1 Ketones present 5 1 Leukocyte esterase present 2 8 WBC present 2 9 Mucus present 3 8 Protein present 1 4 3 Supplemental Table S2. Detailed adverse events (continue-) Gr.1 Gr.2 Gr.3 Gr.4 Gr.1 Gr.2 Gr.3 Gr.4 Renal/Genitourinary (continued) Yeast present 1 Epith cells present 2 6 Specific gravity high 1 2 Cast present 1 3 Crystals present 1 Glucose present 1 RBC present 1 Blood present 1 Syndromes Flu-like syndrome 2 7 1 Vascular Thrombosis/thrombus/embolism 1 Ocular/Visual Ophthalmoplegia/diplopia (double vision) 1 Unrelated Related Supplemental Table S2. Detailed adverse events (continue-) 4
https://openalex.org/W2936296385	https://europepmc.org/articles/pmc6434866?pdf=render	English	null	HPV testing for cervical cancer screening: technical improvement of laboratory logistics and good clinical performance of the cobas 6800 in comparison to the 4800 system	BMC women's health	2,019	cc-by	5,933	Open Access HPV testing for cervical cancer screening: technical improvement of laboratory logistics and good clinical performance of the cobas 6800 in comparison to the 4800 system Helena Frayle1†, Silvia Gori1†, Martina Rizzi1, Bianca Nives Graziani2, Elisa Vian3, Paolo Giorgi Rossi4 and Annarosa Del Mistro1* Annarosa Del Mistro1* Abstract Background: European guidelines for cervical cancer screening now recommend the use of clinically validated assays for high-risk HPV-DNA sequences as primary test in women older than 30 years, performed in centralized laboratories, and run on systems providing automated solutions for all steps. Methods: We conducted a comparison study, according to the international guidelines, nested within the organized population-based cervical screening program, between the cobas 4800 and 6800 systems (Roche Diagnostics), to evaluate accuracy and reproducibility of HPV test results and laboratory workflow. In Italy implementation of HPV cervical screening is under way on a regional basis; in Veneto it started in June 2015, following a piloting phase; the assay in use in the three centralized laboratories is the cobas 4800 HPV test, run on the cobas 4800 system. Comparison of HPV results with a new version of the assay (cobas 6800/8800 HPV) run on the cobas 6800 system, and intra- and inter-reproducibility analyses have been conducted in samples collected in PreservCyt medium (Hologic) from women without and with a subsequent diagnosis of high-grade lesion. Results: Samples from women older than 30 years attending organized cervical cancer screening were used. Clinical sensitivity and specificity were evaluated on 60 cases and 925 controls, respectively; intra-laboratory reproducibility and inter-laboratory agreement by the 6800 system were evaluated on 593 and 460 specimens, respectively. Our results showed a very high agreement (> 98%) for overall qualitative results between the two systems; clinical sensitivity and specificity of the HPV assay run on 6800 were non-inferior to those of the HPV assay run on 4800 (p = 0,0157 and p = 0,0056, respectively, at the recommended thresholds of 90 and 98%); kappa values of 0.967 and 0. 969 were obtained for intra- and inter-laboratory reproducibility analyses in the 6800 system. The 6800 platform displayed several technological improvements over the 4800 system, with higher throughput and laboratory productivity, and lower operator’s hands-on time. Conclusions: The new cobas 6800/8800 HPV assay run on the 6800 instrument is suitable for use in large centralized laboratories included within population-based cervical cancer screening programs. Keywords: Cervical cancer screening, HPV testing, Automation, Technical improvement, Clinical performance * Correspondence: annarosa.delmistro@iov.veneto.it †Helena Frayle and Silvia Gori contributed equally to this work. 1Immunology and Molecular Diagnostic Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, Via Gattamelata, 64, 35128 Padova, Italy Full list of author information is available at the end of the article Frayle et al. BMC Women's Health (2019) 19:47 https://doi.org/10.1186/s12905-019-0743-0 Frayle et al. BMC Women's Health (2019) 19:47 https://doi.org/10.1186/s12905-019-0743-0 Clinical samples and HPV testing by cobas 4800 Clinical samples and HPV testing by cobas 4800 All samples were previously tested by the cobas 4800 HPV assay, based on real-time polymerase chain reac- tion (PCR) technology, by use of the 4800 system, that provides full sample preparation (cobas × 480) and HPV detection (cobas z 480 analyzer); the two instruments are physically separated and require the manual transfer of the reaction plate. The method detects 14 HPV types (the 12 designed as high-risk by the IARC, plus types 68 and 66), provides individual HPV genotype results for HPV16 and HPV18, while detecting the other 12 types as a pool (other HR), and includes an internal quality control (beta-globin) for each sample (in the screening report only the result for all hrHPV types as a pool is in- cluded). The primary vial is mixed and decapped/re- capped by the p480 v1 instrument. y In Italy, the Ministry of Health has introduced the im- plementation of HPV testing for cervical screening in the National Preventive Plan 2014–2018; cancer screen- ings are managed at regional level, and all regions are expected to comply by the year 2018. In the Veneto re- gion, the use of DNA HPV testing in primary cervical screening was piloted in five organized programs from April 2009 to May 2015 [7–9], and fully implemented in all programs since June 2015. The HPV assay actually in use is the clinically validated cobas 4800 HPV test (Roche Diagnostics) [10, 11], run on the cobas 4800 sys- tem. More recently, a new version of the assay (cobas 6800/8800 HPV) for use on the cobas 6800 and cobas 8800 systems [12] has been developed and CE/IVD (Conformité Européenne/in vitro diagnostics) labeled. Residual material of cervical samples collected in Pre- servCyt medium (Hologic, Bedford, MA, USA) from women older than 30 years, attending cervical cancer screening was used. Consecutive specimens were col- lected during February 1 through May 31, 2017 and dur- ing December 1, 2017 through February 15, 2018, and stored for no longer than 6 months before testing by 6800; selected samples were also included, in order to increase the number of cases (25 samples obtained from women with high-grade lesions and stored for up to 3 years at 4 °C before testing for the present study) and of HPV-positive samples (to provide a 25–30% prevalence of high-risk HPV for the reproducibility analyses). Clinical samples and HPV testing by cobas 4800 The 25 samples stored longer than 6 months were re-tested by cobas 4800 to verify amplificability and consistency of results; all samples gave a valid result (1 was invalid only for the HPV channels previously negative) with overlap- ping qualitative results and Ct (cycle threshold) values, and were deemed suitable for inclusion in the study. The aim of our study is the evaluation of the cobas HPV assay performed on the new 6800 platform in com- parison to the 4800 for use in cervical cancer screening. © 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. Frayle et al. BMC Women's Health (2019) 19:47 Page 2 of 7 Frayle et al. BMC Women's Health (2019) 19:47 Page 2 of 7 Aim, design and setting of the study The study was nested within the organized population- based cervical cancer screening, and carried out in one of the three centralized HPV laboratories of the Veneto region (Italy), serving five programs. The HPV screening protocol is applied to women older than 30 years and in- cludes HPV testing and cytology triage of HPV-positive samples, followed by immediate colposcopy in case of ASC-US (Atypical Squamous Cells of Undetermined Sig- nificance) or more severe diagnosis, or HPV retesting at 1-yr recall in case of negative cytology [4]. Women test- ing HPV-negative will be re-invited at 5-yrs interval. For ethical reasons, all samples were anonymized before HPV analysis. The study has been approved by the local Ethical Committee (EC code 2017–07 plus EM 193/2017). Background The study design aimed at evaluating clinical sensitivity and specificity and assay reproducibility by the cobas 6800 HPV assay in comparison to the cobas 4800 assay, accord- ing to the Meijer’s criteria [5], as well as technical per- formance and laboratory workflow of the two platforms. DNA testing for HPV oncogenic types [1] as primary test in organized cervical cancer screening is more ef- fective than cytology in women above 30 years of age [2]. European [3] and Italian [4] guidelines recommend the use of clinically validated HPV assays [5], to be per- formed in centralized laboratories. The instrumentation for the study (p 480 v2 and cobas 6800) was temporarily provided by Roche Diagnostics. The inter-laboratory analyses were performed at the Microbiology and Virology Unit of the General Hospital of Treviso, where a cobas 6800 system is in use for other assays, and the HPV dedicated software was implemented. In order to allow testing in large centralized laborator- ies and to ensure diagnostic accuracy, the assays must be run on systems providing automated solutions for both pre-analytical and analytical phases, starting from specimen primary tubes. Operator interaction, test throughput, work- flow and system maintenance requirements are important determinants and substantial differences among some avail- able automated systems have been shown [6]; for most of the validated assays, at present, two or more diagnostic sys- tems are necessary to guarantee the requested workload of a centralized laboratory. Technological improvements of the instrumentations in use with some of the validated as- says have been released and/or are on the way; their clinical performance, consistency of results and operational charac- teristics are best evaluated by real-world studies. Sample processing and cobas 6800 HPV assay Primary vials were initially processed by the p 480 v2 (with software 2.1.1) instrument, that performed spin-mixing, Frayle et al. BMC Women's Health (2019) 19:47 Page 3 of 7 Page 3 of 7 cap removal, barcode alignment and transfer of 2-ml sam- ple aliquots to secondary vials, and recapping with the ori- ginal primary vial cap. positive results, and 3 with persistent HPV positivity at 1-yr recall) or during follow-up (17 cases). The controls’ group comprised 925 women (mean age 46 yrs.; median age 45 yrs.; range 30–68 yrs) with no or low-grade lesions. By the cobas 4800 HPV assay, all the cases and 67/925 (7.2%) con- trols were HPV-positive. The secondary vials were then manually transferred to the cobas 6800 system, a fully automated unit that pro- vides full sample preparation and HPV detection without further intervention by the operator. A valid cobas 6800 HPV test result has been obtained for all but two samples (both from cases, comprising the partially invalid at 4800 re-testing, excluded from the study); 59/60 (98.4%) cases and 73/925 (7.9%) controls gave a positive result. A concordant result was recorded in 59/60 (98.4%) cases and in 915/925 (98.9%) controls. In Table 1 the qualitative results (positive/negative), as well as the HPV type distribution by hierarchical categorization, of all the samples are reported. Both cobas HPV real-time PCR assays detect the same 14 types of HPV, use the same primers and probes, and provide partial genotyping for HPV16 and HPV18. The two assays do however differ in their Thermal Cycling (CT) profile (the cobas 6800/8800 runs a universal ther- mal cycling profile to allow for mixed batching of differ- ent PCR tests), as well as in the elution sample volume amplified (50 μl on 6800/8800 vs 150 μl on 4800 out of a 400 μl aliquot of extracted nucleic acids). Both HPV as- says were performed according to the manufacturer’s in- structions. Each plate, besides the 2 assay’s controls, contained 92 clinical specimens and 2 additional samples (selected from 10 internal and 5 external quality con- trols, and 2 clinical samples previously found to be in- valid by the cobas 4800 HPV test). Among the 60 women with a CIN2+ diagnosis and a cobas 6800 valid result, the HPV test was positive in all but one sample (diagnosed as CIN2 and 4800-positive with a 39,7 Ct value). Statistical analyses Clinical sensitivity and clinical specificity were evaluated on samples from women with a histologically confirmed high-grade lesion (CIN2+) and on samples from women with no or low-grade lesions, respectively; the results with the 6800 assay were compared to those with the 4800 by a non-inferiority score test, according to the thresholds (relative sensitivity of at least 90% and relative specificity > 98%) recommended by Meijer et al. [5]. The HPV results obtained by the two systems were assessed using overall percentage agreement of qualitative (posi- tive/negative) results and type (HPV16, HPV18, other HPV) agreement; complete and partial type concordance were evaluated also by hierarchical categorization (i.e., HPV16 alone and with any other type; HPV18 alone and with any non-16 other type; non-16/non-18 other HPV types only). For discordant results, the cycle threshold (Ct) values were also analyzed. The intra-laboratory re- producibility and the inter-laboratory agreement were evaluated by calculating the kappa values and 95% confi- dence intervals (95%CI) by bootstrap analysis (replica- tions = 1000; bias corrected); we also report the squared correlation coefficient R2 (variation in Y explained by X/ variation in Y) between the Ct values. Overall, HPV type analysis of the 124 samples positive by both systems (cases plus controls) showed complete Table 1 Comparison of HPV test results on cobas 4800 and cobas 6800 systems on samples from women with no or low grade lesions (controls, N = 925) and women with CIN2+ lesions (cases, N = 60); HPV prevalence by the cobas 4800 assay was 7.2% among controls and 100% among cases Cobas 4800 HPV test results Cobas 6800 HPV test results Total Positive Negative HPV16a HPV18b Other HPV CONTROLS (<CIN 2) Negative 850 4 3 1 858 Positive HPV16a 13 13 HPV18a,b 5 5 Other HPV 2 2 4 41 49 Total 852 19 12 42 925 CASES (CIN 2+) Positive HPV16a 28 28 HPV18a,b 4 4 Other HPV 1 27 28 Total 28 4 27 60 asingle and mixed infections are included bHPV16/18 mixed infections are counted among HPV16-positives Table 1 Comparison of HPV test results on cobas 4800 and cobas 6800 systems on samples from women with no or low grade lesions (controls, N = 925) and women with CIN2+ lesions (cases, N = 60); HPV prevalence by the cobas 4800 assay was 7.2% among controls and 100% among cases Sample processing and cobas 6800 HPV assay Of the 10 discordant samples among the controls, 2 were 4800-positive/6800-negative and 8 were 4800-negative/6800-positive (Table 1). Reactivity in only one of the three HPV channels was recorded in all discord- ant specimens, with a median Ct value of 38,05 (range 37,7-38,4) for the 4800-positive/6800-negative samples, and of 36,11 (range 31,46-37,47) for the 4800-negative/ 6800-positive ones (Table 2, upper panel). asingle and mixed infections are included bHPV16/18 mixed infections are counted among HPV16-positives Results 1; a linear correlation was found for all the data; coefficients for cobas 6800 intra- and interlaboratory data were higher (R2 = 0,92) than those for 4800 vs 6800 comparison (R2 = 0,78). In each plate, two control samples were included. The 10 (8 HPV-positive, 2 HPV-negative) internal quality controls (IQC), each tested 2–6 times, were always con- cordant but twice for the same HPV18-positive sample that on two-out-of-three repetitions was judged as nega- tive; additional in-depth evaluation of its analytical data disclosed that for one of the two negative results the Ct for the HPV18 channel was very high and that the mean fluorescence intensity was very weak. The 5 (4 HPV-positive, 1 HPV-negative) external quality controls (EQC) were concordant in 3 HPV-positive cases and in- valid in the other 2 (one due to insufficient material, and one to a negative beta-globin result). The 2 samples re- sulted invalid on two different runs with the 4800 assay, both gave a valid result on the 6800 assay. D 642: sample from a woman with a CIN2 lesion concordance in 118 (95.2%) specimens and partial con- cordance in the remaining 6. The results of the partially discordant specimens are detailed in Table 2, lower panel. Reactivity to a single channel was detected by the 4800 assay in all cases; this was confirmed by the 6800 assay, which additionally displayed reactivity with an- other HPV channel. Of note, on the 6800 platform the Ct values for the concordant results were always lower than those for the additional positivity, indicative of a higher viral load of the concordantly detected type. concordance in 118 (95.2%) specimens and partial con- cordance in the remaining 6. The results of the partially discordant specimens are detailed in Table 2, lower panel. Reactivity to a single channel was detected by the 4800 assay in all cases; this was confirmed by the 6800 assay, which additionally displayed reactivity with an- other HPV channel. Of note, on the 6800 platform the Ct values for the concordant results were always lower than those for the additional positivity, indicative of a higher viral load of the concordantly detected type. Comparison of the workflow by the two systems has shown higher laboratory operational performances of the 6800 over the 4800, and of the p 480 v2 in relation to the p 480 v1, respectively. Results The cases’ group comprised 62 samples from women (mean age 41 yrs.; median age 40 yrs.; range 30–59 yrs) with a histologically confirmed high-grade lesion (32 CIN2, 27 CIN3, 2 squamous cell carcinomas, 1 adenocarcinoma) di- agnosed either at baseline (42 with HPV-positive/cytology Frayle et al. BMC Women's Health (2019) 19:47 Page 4 of 7 Page 4 of 7 Table 2 Comparison of results between cobas 4800 and cobas 6800 assays; samples with discordant results (upper panel) and samples with partial discordant results (lower panel). Qualitative results and Cycle threshold (Ct) values of HPV-positive specimens are reported Sample ID Cobas 4800 Cobas 6800 HPV channel (Ct) HPV channel (Ct) 722 HPV HR (37,7) HPV NEG 731 HPV HR (38,4) HPV NEG 642a HPV HR (39,7) HPV NEG 13 HPV NEG HPV 18 (37,47) 18 HPV NEG HPV 16 (36,84) 297 HPV NEG HPV HR (31,46) 426 HPV NEG HPV 16 (35,63) 690 HPV NEG HPV 16 (35,43) 1018 HPV NEG HPV 16 (36,80) 1195 HPV NEG HPV 18 (35,67) 1198 HPV NEG HPV 18 (36,55) 170 HPV HR (22,6) HPV HR (17,6) + HPV 16 (35,3) 273 HPV HR (26,5) HPV HR (19,6) + HPV 16 (35,3) 353 HPV HR (22,6) HPV HR (19,2) + HPV 18 (34,7) 357 HPV HR (25,1) HPV HR (19,9) + HPV 18 (35,0) 459 HPV HR (24,1) HPV HR (21,2) + HPV 18 (33,9) 907 HPV HR (22,9) HPV HR (19,1) + HPV 18 (35,3) aID 642: sample from a woman with a CIN2 lesion Table 2 Comparison of results between cobas 4800 and cobas 6800 assays; samples with discordant results (upper panel) and samples with partial discordant results (lower panel). Qualitative results and Cycle threshold (Ct) values of HPV-positive specimens are reported type-specific agreement was observed in 169/172 (98.3%) (Table 3). The inter-laboratory reproducibility was evaluated on 460 samples (Table 4). Overall qualitative agreement was recorded for 456/460 (99.1%), with a kappa value of 0.969 (95%CI 0.941–0.990). All discordant samples displayed single reaction to one HPV channel (1 for HPV16 and 3 for other HPV). Type-specific agreement was observed in 117/119 (98.3%) samples HPV-positive in both runs. Scatter plots of the Ct values of the HPV-positive sam- ples are reported in Fig. Results The most important differ- ences are summarized in Table 5. Of note, the 6800 unit contains an on-board refrigerator for reagent storage and additional storage space for consumables, thus im- proving both loading and long-term use of reagents and Table 3 Intra-laboratory reproducibility analysis by the cobas 6800 HPV assay. Overall (i.e., hrHPV positive/negative) and type-specific HPV test agreement were 99% (587/593 samples) and 98.3% (169/ 172 samples), respectively. Kappa value = 0.967 (95%CI 0.942–0.985) Cobas 6800 HPV test results [1] Cobas 6800 HPV test results [2] Total Positive Negative HPV16a HPV18b Other HPV Negative 415 415 Positive HPV16a 2 49 1 52 HPV18a,b 13 13 Other HPV 4 2 107 113 Total 421 49 15 108 593 asingle and mixed infections are included bHPV16/18 mixed infections are counted among HPV16-positives Clinical sensitivity and specificity values of the cobas 6800 HPV assay were compared to those of the 4800 assay, according to the recommendations for the clinical valid- ation of new HPV-DNA assays [5]; the score values of the 6800 for both sensitivity (98% at 0.90 threshold) and specifi- city (99% at 0.98 threshold) were non-inferior to those of the 4800 (p = 0,0157 and p = 0,0056, respectively). The intra-laboratory reproducibility of the 6800 assay was evaluated on 593 samples, 178 (30%) resulted HPV-positive at first testing. Overall qualitative agree- ment was recorded for 587/593 (99%), with a kappa value of 0.967 (95%CI 0.942–0.985). All 6 discordant samples (positivity for HPV16 in 2 and for other HPV in 4; median Ct values 34) were negative on the second testing. Among the samples HPV-positive in both runs, asingle and mixed infections are included bHPV16/18 mixed infections are counted among HPV16-positives Frayle et al. BMC Women's Health (2019) 19:47 Page 5 of 7 Table 4 Inter-laboratory agreement analysis by the cobas 6800 HPV assay. Overall (i.e., hrHPV positive/negative) and type- specific HPV test agreement were 99.1% (456/460 samples) and 98.3% (117/119 samples), respectively. Kappa value = 0.969 (95%CI 0.941–0.990) Cobas 6800 HPV test results [1] Cobas 6800 HPV test results [3] Total Positive Negative HPV16a HPV18b Other HPV Negative 337 1 338 Positive HPV16a 1 29 1 31 HPV18a,b 7 7 Other HPV 2 1 81 84 Total 340 29 8 83 460 asingle and mixed infections are included bHPV16/18 mixed infections are counted among HPV16-positives asingle and mixed infections are included bHPV16/18 mixed infections are counted among HPV16-positives consumables. Results An additional improvement of the 6800 system is the automatic reporting of the Ct values for the HPV results and the internal control for the samples and the kit positive control on the assay report; the Ct values for the three HPV channels of the kit control were more uniform (median ~ 35 for all) than what re- cording with the 4800 system (median ranges comprised from ~ 37 to ~ 39). Finally, taking into account both the preparation and the analysis of the specimens, the amount of liquid and solid waste produced is sensibly reduced on the p 480 v2/6800 platform in comparison to the p 480 v1/4800 one (Table 5), as a result of import- ant technological differences in all the steps. In particu- lar, among others, while recapping by the p 480 v1 is done with new caps, the original caps are used by the p 480 v2, and while 11 tips/sample are used by the 4800, a unique tip/sample is used by the 6800. Discussion The cobas HPV test detects 14 high-risk HPV types, is based on real-time PCR technology, has been clinically validated [10, 11] and provides partial genotyping for HPV16 and HPV18, while detecting the other 12 HPV types as a pool. More recently, it has been released for use on the newly launched cobas 6800 platform (already in use for other molecular assays). In this study we com- pared the analytical and clinical performance, as well as other system features, of the cobas 4800 and 6800 plat- forms for HPV testing in cervical cancer screening, ac- cording to the Meijer’s criteria [5]. Compared to the cobas 4800 assay, the results by cobas 6800 showed an agreement > 98% for overall qualitative results between the two systems, a non-inferior clinical sensitivity and specificity, and excellent intra- and inter-laboratory re- producibility. These figures are in line with the results previously obtained in the studies conducted for the Frayle et al. BMC Women's Health (2019) 19:47 Page 6 of 7 Frayle et al. BMC Women's Health Page 6 of 7 of debate [17, 18]. In our study, HPV16 (either alone or mixed with other types) was detected in 19,4% (13/67) and in 26% (19/73) of the specimens by cobas 4800 and 6800, respectively, while complete type concordance was observed among the women with a diagnosis of CIN2+. (See figure on previous page.) Fig. 1 Scatter plots of the Ct values for the three HPV channels. Panel a: all HPV-positive samples by both systems, results on cobas 4800 vs cobas 6800; linear correlation, R2 = 0,78. Panel b: cobas 6800 intra-laboratory reproducibility; linear correlation, R2 = 0,92. Panel c: cobas 6800 inter-laboratory reproducibility; linear correlation, R2 = 0,92. Within each panel, the trendlines for the three channels overlapped. Ct = cycle threshold g g According to the European and national guidelines, HPV testing for cervical cancer screening must be cen- tralized in large reference laboratories and performed by using clinically validated assays run on automated sys- tems. As a consequence, the degree of automation for all the steps of the process (from sample preparation, start- ing from the original sample vial, to validation of the re- sults) and the laboratory productivity are essential components to assure high-quality results and high throughput. Discussion In our study, we have shown that the work- flow and several instrumentation features are sensibly improved on the new platform, as already shown also for other assays [19]. Among others, it is a compact unit comprising the thermal cycler for the real-time PCR (which implies direct transfer of the reaction plate with- out operator interventions); reagents and consumables are provided in a cartridge format (that avoid loading/ unloading operations at each plate run); it is provided by an on-board refrigerated storing compartment for re- agents (allowing their subsequent use); it can accommo- date a larger number of samples (up to 280); it has shorter turn-around time for assay results and hands-on time, and is user-friendly. clinical validation of the cobas 4800 assay [10, 11]. The Ct values (that are inversely proportional to the viral load) obtained on the 6800 system have always been lower than those recorded by the 4800, most probably due to the different amount of target sequences analyzed by the two instruments (i.e., 25/150 μl by 4800 vs 25/ 50 μl by the 6800). Overall, the minor differences that emerged at the analytical level (i.e., Ct values) had no major impact on the clinical performance. Differences in semi-quantitative results in this setting are relevant only if they actually determine a change in the positive/nega- tive result, i.e., if the value passes the threshold. Comparison of HPV type distribution among the sam- ples positive by both systems, and on 6800 reproducibil- ity assays, showed a very high complete concordance for type(s) assignment (95.2% in 4800/6800 comparison, and 98.3% in both 6800 intra- and inter-reproducibility as- says), in line with previous studies [10, 11, 13]. HPV16 (and to a lesser extent HPV18) has been shown to have a stronger association than other high-risk types for CIN2+ and invasive cancer [14]; as a consequence, partial genotyping for the management of HPV-positive women in the screening programs has recently been pro- posed as a triage test in the Australian [15] and Dutch [16] protocols, although its clinical value is still a matter (See figure on previous page.) Fig. 1 Scatter plots of the Ct values for the three HPV channels. Panel a: all HPV-positive samples by both systems, results on cobas 4800 vs cobas 6800; linear correlation, R2 = 0,78. Panel b: cobas 6800 intra-laboratory reproducibility; linear correlation, R2 = 0,92. Panel c: cobas 6800 inter-laboratory reproducibility; linear correlation, R2 = 0,92. Within each panel, the trendlines for the three channels overlapped. Ct = cycle threshold Competing interests The authors declare that they have no competing interests. 15. Simms KT, Hall M, Smith MA, Lew JB, Hughes S, Yuill S, et al. Optimal management strategies for primary HPV testing for cervical screening: cost- effectiveness evaluation for the national cervical screening program in Australia. PLoS One. 2017;12(1):e0163509. Abbreviations ASC-US: Atypical Squamous Cells of Undetermined Significance; CE/ IVD: Conformité Européenne/in vitro diagnostics; CI: Confidence Intervals; CIN: Cervical Intraepithelial Neoplasia; Ct: Cycle threshold; EQC: External quality control; HPV: Human papillomavirus; IARC: International Agency for Research on Cancer; IQC: Internal quality control; PCR: Polymerase chain reaction 4. Ronco G, Biggeri A, Confortini M, Naldoni C, Segnan N, Sideri M, et al. Health Technology Assessment report: HPV DNA based primary screening for cervical cancer precursors. Epidemiol Prev. 2012;36(3–4 Suppl 1):1–72. Italian. 5. Meijer CJ, Berkhof J, Castle PE, Hesselink AT, Franco EL, Ronco G, et al. Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older. Int J Cancer. 2009; 124:516–20. Funding h b g The cobas 6800 HPV assays performed in this study were funded entirely by Roche Diagnostics, Italy; no payments were made to any of the investigators. The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication. 7. Zorzi M, Del Mistro A, Farruggio A, de’ Bartolomeis L, Frayle Salamanca H, Baboci L, et al. Use of high-risk human papillomavirus DNA test as primary test in a cervical cancer screening programme: a population-based cohort study. BJOG. 2013;120:1260–7. 8. Del Mistro A, Frayle H, Ferro A, Callegaro S, Del Sole A, Stomeo A, et al. On behalf of the Veneto HPV-screening working group. Cervical cancer screening by high risk HPV testing in routine practice: results at one year recall of high risk HPV-positive and cytology-negative women. J Med Screen. 2014;21:30–7. Author details 1 1Immunology and Molecular Diagnostic Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, Via Gattamelata, 64, 35128 Padova, Italy. 2Pathology Unit, Ospedale di Santorso, Via Garziere, 42-36014 Santorso (VI), Italy. 3Microbiology and Virology Unit, Clinical Pathology Department, Ospedale Ca’ Foncello, Piazza Ospedale, 1-Treviso, Italy. 4Epidemiology Unit, AUSL Reggio Emilia, IRCCS, Reggio Emilia, Italy. 1Immunology and Molecular Diagnostic Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, Via Gattamelata, 64, 35128 Padova, Italy. 2Pathology Unit, Ospedale di Santorso, Via Garziere, 42-36014 Santorso (VI), Italy. 3 17. Rijkaart DC, Berkhof J, van Kemenade FJ, Coupe VM, Hesselink AT. Rozendaal, et al. evaluation of 14 triage strategies for HPV DNA-positive women in population-based cervical screening. Int J Cancer. 2012;130:602–10. 3Microbiology and Virology Unit, Clinical Pathology Department, Ospedale Ca’ Foncello, Piazza Ospedale, 1-Treviso, Italy. 4Epidemiology Unit, AUSL Reggio Emilia, IRCCS, Reggio Emilia, Italy. 18. Wentzensen N, Arbyn M, Berkhof J, Bower M, Canfell K, Einstein M, et al. Eurogin 2016 roadmap: how HPV knowledge is changing screening practice. Int J Cancer. 2017;140:2192–200. 19. Vermehren J, Stelzl E, Maasoumy B, Michel-Treil V, Berkowski C, Marins EG, et al. Multicenter comparison study of both analytical and clinical performance across four Roche hepatitis C virus RNA assays utilizing different platforms. J Clin Microbiol. 2017;55:1131–9. Received: 11 May 2018 Accepted: 14 March 2019 Authors’ contributions 9. Zorzi M, Frayle H, Rizzi M, Fedato C, Rugge M, Penon MG, et al. A 3-year interval is too short for re-screening HPV negative women: a population- based cohort study. BJOG. 2017;124:1585–93. HF, SG, and ADM contributed to the conception and design of the study, analysis and interpretation of data, and manuscript preparation. MR, BNG, EV and PGR contributed to data acquisition and analysis. All authors read and approved the final manuscript. 10. Heideman DA, Hesselink AT, Berkhof J, van Kemenade F, Melchers WJ, Fransen DN, et al. Clinical validation of the cobas(R)4800 HPV test for cervical screening purposes. J Clin Microbiol. 2011;49:3983–5. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Conclusions In: Anttila A, Arbyn M, De Vuyst H, Dillner J, Dillner L, Franceschi S, Patnick J, Ronco G, Segnan N, Suonio E, Törnberg S, von Karsa L, editors. European guidelines for quality assurance in cervical cancer screening. Second edition, Supplements. Luxembourg: Office for Official Publications of the European Union; 2015. p. 1–68. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 16. Luttmer R, De Strooper LMA, Steenbergen RDM, Berkhof J, Snijders PJF, Heideman DAM, Meijer CJLM. Management of high-risk HPV-positive women for detection of cervical (pre)cancer. Exp Rev Mol Diagn. 2016;16:961–74. Acknowledgments h k h d g We thank the medical and technical teams at Roche Diagnostics in Monza, Italy, for their support. In particular, we thank M. Fumiani, M. Griffante, C. Calvi, P. Maceratesi for their help. 6. Ratnam S, Jang D, Gilchrist J, Smieja M, Poirier A, Hatchette T, et al. Workflow and maintenance characteristics of five automated laboratory instruments for the diagnosis of sexually transmitted infections. J Clin Microbiol. 2014;52:2299–304. Consent for publication Not applicable. 14. Schiffman M, Glass AG, Wentzensen N, Rush BB, Castle PE, Scott DR, et al. A long-term prospective study of type-specific human papillomavirus infection and risk of cervical neoplasia among 20,000 women in the Portland Kaiser cohort study. Cancer Epidemiol Biomark Prev. 2011;20:1398–409. Ethics approval and consent to participate 11. Lloveras B, Gomez S, Alameda F, Bellosillo B, Mojal S, Muset M, et al. HPV testing by cobas HPV test in a population from Catalonia. PLoS One. 2013;8:e58153. The study has been approved by the IOV-IRCCS Ethical Committee (EC code 2017–07 plus EM 193/2017). The women who participate to the screening program give a written consent, and an additional specific consent for the study was deemed unnecessary, as all the samples were anonymized and the results of the study didn’t have any consequence for the women’s management. 12. Cobb B, Simon CO, Stramer SL, Body B, Mitchell PS, Reisch N, et al. The cobas 6800/8800 system: a new era of automation in molecular diagnostics. Expert Rev Molec Diagn. 2017;17:167–80. 13. Engesaeter B, van Diermen Hidle B, Hansen M, Moltu P, Staby KM, Borchgrevink-Persen S, et al. Quality assurance of human papillomavirus (HPV) testing in the implementation of HPV primary screening in Norway: an inter-laboratory reproducibility study. BMC Infect Dis. 2016;16:698. Conclusions Our data on the HPV assay performed in a cervical screening context on the new cobas platform composed by the p 480 v2 plus the 6800 instruments, have shown high consistency of results with the cobas 4800 system in Table 5 Comparison of workflow efficiencies and throughput on the cobas 4800 and cobas 6800 systems (including p 480 instruments) p 480 v1 + 4800 p 480 v2 + 6800 Preanalytical step: - samples’ aliquoting performed by × 480 performed by p 480 Analytical steps: - instrument loading (reagents) single-use vials with pouring (10 min) re-usable reagent cartridges, no preparation needed - reagent on-board stability discarded after run 90 days - user interaction vials loading/unloading at each run cartridge loading - unloading when empty/expired - instrument loading (samples) 94 uncapped primary vials up to 280 secondary tubes - throughput (per 8 h) 192 384 - time to first 96 results 4.9 h < 3.5 h - time to each additional 96 results 160 min 90 min - hands-on-time (8 h) 60 min 30 min Instrumentation maintenance daily monthly Amount of waste produced ~ 2.6 l of liquids ~ 1.7 l of liquids / ~ 40% less of sol Frayle et al. BMC Women's Health (2019) 19:47 Page 7 of 7 Frayle et al. BMC Women's Health (2019) 19:47 Page 7 of 7 Frayle et al. BMC Women's Health use and high intra- and inter-reproducibility, together with higher performance in workflow and laboratory productiv- ity, thus demonstrating its suitability for use in large cen- tralized laboratories included within population-based cervical cancer screening programs. 2. Ronco G, Dillner J, Elfström M, Tunesi S, Snijders PJ, Arbyn M, et al. International HPV screening working group. Efficacy of HPV-based screening for preventing invasive cervical cancer: follow-up of European randomised controlled trials. Lancet. 2014;383:524–32. 3. Ronco G, Arbyn M, Meijer CJLM, Snijders PJF, Cuzick J. Screening for cervical cancer with primary testing for human papillomavirus. S1. In: Anttila A, Arbyn M, De Vuyst H, Dillner J, Dillner L, Franceschi S, Patnick J, Ronco G, Segnan N, Suonio E, Törnberg S, von Karsa L, editors. European guidelines for quality assurance in cervical cancer screening. Second edition, Supplements. Luxembourg: Office for Official Publications of the European Union; 2015. p. 1–68. 3. Ronco G, Arbyn M, Meijer CJLM, Snijders PJF, Cuzick J. Screening for cervical cancer with primary testing for human papillomavirus. S1. References 1. Bouvard V, Baan R, Straif K, El Ghissassi F, Benbrahim-Tallaa L, Guha N, et al. On behalf of the WHO IARC in Cancer monograph working group. A review of human carcinogens –part B: biological agents. Lancet Oncol. 2009;10: 321–2.
https://openalex.org/W2435568008	https://revistas.ecosur.mx/sociedadyambiente/index.php/sya/article/download/15/26, https://www.redalyc.org/pdf/4557/455745076003.pdf	es		La lucha en torno a la minería en Manantlán	Sociedad y ambiente	2,013	cc-by-sa	14,031	Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 La lucha en torno a la minería en Manantlán1 The Struggle Around Mining in Manantlan Darcy Tetreault* Resumen Este artículo pretende aclarar la siguiente paradoja: ¿cómo ha sido posible que en la Sierra de Manantlán la extracción minera ha logrado desposeer y empujar a las comunidades locales a una posición marginal en el control de gran parte de su territorio, de su vida económica, social y política? Se hace un recorrido histórico con el objetivo de analizar las condiciones estructurales que han facilitado el saqueo de minerales y maderas preciosas. Se argumenta que las actividades mineras en Manantlán evidencian un proceso de acumulación por desposesión en tanto privan a la población local de los recursos naturales y paisajes culturales que sustentan sus medios de vida, bienestar social y cosmovisión indígena, y que las tácticas políticas del Consorcio Minero Benito Juárez-Peña Colorada reflejan la “reciprocidad negativa”, pues se orientan a sacar los minerales a cambio de nada o muy poco para la población local. Se muestra cómo el movimiento de resistencia local corresponde al prototipo de “ecologismo de los pobres”, con demandas que tienen que ver no sólo con la distribución de los costos y beneficios de la minería, sino también con la autonomía y la autodeterminación. Palabras clave: minería, acumulación por desposesión, reciprocidad negativa, ecologismo de los pobres, autonomía indígena. * Investigador en la Universidad Autónoma de Zacatecas. Correo electrónico: darcytetreault@yahoo.com Este trabajo se basa en investigaciones llevadas a cabo por el autor en y sobre la Sierra de Manantlán desde el año 2000. El grueso de las investigaciones de campo se llevaron a cabo entre 2000 y 2006. Durante este período, se aplicaron diversas técnicas de investigación (por ejemplo, observación participativa, entrevistas abiertas y semiestructuradas, aplicación de encuestas) para recolectar información sobre el conflicto minero y, de manera general, los pequeños proyectos de desarrollo local y las acciones de protesta y demanda para superar los problemas de pobreza y degradación ambiental en el ámbito local. La historia de la región fue reconstruida con la revisión de textos relevantes, los más significativos están incluidos en las referencias del presente artículo; además se tomaron en cuenta los testimonios de los ancianos que participan en el Consejo de Mayores en Ayotitlán. La política minera mexicana y la historia del Consorcio Minero Benito Juárez-Peña Colorada también fueron investigadas a través de la revisión de textos, y se complementó con las observaciones directas en el campo. El análisis de la dinámica actual de la lucha en torno a la minería en Manantlán se basa en entrevistas con informantes clave y de una revisión sistemática de fuentes hemerográficas. 1 48 La lucha en torno a la minería en Manantlán Abstract This article seeks to shine light on the following paradox: How is it possible that mining extraction in the Sierra of Manantlan has managed to dispossess local communities and push them to a marginal position in the control of a large part of their territory and of their economic, social and political life? A historical review is provided, with the objective of analyzing the structural conditions that have facilitated the plunder of minerals and precious wood. It is argued that mining activities in Manantlan exhibit a process of “accumulation by dispossession” insofar as they deprive the local population of the natural resources and cultural landscapes that sustain its livelihoods, social wellbeing and indigenous cosmovision. Likewise, it is argued that the political tactics employed by the Mining Consortium Benito Juárez-Peña Colorada reflects “negative reciprocity” insofar as they are oriented towards obtaining minerals in exchange for little or nothing for the local population. The article also demonstrates how the local resistance movement corresponds to the prototype of “the environmentalism of the poor”, with demands that have to do with, not just with the distribution of the costs and benefits of mining, but also autonomy and self-determination. Key words: mining, accumulation by dispossession, negative reciprocity, environmentalism of the poor, indigenous autonomy. Introducción En la Sierra de Manantlán, en una zona fronteriza, disputada por Colima y Jalisco (véase la Figura 1), se encuentran algunos de los yacimientos de hierro más importantes del país. Durante cuatro décadas empresas mineras privadas, y en el pasado paraestatales, han explotado estos minerales mediante minas a cielo abierto, con la consabida degradación ambiental de una región que se reconoce internacionalmente por sus altos niveles de biodiversidad. Esta misma zona es el territorio ancestral de los nahuas y otomíes que han vivido en la Sierra de Manantlán y en los valles circundantes desde tiempos inmemoriales. Estos grupos indígenas, que se autodefinen en el presente como nahuas, han sostenido una larga defensa de su territorio y de sus recursos naturales. Actualmente, la lucha socioambiental más aguda, en la Sierra de Manantlán, gira en torno a la minería; la empresa minera de mayor presencia es el Consorcio Minero Benito Juárez-Peña Colorada (CMBJPC); además de la Gan-Bo Minera Internacional y la Minera del Norte (antes Minera Monterrey). Los activistas locales han tomado varias acciones para contener la expansión de las explotaciones mineras, obligar a las compañías a internalizar los costos ambientales e insistir que se derramen más beneficios económicos y sociales en el ámbito local. Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 49 Figura 1: Ubicación de la mina Benito Juárez Peña Colorada Fuente: Elaboración propia con el apoyo técnico del maestro Antonio Reyes Cortés En este artículo se recurre al modelo explicativo marxista de acumulación por desposesión y al concepto de “reciprocidad negativa” para responder la siguiente pregunta: ¿cómo ha sido posible que en la Sierra de Manantlán la extracción minera ha logrado desposeer y empujar a las comunidades locales a una posición marginal en el control de gran parte de su territorio, de su vida económica, social y política? Para abordar esta problemática se hace un breve recorrido histórico con el objetivo de analizar las condiciones estructurales que facilitaron el saqueo de recursos naturales desde el período colonial, con un enfoque en las leyes y maniobras políticas que facilitaron la penetración del capital minero en la región desde los años sesenta hasta el presente. Se argumenta que la explotación minera en Manantlán exhibe un proceso de “acumulación por desposesión”, pues priva a la población local de los recursos naturales y paisajes culturales que sostienen sus medios de vida, bienestar social y cosmovisión indígena. Además, se observa que este proceso refleja la “reciprocidad negativa” en tanto el CMBJPC ha intentado extraer los minerales impunemente, sin dar nada a cambio, a través de la captura política de diversas instituciones locales. Sociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 50 La lucha en torno a la minería en Manantlán Por otra parte, se analiza el movimiento de resistencia, tomando en cuenta el perfil de los activistas locales y sus aliados, los conflictos internos entre distintas facciones del núcleo agrario más grande de la zona (Ayotitlán), y las tácticas empleadas para enfrentar y negociar con el capital minero y sus cómplices en el gobierno. En este análisis se observa que el movimiento en Manantlán corresponde en gran medida al prototipo de “ecologismo de los pobres”, tal como se construye por Joan Martínez Alier (1997; 2012). Además, las demandas de los activistas indígenas de Manantlán tienen que ver no sólo con cuestiones distributivas (en términos ecológicos y económicos), sino también con la autonomía y la autodeterminación de los pueblos indígenas, y el respeto de los derechos humanos, agrarios e indígenas de la población local. Acumulación por desposesión y reciprocidad negativa De acuerdo con Harvey (2004), la acumulación por desposesión es equivalente al proceso de “acumulación primitiva” desarrollado por Marx en el primer tomo de El Capital. Este proceso, también conocido como “acumulación originaria”, describe la emergencia del sistema capitalista. Consiste en la separación del productor (el campesino) de sus medios de producción (la tierra), una condición necesaria para la transición del feudalismo al capitalismo: la proletarización del campesinado.2 Mientras que Marx vio este proceso sólo en las etapas iniciales del capitalismo, Harvey, siguiendo a Rosa Luxemburg (2003), argumenta que se puede observar a lo largo de la trayectoria del capitalismo. En esta visión, la acumulación por desposesión incluye, entre otros procesos, “la mercantilización y privatización de la tierra y la expulsión forzosa de las poblaciones campesinas; la conversión de diversas formas de derechos de propiedad —común, colectiva, estatal, etc.— en derechos de propiedad exclusivos; la supresión del derecho a los bienes comunes; la transformación de la fuerza de trabajo en mercancía y la supresión de formas de producción y consumo alternaMarx recurre al caso “clásico” de Inglaterra para ilustrar el proceso de acumulación primitiva. De acuerdo con su análisis, la servidumbre había prácticamente desaparecido en Inglaterra antes de que se iniciara el proceso de acumulación originaria; es decir, la gran mayoría de la población consistía en “campesinos propietarios libres”, con derechos de propiedad escondidos por debajo de los títulos feudales. A partir de finales del siglo XV, los grandes señores feudales empezaron a apropiarse de sus tierras cultivables y comunales, convirtiéndolas en pastizales para criar ovejas, cuya lana se vendía a los manufactureros flamencos. El proceso recibió un empuje durante el siglo XVI por la Reforma, que conllevó la expropiación de las tierras de la Iglesia, incluyendo las que se apartaron para los pobres. Durante los primeros 150 años del proceso, la monarquía y el parlamento aprobaron leyes para restringir la privatización de las tierras de los campesinos, pero era en vano. Para mediados del siglo XVIII el campesinado (yeomanry) había desaparecido por completo y para finales del mismo siglo, las tierras colectivas de los jornaleros rurales también habían desaparecido. Además, en ese siglo, la ley misma, en la forma de las Leyes de Cercamiento de Tierras Comunales, se convierte en un instrumento para robar las tierras colectivas. Desde luego, este proceso no procedió por su propia inercia o de manera pacífica, como lo imaginaba Adam Smith, sino que fue impulsado por los grandes señores feudales, a través de “actos individuales de violencia”, y posteriormente por los grandes terratenientes que los reemplazaron, con el apoyo del Estado, por medio de formas institucionalizadas de robo, fraude y violencia. Así, en las palabras de Marx, la historia de la acumulación originaria “está escrita en los anales de la humanidad con letras de sangre y fuego” (Marx, 2000: 264). 2 Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 51 tivas; los procesos coloniales, neocoloniales e imperiales de apropiación de activos, incluyendo los recursos naturales” (Harvey, 2004: 113). Desde esta perspectiva teórica, existen dos procesos de acumulación capitalista orgánicamente entrelazados y dialécticamente relacionados que funcionan de manera simultánea en cualquier momento dado: la producción expandida, con base en la explotación de la fuerza de trabajo y realizada, por lo menos formalmente, bajo condiciones de “paz, propiedad e igualdad”; y la acumulación por desposesión, en donde “aparecen sin disimulo […] la violencia, el engaño, la opresión y la rapiña” (Luxemburg, citado en Harvey, 2004: 111-112). El primero es un aspecto interno del sistema capitalista, dando lugar a “las crisis de sobreacumulación”; es decir, situaciones en las cuales se acumula capital excedente a tal grado que una buena parte queda sin ser aprovechado y sin ninguna aplicación redituable a la vista. El segundo es una respuesta y solución temporal a una crisis de sobreacumulación, en tanto proporciona una salida para la inversión lucrativa de capital al alimentarse de factores de producción que están fuera de la esfera de la lógica capitalista estricta, por ejemplo, la tierra y los recursos naturales usados para la agricultura de subsistencia y los bienes públicos en forma de reservas minerales. Tal como observa Harvey, la acumulación por desposesión es especialmente brutal en la periferia de la economía global e invariablemente respaldada y promovida por el Estado. La “reciprocidad negativa”, por su parte, es un concepto rescatado por Garibay y Balzaretti (2009) para explicar los mecanismos de desposesión en el ámbito local. Como tal, puede ser visto como la forma etnográfica del proceso de acumulación por desposesión, incluso respecto al engaño, robo y saqueo. Con referencia al trabajo de Marshall Sahlins (1977), Garibay y Balzaretti (2009: 92) definen la reciprocidad negativa como “la intención de tomar algo impunemente sin dar nada a cambio”; y lo contrasta con dos otras formas de reciprocidad: el acto de dar sin esperar recibir inmediatamente (reciprocidad generalizada) y el acto de dar en un esfuerzo por obtener algo equivalente (reciprocidad balanceada). Además, con referencia a Lomnitz (2005), los mismos autores distinguen entre dos tipos de reciprocidad negativa: la “simétrica”, que sucede entre actores autónomas mutuamente amenazantes; y la “asimétrica”, en la que un actor subordina al otro y le impone un régimen de coacción (Ibid.: 93). A través de un estudio de caso, Garibay y Balzaretti (2009) ilustran la reciprocidad negativa asimétrica en comunidades mexicanas afectadas por la minería, y describe las tácticas empleadas por el capital minero para realizar la “captura política” de las instituciones locales, por ejemplo, los gobiernos estatales y municipales, los comisariados ejidales, las franquicias de partidos políticos y las organizaciones civiles. Observan que estas tácticas provocan divisiones internas entre la población local, dando lugar a tres posibles posturas: la “ética fuerte”, que rechaza la ocupación y destrucción minera del territorio; la “ética negociada”, que acepta un acuerdo con la compañía minera basado en la indemnización, y la “ética débil”, que se rinde a la Sociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 52 La lucha en torno a la minería en Manantlán imposición de un régimen de alienación del territorio y a la disolución de la comunidad en grupos clientelares. Veamos en las siguientes secciones cómo la acumulación por desposesión corresponde a la larga historia de explotación de recursos naturales en la Sierra de Manantlán, particularmente de minerales; y también cómo la actual dinámica política en torno a la minería conforma a la reciprocidad negativa, bajo relaciones asimétricas entre el CMBJPC y los pobladores locales que luchan por detener su expansión (ética fuerte) y negocian por mayor indemnización (ética negociada). Breve recorrido histórico Cuando los españoles llegaron a la Sierra de Manantlán a principios del siglo XVI, era habitada por indígenas otomíes. El primer contacto con los españoles fue entre 1524 y 1525, cuando Francisco Cortés de San Buenaventura dirigió una expedición hacia el interior de los actuales estados de Jalisco y Nayarit. Arribaron por la Villa de Colima y cruzaron la Sierra de Manantlán que los condujo al valle de Autlán. Poco tiempo después la población indígena de la región fue diezmada, no sólo por las enfermedades europeas, sino por la brutalidad de los españoles. Para 1540, “las tierras bajas de Alima y la región de Cihuatlán, casi habían sido evacuadas por los indios, así como las faldas de los declives del volcán alrededor de la villa de Colima” (Sauer, 1976: 113). Se estima que más del 80% de la población indígena de tierras cálidas desapareció en este corto tiempo (Ibid.). La población del valle de Autlán fue un poco menos afectada, sólo se redujo entre el 65 y 78% (Laitner-Benz, 1992: 325), probablemente porque estaba más lejos de las principales minas de oro, donde se explotaba la mano de obra indígena esclavizada.3 A todas luces, la población de las montañas fue afectada mucho menos, debido a su aislamiento. Por otra parte, la evidencia disponible sugiere que los indígenas de los valles huyeron a la Sierra de Manantlán. Según Sauer (1976: 116), “la sobrevivencia indígena era […] favorecida por el medio ambiente. Las comunidades de las montañas se encontraban relativamente en buenas condiciones; pero las tierras bajas decayeron”. Asimismo, Mejía observa que “algunos de ellos [indígenas de Colima y Amula] huyeron de sus poblados originales a lugares distantes de la influencia española donde refundaron muchos de los antiguos pueblos, manteniendo, en parte, la anterior organización política y administrativa” (Mejía, 2008: 61). De esta manera, para finales del siglo XVI la Sierra de Manantlán empezó a perfilarse como región de refugio, incluso para los nahuas de lo que en el presente es Colima. En esa época hubo minas de oro en la sierra al norte del bajo Río Grande de Colima (hoy llamado Río Armería), en las antiguas Provincias de Tepetitango y Colimotl, y también en la Provincia de Motín (Sauer, 1976: 106-107), en el actual estado de Michoacán, cerca de su límite con el estado de Colima. 3 Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 53 Durante el período colonial, los españoles consolidaron su presencia en la región con la fundación de haciendas. Ahuacapán, en el valle de Autlán, era una de las más grandes, con una extensión que llegó hasta el estado de Colima. Ésta se fundó a mediados del siglo XVII e introdujo la ganadería. Con el tiempo, se fundaron muchas otras, hasta que empezaron a presionar los territorios indígenas en la Sierra de Manantlán. Éste es el primer momento histórico en que se puede observar un proceso de acumulación por desposesión, en tanto que los indígenas de Manantlán perdieron territorio para dar lugar a la crianza de ganado para el mercado. En este contexto, como parte de un esfuerzo por controlar y administrar el repartimiento de las propiedades en todas partes de la Nueva España, la Corona española extendió, en 1696, un título de propiedad a la “República de Ayotitlán”. Se calcula que dicho título otorgó a los indígenas de Ayotitlán en la Sierra de Manantlán una extensión de 450 000 hectáreas (Robertson, 2002). En el transcurso de los siguientes doscientos años, el tamaño de Ayotitlán disminuyó drásticamente, a medida que las haciendas vecinas expandieron sus actividades agropecuarias comerciales. Este proceso se aceleró después de la Independencia, cuando una serie de leyes locales (promulgadas a partir de la década de 1830) inició en la región un proceso temprano de desamortización de bienes comunales. De esta manera se establecieron varias haciendas, incluso Ixcuintla, en el municipio de Autlán; San Pedro, en Tolimán; El Zapotillo y La Resolana, en Casimiro Castillo; la hacienda Cuautitlán, en el municipio con el mismo nombre, y las haciendas de Platanarillo, El Saúz y Cerro Grande en Minatitlán (IMECBIO, 2000a). Debido a la expansión de estas haciendas y de ranchos más pequeños, cuando la Revolución estalló en 1910, el territorio de Ayotitlán se había reducido a aproximadamente 8 200 hectáreas (Robertson, 2002: 87) Hubo avances en la lucha por la tierra durante el siglo XX, dando lugar a varios núcleos agrarios en la región, entre los más importantes destacan el ejido de Ayotitlán y las comunidades indígenas de Cuzalapa, Chacala y Teutlán (en Jalisco), y Zacualpán (en Colima). Sin embargo, las contradicciones internas de la reforma agraria resultaron en demoras, omisiones y empalmes territoriales, que a su vez exacerbaron conflictos relacionados con la posesión de la tierra y sobre los derechos de usufructo. En el caso de Ayotitlán, el núcleo agrario más grande de la región, los pobladores locales solicitaron la restitución de sus tierras en 1921. Treinta y cinco años después, las autoridades agrarias revirtieron el proceso de Restitución de Tierras Comunales y lo transformaron en otro de Dotación Ejidal, argumentando que era imposible emitir dictamen sobre la autenticidad del título virreinal fechado en 1757. No fue hasta 1963 que se emitió la resolución presidencial que otorgó 50 332 hectáreas a 783 jefes de familias en Ayotitlán. Luego, pasaron 14 años más hasta que el Departamento de Asuntos Agrarios y Colonización (DAAC) ejecutara esta resolución, y por si fuera poco, sólo se enSociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 54 La lucha en torno a la minería en Manantlán tregaron 34 700 hectáreas.4 Hasta la fecha, no se han entregado las 15 632 hectáreas restantes. Además, no se ha ejecutado la ampliación de 10 350 hectáreas que fue otorgada a través de una resolución presidencial publicada en el Diario Oficial de la Federación, el 21 de agosto de 1974. En parte, estas irregularidades pueden ser explicadas por el imperativo desarrollista de entregar los recursos naturales más valiosos del país a empresas privadas y paraestatales, en consonancia con el objetivo de maximizar el crecimiento del Producto Interno Bruto (PIB) y alimentar el proceso de industrialización. De acuerdo con este imperativo, la reforma agraria en Manantlán fue entorpecida, primero, por los intereses de compañías forestales, y más tarde por los del capital minero. Así, durante la fase de Industrialización por Sustitución de Importaciones (ISI, de 1940 a 1982), cuando se abrió la Sierra de Manantlán al extractivismo industrial a través de la construcción de carreteras y terracerías, caciques notorios como Longinos Vázquez, Antonio Correa y Guadalupe Michel ganaron acceso a los bosques. Con la complicidad de diversos actores gubernamentales, crearon títulos fraudulentos, provocaron el aplazamiento de la reforma agraria local e ignoraron por completo las normas gubernamentales diseñadas para limitar y regular la tala de árboles (Rojas, 1996). Los inconformes fueron reprimidos, incluso con una matanza en 1951 y con la erradicación de un poblado llamado Tenamaxtla, por órdenes del notorio general Marcelino García Barragán, según los testimonios de los ancianos. De esta manera, durante décadas las compañías privadas talaron los bosques de la sierra a un ritmo desmesurado, que dejó atrás una huella de destrucción ambiental, conflictos internos y represión, lo que se tradujo en otra manifestación de acumulación por desposesión. Ante esta situación, a finales de los años setenta, en un contexto nacional caracterizado por un auge en la movilización de campesinos y pueblos indígenas, los líderes tradicionales de Manantlán se organizaron para defender su territorio ancestral, proteger sus medios de vida y hacer valer sus derechos agrarios, indígenas y humanos. Con el apoyo de los maestros de la primaria del pueblo Telcruz, quienes tenían lazos con la Alianza Campesina Revolucionaria (ACR), y de un sacerdote influido por la teología de liberación, conocido localmente como “el padre Tomás”, recurrieron a acciones directas para expulsar a los taladores, incluso al bloqueo de caminos y al sabotaje de maquinaria (Tetreault, 2009). El Estado respondió violentamente, hasta que un grupo de conservacionistas de la Universidad de Guadalajara ganó el apoyo político necesario para crear la Reserva de la Biosfera de la Sierra de Manantlán (RBSM), con base en argumentos sobre la necesidad de proteger los altos niveles de biodiversidad en la región después del descubrimiento de la especie Zea Diploperennis, un pariente silvestre del maíz con propiedades genéticas valiosas. Según la información disponible en el sitio de Internet del Instituto Nacional de Estadística y Geografía (INEGI), la superficie del ejido de Ayotitán fue medida y registrada en 41 427.49 hectáreas por el Programa de Certificación de Derechos Ejidales y Titulación de Solares Urbanos (PROCEDE). Esta cifra difiere del cálculo anterior —ampliamente difundido (por ejemplo, en AJDH, 1990)— que estima la superficie entregada en 34 700 hectáreas. 4 Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 55 De esta manera, en 1987 se puso fin definitivo a la tala comercial de árboles (no clandestina), sin afectar las actividades mineras que quedaron en la zona de influencia de la reserva, en la franja fronteriza disputada por Jalisco y Colima. La penetración de capital minero En efecto, el control de la riqueza en recursos minerales está detrás de la disputa fronteriza entre estas dos entidades federativas. La confusión fue sembrada a mediados del siglo XIX, cuando el gobierno del emperador Maximiliano (1864-1867) trató de llevar a cabo una reestructuración completa del territorio nacional, al sustituir la base de organización territorial existente por “departamentos imperiales” (Gobierno de Jalisco, 2002). Aunque esta iniciativa murió con el derrocamiento del imperio, generó cierta ambigüedad sobre la frontera entre las dos entidades federativas. Sea como fuere, en 1920, el hacendado colimense Carlos Fernández, al enterarse de las riquezas minerales ubicadas al oeste del río Marabasco, reclamó la zona como suya y ordenó a sus sicarios matar a trece de los indígenas que rehusaron alejarse.5 Cinco décadas después, el estado de Colima otorgó derechos de usufructo al Consorcio Minero Benito Juárez-Peña Colorada (CMBJPC) y a otras compañías mineras para explotar los yacimientos ferruginosos. De acuerdo con Rojas (1996: 83), este proceso se llevó a cabo de la siguiente manera: En noviembre de 1971 el ejecutivo del estado de Colima emitió un decreto donde declara de utilidad pública el establecimiento de una zona industrial y se expropian para este efecto cinco fracciones de terrenos rústicos considerados como pequeñas propiedades (supuestamente ubicados en el municipio de Minatitlán, Colima). En diciembre del mismo año se expide otro decreto que autoriza al gobernador de Colima para que celebre contrato de fideicomiso con Nacional Financiera, S.A., a fin de que se lleve a cabo la explotación minera en los terrenos expropiados. Antes de que ambos decretos fuesen publicados ya se habían construido el 30% aproximadamente de las instalaciones mineras de la paraestatal [el Consorcio Minero Benito Juárez-Peña Colorada]. Con esta maniobra el gobierno de Colima extendió de manera arbitraria su jurisdicción 12 kilómetros tierra adentro del territorio en disputa, de modo que se apropió de aproximadamente 3 500 hectáreas del ejido de Ayotitlán (Loeza y Gutiérrez, 1996). Durante las siguientes tres décadas el municipio de Cuautitlán y el gobierno del estado de Jalisco no hicieron nada ante este atropello. No fue sino hasta 1998 cuando el gobierno del estado de Jalisco levantó un juicio ante la Suprema Corte de Justicia de la Nación, lo que dio lugar a la Controversia Constitucional 3/98. La disputa fronteriza sigue vigente hasta la fecha, a pesar de haber pasado ocho años en las manos de la Suprema Corte de Justicia de la Nación, para posteriormente turnarse al Senado. La falta de certeza 5 Crescenciano Brambila, citado en Rojas (1996: 92-93). Sociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 56 La lucha en torno a la minería en Manantlán jurídica sobre el territorio ha servido para proteger los intereses de las compañías mineras, que hacen todo lo posible para no derramar sus ganancias en la zona, ni sufragar los costos ecológicos de sus actividades, maximizando así las ganancias de sus accionistas. La misma incertidumbre jurídica ha funcionado como un pantano burocrático, donde se estancan las exigencias de los indígenas que luchan por poner fin a la destrucción medioambiental y a la constante violación de sus derechos humanos (Brigada Zeferino Padilla Villa, 2007). Para entender la complicidad y la permisividad oficial ante estas irregularidades, hay que recurrir a una explicación estructuralista. En este nivel de análisis, recordemos que durante el período posterior a la segunda guerra mundial México seguía una estrategia de desarrollo capitalista keynesiana, con fuertes intervenciones gubernamentales en la economía; “industrialización por sustitución de importaciones (ISI)” como se conoce generalmente, o “desarrollismo” para connotar cierta obsesión con el objetivo de hacer crecer la capacidad productiva industrial del país. De acuerdo con esta estrategia, el Estado controlaba sectores clave de la economía, o bien a través del capitalismo monopólico paraestatal (como en el sector petrolero) o, en el caso del sector minero, con leyes para restringir la participación del capital extranjero y con medidas para fomentar el dominio de grandes compañías paraestatales. Cuando comenzó a promoverse la minería en Manantlán, la economía todavía no había entrado en crisis y después de dos décadas de haber alcanzado altas tasas de crecimiento económico, hubo mucha confianza en la ISI, es decir, la estrategia de desarrollo promovida y teorizada por la Comisión Económica para América Latina y el Caribe (CEPAL). Desde otro ángulo, la expansión de la capacidad productiva industrial del país iba de la mano con una explosión demográfica, un rápido proceso de urbanización, el ensanchamiento del sector formal de empleo y la construcción de un estado de bienestar universal fragmentado y sectorializado. En este escenario se veía la necesidad de impulsar la extracción de minerales, sobre todo en relación con la siderurgia, que producía materias primas básicas para alimentar el proceso de industrialización (Sariego et al., 1988). A pesar de los mecanismos incorporados en la Constitución de 1917 para nacionalizar el sector minero, a mediados del siglo XX éste todavía estaba controlado por el gran capital extranjero, estadounidense en su mayoría. Así, en 1961 se promulgó la “Ley de la Mexicanización del Sector Minero”, misma que estipulaba que todas las empresas mineras que operaban en México debían tener por lo menos 51% de capital mexicano. En 1975, se realizaron nuevas modificaciones a la Ley Minera para promover mayor participación del Estado en el sector. De esta manera, para 1983 las empresas paraestatales controlaban cerca del 40% de la producción minera en el país (Delgado Wise y Del Pozo Mendoza, 2002: 26). Esto fue la culminación del “proceso de mexicanización”. Los principales beneficiarios eran un puñado de empresarios mexicanos adinerados, quienes lograron el control estratégico de las reservas e infraestructura mineras (Ibid.). Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 57 El CMBJPC nació en 1967, durante el auge del proceso de mexicanización, con la participación de cuatro empresas: Altos Hornos de México, S.A. (AHMSA), una compañía paraestatal que nació en 1942 con capital privado y estatal en Monclova, Coahuila; Fundidora de Monterrey, S.A. (FUMSA), una empresa privada, controlada por la familia Garza Sada del Grupo Monterrey, que sería nacionalizada en 1976; Hojalata y Lámina, S.A. (HYLSA), una empresa privada también controlada por la familia Garza Sada; y Tubos de Acero de México, S.A. (TAMSA), fundada en 1952 por Bruno Pagliai, un empresario de origen italiano que había emigrado a México. Cabe enfatizar que este proyecto público-privado era de interés nacional y, según la Constitución, los yacimientos ferrosos de la Sierra de Manantlán pertenecían a la nación mexicana. Así, no se veía la necesidad de consultar ni mucho menos obtener el permiso de la población indígena local para sacarlo adelante. La burocracia y la administración legal se arreglarían de un modo u otro. Tampoco existían leyes ambientales con suficientes atribuciones para controlar la destrucción ambiental causada por la minería (Simonian, 1999) y el movimiento socioambiental e indígena en la Sierra de Manantlán apenas estaba cobrando fuerza para enfrentar a los madereros. Así, el desarrollo de la minería en Manantlán procedió sin impedimentos, sin indemnizaciones para la población local y con consecuencias ambientales devastadoras. Nuevamente, esto puede ser visto como una forma de acumulación por desposesión. Los trabajadores que encontraron empleo en este proyecto venían de otras partes del país, pues se requería mano de obra calificada para operar la maquinaría, e ingenieros y otros profesionales para supervisar y administrar la explotación. Para albergar a estos trabajadores, se construyó el enclave Benito Juárez Peña Colorada. De esta manera, desde un principio, la extracción minera no contribuía sensiblemente a la economía local de los indígenas; todo lo contrario, perjudicaba sus actividades agropecuarias por la destrucción de cerros, la acumulación de escombros y la contaminación y el debilitamiento del sistema hidrológico. Al mismo tiempo, como se verá más adelante, los intereses mineros han tenido repercusiones políticas en la región, al cooptar líderes, fomentar el caciquismo, exacerbar divisiones internas y suscitar la hostilidad hacia la inconformidad. El impulso neoliberal Para finales de los años ochenta, como secuela de la mexicanización del sector minero, el CMBJPC era propiedad mayoritaria de Siderúrgica Mexicana (SIDERMEX), creada en 1977 para agrupar a las tres siderúrgicas bajo el control del gobierno: AHMSA, FUMSA y Siderúrgica Lázaro Cárdenas Las Truchas, S.A. (SICARTSA). Esta agrupación paraestatal tenía 55% de las acciones de Peña Colorada, HYLSA tenía 28.5% y TAMSA, 16.5% (Hufbauer y Schott, 1992: 245). Sin embargo, esta Sociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 58 La lucha en torno a la minería en Manantlán situación estaba a punto de cambiar. La crisis de la deuda externa (1982-1988) había creado las condiciones para la adopción de la agenda neoliberal. Carlos Salinas de Gortari llegó a la presidencia en 1988, a través de elecciones manchadas por fraude, con intenciones de profundizar las reformas estructurales neoliberales. En este contexto, la privatización del sector minero se presentó como una solución al estancamiento de la producción minera a nivel nacional y a los problemas financieros de las compañías paraestatales. Así, en marzo de 1990, el ejecutivo federal anunció sus planes para privatizar AHMSA y SICARTSA. En octubre de 1991 el Grupo Alfa pagó 42 millones de dólares por tres plantas de la División Sur de AHMSA, y en noviembre del mismo año el Grupo Acerero de Norte (GAN) —propiedad de los empresarios mexicanos Alonso Ancira Elizondo y Xavier Autrey Maza— pagó 145 millones de dólares por el resto de AHMSA, incluido 30.37% de las acciones de Peña Colorada (Chávez Quezada, 1994: 80). De acuerdo con Sacristán Roy (2006: 56), con referencia a la privatización de AHMSA y SICARTSA, “el resultado de la gestión de venta fue que las empresas prácticamente se regalaron”. Según sus cálculos, se vendieron por un total de 755 millones de dólares, frente a un valor estimado en por lo menos 6 000 millones de dólares. Esta experiencia no es una anomalía, sino más bien refleja el modus operandi del gobierno de Carlos de Salinas en la venta del patrimonio minero. Sobre esta línea, Delgado Wise y Del Pozo Mendoza (2002: 33) mencionan que la privatización de la industria minera paraestatal se llevó a cabo a un ritmo vertiginoso y que “estas ventas no se hicieron con la debida transparencia e imparcialidad (como desde los círculos oficiales se quiso dar a entender), sino que operaron como un mecanismo de colosales transferencias de recursos públicos a favor del ‘selecto club de consorcios mineros’ que emerge de la mexicanización”. De esta manera, el ejecutivo federal entregó los recursos mineros del país al gran capital privado mexicano, antes de abrir el sector a la inversión extranjera directa. La liberalización del sector minero empezó en 1990, con la promulgación de una nueva Ley Minera que permitía mayor participación extranjera en la exploración y explotación de los minerales. En 1992, se realizaron más modificaciones para permitir 100% de propiedad extranjera en las empresas mineras. Estos cambios, sin embargo, no tenían tanta fuerza hasta que fueron complementados por la Ley de Inversión Extranjera en 1996. Finalmente, en 1999, se realizaron algunas modificaciones a la Ley Minera para simplificar los procedimientos administrativos y allanar la entrada de capital extranjero, incluso en la Sierra de Manantlán, antes del boom minero global, a principios del siglo XXI. Además, coincidió con la alternancia de un gobierno federal panista que pretendía representar los intereses de la clase empresarial, lo que se tradujo en una acelerada entrega de concesiones mineras en todas partes del país (López y Eslava, 2011). Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 59 La Sierra de Manantlán no ha estado exenta de este proceso de desposesión. A pesar de ser un área protegida, la Secretaría de Economía ha otorgado 16 concesiones mineras que cubren prácticamente toda la RBSM, salvo una amplia porción del Cerro Grande y otra área de Cuautitlán (IMECBIO, 2000b: 180). Aunque el Programa de Manejo de la RBSM prohíbe las actividades mineras sin la autorización previa de la Secretaría del Medio Ambiente y Recursos Naturales (Semarnat), el artículo 6 de la actual Ley Minera declara que estas actividades “serán preferentes sobre cualquier otro uso o aprovechamiento del terreno”. Esta contradicción ha provocado la proliferación de explotaciones ilegales en la Sierra de Manantlán desde el año 2000 por compañías mineras que no cuentan con los permisos de impacto ambiental o de cambio de uso de suelo. Además, grupos armados, y con maquinaria, han llegado a la sierra para sacar minerales y madera (El Informador, 2012). Se estima que alrededor de cuatro millones de hierro de contrabando pasaban, anualmente, por los puertos de Manzanillo y Lázaro Cárdenas, con destino a China, hasta que las autoridades pusieron una especie de “candado” en 2011: exigieron que los exportadores mostraran documentos para verificar los permisos de extracción (Guillén, 2013b). No obstante, el saqueo clandestino sigue en marcha (Ibid.), y “los planes de inversión china en los proyectos de hierro en México todavía son confidenciales” (Global Business Reports, 2011: 64). Desde otro ángulo, la liberalización del sector minero abrió las puertas para la venta del CMBJPC a dos compañías transnacionales gigantescas: Mittal Steel, cuyo principal accionista es el multimillonario Lakshmi Mittal, de nacionalidad india, y Ternium, de capital argentino-italiano. Cada una posee 50% del CMBJPC. Actualmente, estas dos compañías monopolizan la producción de hierro en México, junto con el GAN. De hecho, de los 13 811 millones de toneladas de pelet de hierro producido en México en 2011, el CMBJPC produjo 4 003; Ternium y Mittal Steel produjeron en otras minas 1 699 y 3 976 millones de toneladas, respectivamente; y GAN produjo el resto (Camimex, 2012: 18). Así, la producción del CMBJPC representa casi 30% del total del país y todavía cuenta con 18.8% de las reservas ferrosas nacionales (Camimex, 2010: 15). Los impactos ambientales del proceso de extracción Como se mencionó anteriormente, las instalaciones mineras del CMBJPC se empezaron a construir en 1971, y la extracción de minerales comenzó en 1975. En esta primera fase, la infraestructura estaba conformada por una planta de molienda y concentración al lado de la mina, y un ferroducto de 44 km de longitud para transportar el mineral hasta una planta peletizadora ubicada en Manzanillo, uno de los principales puertos marítimos de la costa del Pacífico (Sariego et al., 1988: 282). Al principio, la planta peletizadora tenía una capacidad de producir 1 500 000 toneladas anuales de pelets. En 1978, en el mismo sitio, se terminó la construcción de una segunda planta peletizadora, duplicando así la capacidad productiva del proceso de beneficio. Sociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 60 La lucha en torno a la minería en Manantlán Como ya se mencionó, en aquel entonces no había leyes ambientales que obligaran al consorcio a minimizar los impactos ambientales, mucho menos para presentar un Estudio de Impacto Ambiental (EIA). Así, durante los primeros años de operación, el CMBJPC explotó los yacimientos ferruginosos de la zona sin preocuparse, en absoluto, ni por el medio ambiente ni la población local, de acuerdo con la lógica de minimizar costos y maximizar ganancias, en el marco de una visión desarrollista orientada a la ISI. Conforme iba creciendo la extensión de la mina a tajo abierto y los montones de desechos, los problemas de la población local se multiplicaron y agudizaron. Los más afectados eran los habitantes de los poblados adyacentes a la mina, particularmente La Astilla y Las Pesadas, que forman parte del ejido de Ayotitlán; pero también las comunidades aguas abajo del río Marabasco. La presencia de la mina ha tenido repercusiones políticas y sociales para todos los pobladores de la Sierra de Manantlán. En 1990, 21 ciudadanos de Las Pesadas interpusieron una denuncia con el apoyo de la Academia Jalisciense de Derechos Humanos, en relación con la ocupación de un nuevo terrero, de 177 hectáreas, para los desechos del CMBJPC (AJDH, 1990). Los afectados señalaron que la maquinaria pesada y los terreros de dicho consorcio habían destruido sus caminos de herradura y sepultado sus milpas, cafetales y pastizales; además de haber tapado el ojo de agua del que dependía la comunidad para el consumo humano; también se quejaron del ruido que provoca la maquinaría y la dinamita. Asimismo, denunciaron que el presidente municipal de Minatitlán había ordenado el desalojo del poblado y que la Policía Judicial del estado de Colima los hostigaba y amenazaba. Según sus propios testimonios “[h]aber nacido aquí en Las Pesadas significa estar condenado a la miseria, la represión, el terror y el exterminio” (AJDH, 1990: 3).6 En respuesta a esta denuncia, ese mismo año, el CMBJPC invitó a un equipo de investigadores de la Facultad de Química y del Instituto de Geografía de la Universidad Nacional Autónoma de México a realizar un estudio sobre las afectaciones al ambiente producidas por la mina Peña Colorada. Dos investigadoras de la Universidad de Guadalajara, Alicia Loeza Corichi y Raquel Gutiérrez Nájera, lograron obtener una copia del informe de dicho estudio y realizaron un análisis Ésta era la primera de una serie de denuncias que han enfatizado la violación de los derechos humanos, indígenas y agrarios de la población ubicada en la zona del conflicto limítrofe entre los estados de Colima y Jalisco, donde el CMBJPC lleva a cabo sus actividades mineras. En 1995, la Comisión Nacional de Derechos Humanos emitió la Recomendación 122/95 para señalar la necesidad de establecer un acuerdo entre las dos entidades federativas, investigar y esclarecer las denuncias sobre la violación de derechos humanos, y prestar servicios públicos básicos a la población local. Sin embargo, según el Informe Ejecutivo de la Brigada Zeferino Padilla Villa (2007: 2), el gobierno de Colima “ha rehuido sus responsabilidades en esta materia a cambio de lucrar con el conflicto, utilizando un amplio repertorio de ilícitos y recursos prohibidos como el etnocidio, el férreo control social, las verdades a medias, la xenofobia antijalisciense, la manipulación de la información, y sobre todo, el predominio de un régimen de segregación racial bien montado que oculta discrecionalmente los crímenes contra los derechos humanos de la población civil”. 6 Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 61 crítico. Según sus observaciones, la investigación del equipo interdisciplinario estaba repleta de problemas metodológicos y limitaciones que imposibilitaban cuantificar los diversos impactos ambientales de la mina. No obstante, eludía a las afectaciones ambientales relacionadas a los terreros, el uso de grandes cantidades de agua, y los efluentes descargados en el río Marabasco (Loeza y Gutiérrez, 1996).7 Todavía es imposible cuantificar la mayoría de los impactos ambientales que derivan de las explotación minera en la Sierra de Manantlán, pues hacen falta estudios científicos de investigadores que no estén vinculados al CMBJPC. La empresa no permite que investigadores independientes entren al área de la mina, mucho menos que analicen sus documentos internos. No obstante, se puede obtener una idea sobre la naturaleza —y en algunos casos las dimensiones— de las principales formas de degradación ambiental a partir de una descripción del proceso de extracción del mineral, del proceso de beneficio y con el análisis crítico de los pocos informes disponibles. Actualmente, el consorcio realiza nueve actividades principales: 1) barrenación con rotarias para formar los barrenos donde se deposita el explosivo, 2) voladura, la fragmentación de la roca con explosivos que contienen nitrato de amonio, lo cual deja atrás bancos de 14 metros de altura, 3) carga y acarreo, se usa maquinaria pesada (cargadores frontales con capacidad de por los menos 10 metros cúbicos y camiones con capacidad de 100 toneladas o más), el material con valor económico se traslada a la trituradora para empezar el proceso de beneficio; mientras, el material “estéril” se lleva a los sitios de depósito llamados “terreros”, 4) trituración, reducción de los minerales con una trituradora, 5) preconcentración, por medios magnéticos y a través de poleas y cobbers se eliminan los materiales de poco valor, 6) molienda y concentración, primero se muele el mineral en agua para formar una pulpa, luego, con magnetismo, se separa el material ferroso, y los residuos se depositan en una presa de jales por tiempo indefinido, 7) transporte, hasta la planta peletizadora en Manzanillo, a través de tuberías dobles de 30 centímetros de diámetro, el mineral es impulsado con agua y se aprovecha la gravedad, 8) fabricación de pelets, consiste en la eliminación del exceso de agua, la aglomeración de pequeñas bolas conocidas como “pelets” o “pellas”, el mineral se rueda sobre un plano inclinado, y 9) endurecimiento de los pelets en un horno que alcanza 1 300 °C (Ternium, s/f). Actualmente, la planta peletizadora del CMBJPC de Manzanillo tiene una capacidad de 4 100 000 toneladas anuales. Los impactos ambientales más visibles de este proceso es la decapitación del Cerro de Los Juanes, donde se ubica la principal mina de cielo abierto del CMBJPC, y la acumulación de los esLa Ley General de Equilibrio Ecológico y Protección al Ambiente (LGEEPA), promulgada en 1988, sólo obligaba a las industrias extractivas a realizar los EIA para las nuevas obras o los nuevos procesos productivos; no para los que ya estaban en marcha. Por tanto, el CMBJPC sólo buscó en 1990 patrocinar estudios ambientales selectos, en el marco de una Carta de Compromiso firmada con la entonces Secretaría de Desarrollo Urbano y Ecología (SEDUE), en un esfuerzo por establecer una relación de cooperación con las agencias gubernamentales encargadas de cuidar el medio ambiente. 7 Sociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 62 La lucha en torno a la minería en Manantlán combros en los alrededores. Con base en el análisis de imágenes de satélite, se puede calcular que esta destrucción abarca una superficie de 5 mil hectáreas. Es una destrucción absoluta: deforestación, erosión, pérdida de hábitat para flora y fauna, alteración climática en una de las principales cuencas hidrológicas de la región, y contaminación tóxica derivada de los residuos de la maquinaria, los explosivos, el polvo y las mismas rocas fragmentadas. Es importante mencionar que el Cerro de Los Juanes formaba parte del paisaje cultural de los pobladores originarios de la Sierra de Manantlán; era un lugar donde pasaban peregrinaciones que llevaban imágenes veneradas. Las rocas fragmentadas y amontadas en los terreros sólo pueden considerase “estériles” en tanto no tienen valor económico. Es probable que contengan pequeñas cantidades de metales pesados que se emitan paulatinamente a la atmósfera. Los metales pesados que con frecuencia se encuentran en los desechos de la minería de hierro son antimonio, arsénico, berilio, cadmio, cromo, plata, plomo, níquel y selenio (USEPA, 1994: 1-14). Éstas son sustancias tóxicas que constituyen una seria amenaza a la salud de los seres humanos y otros organismos. Pueden estar presentes también en la presa de jales. El único estudio disponible al público sobre la toxicidad de los jales del CMBJPC se llevó cabo por la Consultaría e Investigación en Medio Ambiente, S.C. (CIMA) en 2010, en relación con el Informe Preventivo que la empresa minera tenía que producir, de acuerdo con la NOM-141-SEMARNAT-2003, para construir una nueva presa de jales, pues la anterior estaba a punto de llegar a su máxima capacidad. La CIMA no detectó la presencia de elementos tóxicos. Sin embargo, los resultados son poco confiables, en primer lugar, porque no se desglosa la metodología empleada y, en segundo, debido a los intereses implicados en la relación cliente-patrón entre la CIMA y el CMBJPC; éste paga a aquella para producir un documento que le permita sacar adelante la expansión de sus explotaciones. Se calcula que el CMBJPC genera más de 300 millones de toneladas anuales de desechos sólidos.8 Dejando a un lado la toxicidad de estos materiales, tienen impactos ambientales perjudiciales para la población local en tanto ocupan terrenos anteriormente dedicados a actividades agropecuarias, son susceptibles a la erosión y trastornan los flujos hidrológicos de la región, ya que obstruyen manantiales y cambian el curso de arroyos (Guillén, 2009). Además, la erosión conlleva a problemas de sedimentación, que a su vez perjudican la fauna acuática. Se puede decir lo mismo respecto de los impactos de los múltiples caminos construidos por el CMBJPC. De acuerdo con un ejidatario de Ayotitlán: Cálculo propio basado en los datos contenidos en EcoFor (2003) y Ternium (s/f). En particular, con base en los datos contenidos en la Tabla No. 1 de EcoFor (2003: 15), se calcula que, para producir una tonelada de minerales preconcentrados, el CMBJPC genera 35.8 toneladas de desechos sólidos. Por otra parte, la mina tiene la capacidad de producir 8,621 millones de toneladas/año de minerales preconcentrados (Ternium, s/f: 21). 8 Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 63 Hay varios arroyos que ya no le da igual; se va disminuyendo la producción. Porque antes de que la mina hiciera sus brechas, eran arroyos con todas sus piedras, ahora ya son playas, entonces, se pierden […] Y también se está llevando con agua. Está sacando mucha agua, tubos de casi catorce pulgadas, para mandarlo a Manzanillo. ¿Por qué no en un carro?, mejor en un carro. Pues dejando de traer el agua, que puede ser utilizada para riego y para consumo humano. Hay poblados que no tienen agua en esa zona y la mina sí tiene.9 Otro impacto ambiental es el debilitamiento del cauce del río Marabasco. La molienda de las minerales y su transporte por ferroducto consumen grandes cantidades de agua, lo que priva a la población ubicada aguas abajo del río Marabasco del recurso para sus actividades agropecuarias, incluidos los habitantes de El Platanar y Plan de Méndez. No hay datos disponibles sobre la cantidad de agua que consume el CMBJPC; la Semarnat no la obliga a proporcionarlos o cuando menos a no hacerlos disponibles al público. Cabe señalar, además, que la población río abajo se vio afectada por un derrame de la presa de jales del CMBJPC en octubre de 2012, según las denuncias de los afectados, cuyas casas, parcelas de cultivo y corrales quedaron inundados (Salazar Zenil, 2013). El CMBJPC presume contar con el Certificado de Industria Limpia otorgado por la Procuraduría Federal de Protección al Ambiente (Profepa) y también el reconocimiento ISO 14000. Sin embargo, se puede cuestionar el significado de estos reconocimientos, al observar la estrecha relación de colaboración entre los dirigentes de esta empresa y los funcionarios de Semarnat. Por ejemplo, el CMBJPC presume dar a la Semarnat, delegación Colima, “apoyo en especie” para la protección de cocodrilos de la Laguna de Alcuzahue en el municipio de Tecomán (Camimex, 2009: 8-9). De acuerdo con Efraín Naranjo Cortés, presidente del Comité Estatal de los Derechos Humanos No Gubernamental, “hay un contubernio de la Profepa y la Semarnat con los grandes poderosos”, que se evidencia en el apoyo político y los premios que estas agencias gubernamentales brindan al CMBJPC (Ecología Radical, 2011). Esto refleja la “captura política” requerida para sostener una relación de reciprocidad negativa. En todo caso, el CMBJPC pretende proyectar una imagen de sustentabilidad con la promoción de proyectos de reforestación, preservación de fauna, educación ambiental, etcétera, lejos de su mina a tajo abierto. Conflictos internos, resistencia y negociación Con la creación de la RBSM en 1987, las compañías forestales fueron expulsadas y los activistas locales voltearon su atención hacia la minería. El movimiento socioambiental e indígena en Manantlán experimentó un proceso de empoderamiento por haber logrado correr a los taladores de 9 Entrevista realizada el 3 de noviembre de 2006. Sociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 64 La lucha en torno a la minería en Manantlán la sierra, y si bien tuvieron que ceder cierta medida de control sobre los recursos naturales locales a la dirección de la RBSM, lo cierto es que el nuevo clima político favorecía el “desarrollo sustentable” de los pueblos marginados en la Zona de Amortiguamiento de la Reserva (Tetreault, 2009). Aprovechando esta situación, en 1988 más de cien indígenas hicieron un plantón frente al Palacio de Gobierno de Colima para denunciar el despojo de recursos naturales del CMBJPC. Hasta esa fecha, la mina no había traído consigo ningún beneficio para la población local, sólo problemas ambientales y sociales. En otras palabras, practicaba la reciprocidad negativa. Los activistas exigieron el reconocimiento de sus derechos humanos, presionaron para poder participar en la toma de decisiones sobre el manejo de los recursos naturales y demandaron una indemnización justa. Aunque el Gobierno de Colima no hizo caso a estas demandas, en el marco del Programa Nacional de Solidaridad, el CMBJPC empezó a contribuir al financiamiento de proyectos de desarrollo social, por ejemplo, la electrificación de los principales poblados de la sierra. Por otra parte, los pobladores de Ayotitlán (el núcleo agrario más grande de la sierra y el más perjudicado por la explotación minera) estaban divididos. Las industrias extractivas habían cooptado a las autoridades locales, fomentando la creación de un grupo de caciques internos que controlaba al comisariado ejidal. Este grupo estaba afiliado a la Confederación Nacional Campesina (CNC) y al Partido Revolucionario Institucional (PRI) y gozaba del apoyo de diversas agencias en los tres niveles de gobierno. Por otro lado, los activistas sociales estaban encabezados por los líderes tradicionales de la comunidad y tenía vínculos con la Coordinadora Nacional Plan de Ayala (CNPA) a través de la ACR. Empoderado por su victoria contra los taladores, este grupo empezó a organizarse en un esfuerzo por democratizar el aparato político del ejido. Los caciques internos respondieron con una maniobra que pretendía quitar a los activistas sus derechos ejidales; así, en 1990, se inició una larga batalla legal y política entre los dos grupos mencionados que, de alguna manera, sigue desenvolviéndose hasta la fecha (Tetreault y Hernández, 2011). A partir de 1994, los activistas socioambientales e indígenas de Manantlán empezaron a recibir asesoría de la Unidad de Apoyo a Comunidades Indígenas (UACI), una dependencia de la Universidad de Guadalajara, creada en el mismo año para responder a los cuestionamientos puestos en evidencia por el levantamiento zapatista. Apegándose a los principios de investigación-acción participativa, la UACI ha trabajado con los líderes tradicionales de la comunidad para emprender varias líneas de acción, por ejemplo: rescatar y fortalecer la cultura y la identidad indígenas, defender los derechos (humanos, agrarios e indígenas) de la población local, resolver el conflicto interno sobre los derechos ejidales, atender los pendientes de la reforma agraria y proteger el medio ambiente. Con la ayuda de la UACI, la lucha socioambiental y cultural en Manantlán se vinculó con el movimiento zapatista, a través del Congreso Nacional Indígena. En este marco, Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 65 en diciembre de 1997, se realizó un acto comunitario para celebrar la reconstitución oficial del Consejo de Mayores, un órgano político tradicional que ha servido como punta de lanza en la lucha contra la minería ecológicamente devastadora y socialmente irresponsable. Un año después de la reconstitución del Consejo de Mayores, en un contexto local caracterizado por altos niveles de tensión y conflictividad entre los dos grupos mencionados, el CMBJPC (en manos del gran capital mexicano) pagó casi dos millones de pesos al ejido de Ayotitlán por la firma de un contrato que le permitiría explotar los recursos minerales de la comunidad durante los siguientes 30 años. Además, prometió pagar al ejido 136.6 mil pesos anuales por el derecho de tirar desechos industriales en el territorio de Ayotitlán, así como 40 mil pesos anuales “para gastos del Comisariado Ejidal”. Aunque a primera vista esto puede parecer un avance en las negociaciones con el CMBJPC, en realidad sólo sirvió para legitimar las actividades mineras en la zona, con pocos beneficios para la población local, ya que los caciques internos todavía controlaban el comisariado ejidal y no manejaban los recursos con transparencia. Como dijo Gaudencio Mancilla Roblada, representante legal del Consejo de Mayores de Ayotitlán, “De ese dinero, no sabemos nada, sólo se beneficia unos cuantos” (Rojas, 2005). Además, el contrato no tomó en cuenta los 23 años de explotación previos, y los montos se traducirían en sólo dos centavos por cada mil pesos de ganancia durante los siguientes 12 años (Tetreault y Hernández, 2011). Así, quedó claro que para poder negociar efectivamente con el CMBJPC era necesario resolver primero los conflictos internos y democratizar el aparato político ejidal. En este contexto, los activistas de Manantlán y sus aliados redoblaron sus esfuerzos para resolver el juicio sobre la privación de derechos ejidales. En 2003 aprovecharon el traslado del juicio de Guadalajara a Colima para acudir al nuevo tribunal y proponer que el asunto se resolviera en la comunidad con la participación de todos los involucrados. El magistrado del órgano jurisdiccional emitió un acuerdo favorable a esta propuesta, dando lugar a un proceso llamado “composición amigable”, que se llevó a cabo entre abril y octubre de 2003. Este proceso consistió en 14 sesiones de trabajo, en las que participaron más de 700 ejidatarios de los dos grupos en conflicto, así como representantes de varias agencias gubernamentales. Al final, todos se pusieron de acuerdo con los resultados; los ejidatarios que habían perdido ilegítimamente sus derechos ejidales en 1990, los recuperaron y se extendieron derechos ejidales a algunos de los adjudicados (Tetreault, 2009). De esta manera, se resolvió el juicio de privación; un gran avance en la democratización del ejido, pero no el fin de la lucha. Durante la primera década del siglo XXI, con los precios de los metales a la alza y en un contexto político nacional a favor de la entrega libre de concesiones a compañías transnacionales, el CMBJPC y otras compañías mineras se animaron a expandir sus explotaciones en Manantlán. Sociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 66 La lucha en torno a la minería en Manantlán Así, en el otoño de 2005, el CMBJPC introdujo de manera sorpresiva y furtiva un camino para facilitar la “exploración” de 150 mil toneladas de material ferruginoso cerca de las comunidades de Chanquiáhuitl y Cerro Prieto en el ejido de Ayotitlán. Al mismo tiempo, financió la campaña del ejidatario Jesús Michel Prudencio, un empleado del CMBJPC, para que éste llegara a ser presidente del comisariado de Ayotitlán. Con la compra de votos, la manipulación del padrón ejidal y otras irregularidades, Michel Prudencio ganó las elecciones del 23 de octubre del mismo año, respaldando así los planes de expansión de la minera. Sin embargo, el Consejo de Mayores, junto con sus asesores, se movilizó para no permitirlo; interpuso un juicio agrario para protestar por el fraude cometido en las elecciones ejidales y solicitó un amparo de la justicia federal, con el objetivo de detener la construcción de caminos y otras obras preparatorias. Con esta acción jurídica se logró paralizar los planes del CMBJPC y durante los siguientes meses se dio seguimiento al proceso legal para desconocer a Michel Prudencio como presidente del comisariado, algo que finalmente se logró en junio de 2008. A finales de ese mismo año, el candidato del Consejo de Mayores (Juan Mancilla) ganó las elecciones ejidales y se abrió la posibilidad de enfrentar al CMBJPC desde el espacio institucional del ejido de Ayotitlán (Tetreault y Hernández, 2011). En esa coyuntura, los activistas socioambientales de la Sierra de Manantlán y sus aliados no veían cómo cerrar definitivamente la mina del CMBJPC, ni de apropiarse de ella, para posteriormente explotarla de una manera social y ecológicamente responsable, en beneficio de la comunidad. Los antecedentes represivos y las limitaciones del sistema jurídico apuntaban hacia una estrategia de negociación menos polémica, combinada con acciones directas en momentos oportunos, con los objetivos de detener la expansión de las actividades mineras, mitigar los impactos ecológicos más nocivos, poner fin a la violencia y obtener más recursos para impulsar proyectos de desarrollo social. Con esta visión, entraron en negociaciones con los gerentes del CMBJPC. Durante este proceso de lucha interna y de resistencia a la expansión territorial de las actividades del CMBJPC, los defensores de la comunidad tuvieron que combatir otra invasión, esta vez de la empresa china Gan-Bo Minera Internacional, que se introdujo en el ejido de Ayotitlán en 2007 sin los permisos correspondientes de impacto ambiental y de cambio de uso de suelo, en un lugar conocido como la Piedra Bola. En este caso, el Consejo de Mayores no sólo interpuso un juicio de amparo para detener la intrusión, sino que también movilizó a centenares de miembros de la comunidad para bloquear los trabajos de Gan-Bo e incautar su maquinaria (González García, 2008). La maquinaría se devolvió meses después, cuando las autoridades correspondientes cedieron a la demanda de suspender la concesión. El 8 de noviembre de 2010 se firmó un nuevo acuerdo entre el ejido de Ayotitlán y el CMBJPC que estipula que dicha empresa pagará siete millones de pesos anuales dentro de un plazo Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 67 de diez años, con incrementos correspondientes a la tasa de inflación y con un pago adicional de tres millones de pesos en 2010, etiquetados para repartirse entre los ejidatarios. Los pagos anuales se destinarán a proyectos de desarrollo social, por ejemplo: una beca universal para todos los estudiantes de bachillerato y licenciatura, asistencia adulta comunitaria, un proyecto de viviendas ecológicas, un programa de proyectos productivos y un programa de investigación sobre impactos ambientales. Desgraciadamente, debido a una maniobra política llevada a cabo por los caciques internos del ejido, los 10 millones de pesos correspondientes al año 2010 fueron repartidos sólo entre los ejidatarios, y se dejó fuera a los miembros de la comunidad que no tienen derechos ejidales, que son la mayoría (Tetreault y Hernández, 2011). No obstante, desde entonces se ha conformado un Comité Técnico, con la participación de diversas instancias gubernamentales, para monitorear la administración de estos recursos. Si bien este financiamiento no resolverá los problemas ambientales y sociales provocados por la explotación minera, sí representa un avance en tanto aumenta la indemnización por un factor de 20 respecto al contrato anterior. Durante los últimos años, además de tener que luchar contra las incursiones de compañías mineras transnacionales, los nahuas de Manantlán han tenido que lidiar con grupos armados anónimos que entran de manera subrepticia a la sierra para sacar minerales y maderas preciosas. Los activistas locales, al perseguirlos a los sitios de explotación, han recibido amenazas. Uno de ellos, Celedonio Monroy, fue secuestrado por un grupo de hombres encapuchados y armados el 23 de octubre de 2012 (García Partida, 2012). En el momento de escribir este ensayo, todavía no se han recibido nuevas noticias de él. Ante esta situación, el Consejo de Mayores ha promovido, desde finales de 2012, la creación de una policía comunitaria para vigilar la zona (Guillén, 2012). Sin embargo, las perennes divisiones internas han obstaculizado esta iniciativa hasta la fecha. El 3 de junio de 2013 los mismos activistas del Consejo de Mayores movilizaron aproximadamente a 200 ejidatarios de Ayotitlán para suspender “por la vía de los hechos” las actividades de dos minas ilegales en sitios cerca de Chanquiáhuitl y Cerro Prieto (Guillén, 2013b). Dos semanas después, hombres armados irrumpieron en la casa de Gaudencio Mancilla, el representante legal del Consejo de Mayores, y le dejaron un mensaje a su familia: que no se metiera en los asuntos de las minas. El 22 de agosto del mismo año personas armadas y encapuchadas llegaron nuevamente a su casa y lo secuestraron. Poco tiempo después se descubrió que elementos de la policía estatal de Jalisco, fueron los que torturaron al líder indígena en un esfuerzo por conseguir una confesión sobre su presunta participación en la distribución de armas para defender la comunidad de las incursiones mineras (Ibid.). Antes de pasar a las reflexiones finales es importante mencionar que hay otra organización popular que ha incidido en la lucha en torno a la minería en Manantlán. En 2006 la RJDH impulsó Sociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 68 La lucha en torno a la minería en Manantlán la creación del Frente Regional pro-Manantlán y Cuenca del Marabasco (Fremmar), con el objetivo de “resolver de manera pacífica y efectiva el ancestral conflicto limítrofe y ecológico que enfrentan los estados de Jalisco y Colima” (citado de la constitución jurídica del Fremmar). En la práctica esto se traduce en un enfoque en la defensa de los derechos humanos, sobre todo para los indígenas que viven en la franja fronteriza en disputa. Los siete miembros de su Consejo Directivo han participado en la elaboración de informes y denuncias sobre la violación de derechos humanos en la zona minera. Además, en su afán por promover la reflexión y acción frente a la minería a cielo abierto, han participado en diversos eventos coordinados por la Red Mexicana de Afectados por la Minería (REMA). El Fremmar no demanda el cierre de las minas en Manantlán, sino la mitigación de los impactos ambientales más nocivos y el cese de la violación de derechos humanos, o sea, “una minería sustentable y se nos haga partícipes de los beneficios” (Fremmar, 2009: 33). Reflexiones finales La economía local de los indígenas de Manantlán gira en torno a las actividades agropecuarias para la autosubsistencia. El eje central de estas actividades es la producción de maíz, que se siembra en las laderas de las montañas, en parcelas pequeñas llamadas “milpas”, utilizando el sistema coamil, también conocido como “roza, tumba, quema”. Alrededor de las viviendas se crían gallinas, cerdos, chivos y otros tipos de ganado menor. Además, muchas familias tienen huertas de traspatio, con árboles frutales, verduras, plantas medicinales y especies. Otras actividades económicas tradicionales que todavía existen, aunque en menor medida en comparación con el pasado, son la caza, la pesca y la recolección de frutas y plantas silvestres. También se practican actividades agropecuarias principalmente para fines comerciales. Las más importantes son la producción de café y fruta (zarzamora, plátano, naranja y aguacate), la apicultura y la ganadería bovina. Por otra parte, los pobladores locales obtienen ingresos monetarios de la renta de pastizales, los programas gubernamentales de transferencia directa, y el trabajo temporal en granjas comerciales y centros urbanos cercanos, especialmente en Manzanillo, Colima y Autlán (Tetreault, 2009: 318-319). No cabe duda que, en términos sociales, desde finales de los años ochenta, ha habido avances en la Sierra de Manantlán, y gracias, en gran medida, a la movilización de los activistas locales y su vinculación con movimientos indígenas y socioambientales en el ámbito nacional, se ha ampliado la cobertura de servicios públicos básicos (electricidad, agua entubada, centros de salud, escuelas, etc.; Tetreault, 2008). Además, en la actualidad, los pobladores locales tienen acceso a pequeñas cantidades de crédito para proyectos productivos, los agricultores con derechos ejidales reciben transferencias monetarias del Programa de Apoyos Directos al Campo (Procampo) y la mayoría de las familias que viven en Manantlán es beneficiaria del programa Oportunidades. Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 69 No obstante estos avances, los nahuas de Manantlán siguen viviendo en condiciones de pobreza y marginación. El municipio de Cuautitlán, por ejemplo, donde se ubica el ejido de Ayotitlán, tiene un alto grado de marginación (Conapo, 2012a) y 85.7% de la población vive en condiciones de pobreza (Coneval, 2012). Los indicadores son aún más desalentadores para la población indígena que vive fuera de la cabecera municipal; 35 poblados en el ejido de Ayotitlán tienen un grado de marginación muy alto (Conapo, 2012b), y si el ejido fuera un municipio en sí mismo, sería el centésimo décimo más marginado del país (donde hay 2 456 municipios; Tetreault, 2009). ¿Cómo es posible que en esta región, donde se han llevado a cabo actividades extractivistas lucrativas durante varias décadas, todavía haya tanta pobreza? La respuesta, en términos sencillos, es que las ganancias derivadas de estas actividades económicas no han quedado en la región, mientras que los costos sí. En otras palabras, el extractivismo en Manantlán refleja un proceso de acumulación por desposesión. Como hemos visto, desde los tiempos de la Colonia los indígenas de Manantlán fueron desposeídos de buena parte del territorio y los recursos naturales que sostenían su economía de autosubsistencia, para entregarlos a agentes externos que producían mercancías para el mercado. Aunque la reforma agraria del siglo XX ostensiblemente devolvió los derechos de propiedad colectiva sobre el territorio a los pobladores originarios, esto se hizo de modo tal que se pudiera resguardar los recursos naturales más valiosos para el gran capital. Desde esta perspectiva, las demoras, omisiones e irregularidades asociadas con la reforma agraria y la disputa fronteriza no son tanto problemas institucionales, sino más bien reflejan el imperativo del sistema capitalista de expandirse indefinidamente en búsqueda de recursos de bajo costo para sostener el proceso de acumulación. “¡Acumulad, acumulad! ¡He ahí a Moisés y los profetas!” escribió Marx (2000: 75) respecto de la lógica capitalista. Conforme se expande este sistema, va incorporando cada vez más los recursos naturales de la Sierra de Manantlán y otras regiones en la periferia del sistema, destruyéndolos sobre la marcha e impulsando así un proceso de proletarización. Los nahuas de Manantlán, en el presente, dependen de los ingresos derivados de la migración laboral temporal, así como de las transferencias monetarias directas de los programas gubernamentales para combatir la pobreza, para satisfacer sus necesidades fisiológicas básicas. La minería no genera un número significativo de trabajos para los indígenas, por lo tanto se ven obligados a buscarlos en los centros urbanos cercanos, las zonas turísticas y en los valles agrícolas, donde se paga el salario mínimo o menos. Mientras las ganancias derivadas de las industrias extractivas vuelan de la sierra para concentrarse en las manos del gran capital nacional y extranjero, los costos se observan en la destrucción ambiental masiva y desarticulación social. La explotación forestal de 1940 a 1987 acarreó un Sociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 70 La lucha en torno a la minería en Manantlán deterioro ambiental que todavía puede verse en la reducción de la superficie arbolada, disminución de pinos, bosques secundarios con árboles de menor tamaño, alteraciones en la retención y flujo de aguas superficiales y disminución de ciertas especies de flora y fauna (Jardel, 1998). En cuanto a la minería a cielo abierto, como se ha visto, los costos ecológicos incluyen la demolición de cerros, el debilitamiento del sistema hidrológico, erosión, sedimentación y la alteración de paisajes culturales. La gente que vive cerca de las mineras reportan problemas de salud, entre ellas enfermedades de la piel, esterilidad, daños oculares y constantes diarreas (Fremmar, 2009). A estos costos ecológicos y de salud humana, hay que agregar los sociales y culturales. La penetración del capital extractivista ha ido de la mano de la supresión de formas tradicionales de gobierno, la socavación de una cosmovisión que ve espíritus divinos en la naturaleza (Robertson, 2002) y la provocación de divisiones internas. Además, los nahuas de Manantlán han sido víctimas de matanzas (1920 y 1951) y hasta la fecha sufren de una violación sistemática de sus derechos humanos; hostigamiento, detenciones arbitrarias, golpes, secuestros y al menos 20 ejecuciones de campesinos indígenas desde los años sesenta (Brigada Zeferino Padilla Villa, 2007), incluidos dos miembros del Consejo de Mayores en años recientes. Desde la perspectiva teórica presentada en este trabajo, esto no debe causar sorpresa, pues en consonancia con las observaciones históricas de Marx, la acumulación por desposesión no sucede de manera pacífica; y como nota Harvey (2004), es especialmente brutal en la periferia del sistema capitalista global. Desde otro ángulo, la penetración del capital minero en la Sierra de Manantlán refleja la “reciprocidad negativa” en tanto el CMBJPC ha demostrado “la voluntad de recibir lo más por lo menos” (Garibay y Balzaretti, 2009: 92), en una relación con los pobladores locales caracterizada por la asimetría política. Como se vio, durante más de 20 años esta empresa no indemnizó a los afectados. No fue hasta principios de los años noventa, en respuesta a las denuncias y movilizaciones en Manantlán, que empezó a usar una pequeña parte de sus ganancias para cofinanciar proyectos de desarrollo social. Mientras tanto, para sostener esta reciprocidad negativa, ha empleado una amplia gama de tácticas para efectuar la “captura política” de las instituciones locales, es decir, los gobiernos estatales de Colima y Jalisco, los gobiernos municipales de Minatitlán y Cuautitlán, las delegaciones locales de la Semarnat, el comisariado del ejido de Ayotitlán, etcétera. Como ya se relató, estas tácticas han provocado divisiones internas entre dos grupos de ejidatarios antagónicos, denominados “caciques internos” y “activistas sociales”. El primero ha asumido una postura de “ética débil” al aceptar la imposición del mina y la destrucción ambiental que ésta implica a cambio de recibir dádivas, pagos en efectivo y control del comisariado ejidal; mientras que el segundo grupo, organizado formalmente en el Consejo de Mayores y el Fremmar, ha oscilado entre la “ética fuerte” y la “ética negociada”, dependiendo de las circunstancias y las ventanas de Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 Darcy Tetreault 71 oportunidad, en un esfuerzo por detener la expansión de la explotación minera y por obtener más beneficios para la población local. Desde un punto de vista teórico complementario, el movimiento de resistencia en la Sierra de Manantlán puede ser visto como un ejemplo de lo que Joan Martínez Alier llama el “ecologismo de los pobres” o alternativamente “el ecologismo popular”. Este tipo de ecologismo “dimana de los conflictos distributivos sobre el uso de los recursos ecológicos requeridos para el sustento” (Martínez Alier, 1997: 23). En contraste con el ambientalismo posmaterialista del Norte, que está detrás de la formación de la RBSM con la preocupación medular de conservar la biodiversidad; los objetivos del movimiento popular en Manantlán “son definidos en términos de las necesidades ecológicas para la vida” (Martínez Alier, 2012: 59). Los activistas de Manantlán buscan conservar el medio ambiente no tanto por motivos estéticos o por preocupaciones por la supervivencia de otras especies, sino más bien para proteger sus medios de vida, salud y entorno cultural. Según Martínez Alier (2012: 59), el ecologismo de los pobres trata “de sacar los recursos naturales de la esfera económica, del sistema de mercado generalizado, de la racionalidad mercantil, de la valoración crematística (reducción del valor a costos y beneficios monetarios) para mantenerlos o devolverlos a la oikonomia (en el sentido con que Aristóteles usó la palabra, parecido a ecología humana, opuesto a crematística)” ¿Éste es el caso del movimiento de resistencia en Manantlán? No cabe duda que a los activistas más radicales les gustaría cerrar las minas de una vez por todas, pero otros pobladores locales simplemente quieren un trozo más grande del pastel. Así, no se debe romantizar sobre este movimiento o interpretarlo ipso facto como anticapitalista. Las demandas del Fremmar, por ejemplo, son esencialmente que el capital minero internalice los costos ecológicos más perjudiciales y que genere más beneficios para la población local. Sea como fuere, las cuestiones redistributivas están en el centro de esta lucha y esto corresponde con las teorizaciones de Schlosberg (2007) sobre los movimientos de justicia ambiental. Desde esta perspectiva, las demandas de la justicia ambiental son en primer lugar distributivas, pero no se limitan a ellas; se traslapan con demandas por el reconocimiento, la participación y las capacidades colectivas. Estas dimensiones se perfilan en Manantlán en los esfuerzos por rescatar y fortalecer los aspectos positivos de la cultura tradicional, exigir el reconocimiento de la identidad indígena y demandar el respeto a los derechos humanos. Además, se reflejan en la reconstitución del Consejo de Mayores como un espacio institucional tradicional para el diálogo, y también en la lucha por democratizar el aparato político del ejido de Ayotitlán, como un medio para lograr mayores niveles de participación en la toma de decisiones sobre el manejo de los recursos naturales locales. En última instancia, la lucha en Manantlán es por la autonomía y la autodeterminación; se trata de potenciar la capacidad de la comunidad de funcionar como un espacio sano para la reproSociedad y Ambiente, año 1, vol. 1, núm.2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74 72 La lucha en torno a la minería en Manantlán ducción social y cultural, y para lograrlo, en un mundo regido por el sistema capitalista globalizado y en un país periférico reestructurado alrededor de los preceptos del neoliberalismo, se requiere del fortalecimiento de las redes de solidaridad que vinculan el activismo en Manantlán a luchas socioambientales y étnicas más amplias en los ámbitos nacional e internacional. Referencias Academia Jalisciense de Derechos Humanos (AJDH) (1990). Queja No. 3/90. Asunto: Ranchería Las Pesadas, Ejido Ayotitlán, del Municipio de Cuautitlán, Jalisco. Guadalajara: AJDH. 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Washington: Usepa, 122 pp. Recibido: 20 de agosto de 2013 Aceptado: 24 de octubre de 2013 Sociedad y Ambiente, año 1, vol. 1, núm. 2, julio-octubre de 2013, ISSN 2007-6576, pp. 47-74
https://openalex.org/W1984859189	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0007428&type=printable	English	null	One More Piece in the VACV Ecological Puzzle: Could Peridomestic Rodents Be the Link between Wildlife and Bovine Vaccinia Outbreaks in Brazil?	PloS one	2,009	cc-by	7,178	Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: kroone@icb.ufmg.br One More Piece in the VACV Ecological Puzzle: Could Peridomestic Rodents Be the Link between Wildlife and Bovine Vaccinia Outbreaks in Brazil? Joˆ natas S. Abraha˜o1, Maria Isabel M. Guedes1, Giliane S. Trindade1, Fla´vio G. Fonseca2, Rafael K. Campos1, Bruno F. Mota1, Ze´lia I. P. Lobato3, Andre´ T. Silva-Fernandes1, Gisele O. L. Rodrigues1, Larissa S. Lima1, Paulo C. P. Ferreira1, Cla´udio A. Bonjardim1, Erna G. Kroon1* Joˆ natas S. Abraha˜o1, Maria Isabel M. Guedes1, Giliane S. Trindade1, Fla´vio G. Fonseca2, Rafael K. Campos1, Bruno F. Mota1, Ze´lia I. P. Lobato3, Andre´ T. Silva-Fernandes1, Gisele O. L. Rodrigues1, Larissa S. Lima1, Paulo C. P. Ferreira1, Cla´udio A. Bonjardim1, Erna G. Kroon1* 1 Universidade Federal de Minas Gerais, Instituto de Cieˆncias Biolo´gicas, Laborato´rio de Vı´rus, Belo Horizonte, Minas Gerais, Brazil, 2 Universidade Federal de Minas Gerais Instituto de Cieˆncias Biolo´gicas, Laborato´rio de Virologia Comparada, Belo Horizonte, Minas Gerais, Brazil, 3 Universidade Federal de Minas Gerais, Escola de Veterina´ria Laborato´rio de Vı´rus, Belo Horizonte, Minas Gerais, Brazil Abstract Background: Despite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks – BV), affecting dairy cattle and milkers. Little is known about VACV’s natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated. Methodology/Principal Findings: In this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks. Conclusion/Significance: These data provide new biological and epidemiological information on VAC nteresting question: could peridomestic rodents be the link between wildlife and BV outbreaks? Citation: Abraha˜o JS, Guedes MIM, Trindade GS, Fonseca FG, Campos RK, et al. (2009) One More Piece in the VACV Ecological Puzzle: Could Peridomestic Rodents Be the Link between Wildlife and Bovine Vaccinia Outbreaks in Brazil? PLoS ONE 4(10): e7428. doi:10.1371/journal.pone.0007428 Editor: Joel Mark Montgomery, U.S. Naval Medical Research Center Detachment/Centers for Disease Control, United States o Received June 14, 2009; Accepted September 21, 2009; Published October 19, 2009 Copyright: 2009 Abraha˜o et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. aha˜o et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support was provided by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG) and Ministe´rio da Agricultura, Pecua´ria e Abastecimento (MAPA), Brazil. The funders had no role in study design, data collection and analisys, decision to publish, or preparation of the manuscript. PLoS ONE \| www.plosone.org Introduction Over the last decade, several VACV strains have been isolated from these outbreaks, also known as bovine vaccinia (BV) disease, and biological and molecular studies have shown a high degree of polymorphism among these isolates [15]. During BV outbreaks, cows exhibit lesions on the teats and udders, ranging from roseolar erythema to papules, vesicles, pustules, and crusts [16,17]. Secondary bacterial mastitis is frequently associated with decrease in milk production, leading to economic losses and social impact, mainly in subsistence farming properties [18]. Infected milkers usually present lesions on their hands, apparently transmitted by unprotected contact with infected cattle [13,19]. On some properties, the milking is performed without strict aseptic measures and the unsophisticated infrastructure of some farms allows for contact of cattle with wildlife and other domestic animals [18] such as small ruminants, dogs, cats and rodents. seropositive mammalians [25,26]. Despite all the speculation about VACV circulation in Brazilian forests, the link between wildlife and BV outbreaks has not yet been established. To date, the discovery of potential VACV hosts and reservoirs, as well as the study of viral circulation between wildlife and farms, could help to predict and perhaps prevent future BV outbreaks. In this study, we actively searched for VACV-infected peridomestic rodents (PdR) in BV-affected areas in Brazil and speculated about their role in outbreaks. We describe for the first time the isolation of VACV from a mouse during a BV outbreak. Our data show that this isolate was the same etiological agent of exanthematic lesions observed in the cattle and human inhabitants of a BV- affected area. These results provide new and important epidemi- ological information on VACV and lead to an interesting question: could PdRs be the link between wildlife and BV outbreaks? The circulation of VACV within and/or among farms during BV outbreaks is usually promoted by infected milkers – who spread the virus to the herd by contact with their hands – and by the trade of infected cattle between properties [19]. However, some VACV outbreaks are temporally and spatially distant from previously notified BV areas. Therefore, the focal origin of outbreaks is frequently unknown. Moreover, BV usually occurs during the dry season, when some Brazilian biomes present a scarcity of victuals, leading some wild species to search for food in farm storehouse and corrals. Rats, mice, opossums, foxes, wild dogs and small felids are frequently observed around farming properties [20,21]. Introduction diseases are mainly caused by OPV species, such as (i) Cowpox virus (CPXV) in Europe [6]; (ii) Monkeypox virus (MPXV), which occurs naturally in Africa and was recently introduced in the USA [7]; and (iii) Vaccinia virus (VACV) in Asia and South America [8–10]. The host range of zoonotic OPV remains under investigation, and some naturally infected mammalian species have been described. Serological and molecular approaches have shown that CPXV persists in bank voles, field voles and wood and house mice, while squirrels seem to be the main natural reservoir of MPXV [11]. Despite the historical importance of VACV, there are few data about the origins, natural reservoirs and mechanisms by which the virus persists in nature [12–14]. Although some authors believed that VACV vaccine strains could have spread from humans to domestic animals and adapted to the rural environment [8], recent studies have suggested an independent origin for South American VACV isolates, distinct from the vaccine strains used on this continent during the WHO campaign [13,14]. Thirty years ago, the scientific community celebrated the eradication of smallpox, a highly lethal disease caused by the Variola virus (VARV) [1], a member of Family Poxviridae [2]. This achievement was the result of a coordinated effort by the World Health Organization (WHO), which promoted a world-wide vaccination campaign during the 1960s and 1970s [3]. The smallpox vaccines used in the WHO campaign were, in fact, Vaccinia virus (VACV) strains, a species belonging to the Orthopoxvirus (OPV) genus that presents serological cross-reaction with other OPV, including VARV [1,3]. Despite the immune protection provided by VACV, several cases of adverse manifestations due to vaccination were reported, which led to the suspension of the vaccination campaign after eradication of the disease [4,5]. Many experts believed that the war against the poxvirus had been won. However, in recent years, other poxviruses have emerged and re-emerged, causing exanthematic infections in humans and domestic animals, both in rural and urban areas. These zoonotic Today, VACV infections affecting dairy cattle and milkers, mainly in Southeast Brazil, represent a frequently reported OPV PLoS ONE \| www.plosone.org October 2009 \| Volume 4 \| Issue 10 \| e7428 October 2009 \| Volume 4 \| Issue 10 \| e7428 1 BV: VACV Circulation in PdRs zoonosis. Introduction In theory, some of these species, especially rodents, could be VACV reservoirs. In fact, at least three different VACV strains were isolated in Brazilian forests in the past, away from rural environments: Cotia virus (CTV)/SPAn 232 virus (SAV) was isolated from sentinel mice in Sa˜o Paulo state [22,23]; and BeAn 58058 virus (BAV) was isolated from the blood of an Amazon rodent specimen belonging to the Oryzomys genus [24]. Serological studies using sera from wild animals captured in the Brazilian Cerrado (a savanna-like environment) and animals from the Amazon biome have revealed a high prevalence of OPV- Viral isolation from a peridomestic mouse A total of six house rats (Rattus rattus) and seven house mice (Mus musculus) were trapped in different BV-affected properties. They were identified according to morphological characteristics using the Brazilian Rodents Guide and taxonomical keys (Pan-American Health Organization, 2008), and by mastozoologists from the Zoology Department of our institute. To data, both Rattus rattus and Mus musculus are Old World species, artificially introduced in Brazil. The rodents were captured in the vicinity of the farms, including in corrals, food warehouses, grain plantations and boundaries of adjacent forests. Although none of the rodents presented apparent VACV epidermal lesions or morphological changes in collected organs, neutralizing antibodies against OPV were detected in two serum samples collected from adult mice captured close to a small cornfield, with titers of 1:40 and 1:320 neutralizing units/ml. The inoculation with clarified material from the peritoneum and testicles of the mouse with the highest level of OPV-neutralizing antibodies in chicken embryo fibroblast (CEF) monolayers induced the formation of typical poxvirus cytopathic effects after 72 hours. This isolate, named Mariana virus (MARV), was further multiplied in BSC-40 cells and purified in order to perform biological and molecular characterizations. Ecological and epidemiological data Our investigation was performed in a BV outbreak-affected area around Mariana County (20u229400S - 43u249570W), in Minas Gerais State, Brazil (Figure 1). The region is localized in a biome transition area, between the Mata Atlaˆntica (a tropical rainforest biome on the Brazilian Atlantic coast) and the Cerrado, which features fragments of deciduous seasonal forest alternated with savannas. The region’s altitude is approximately 720 m, and it has a subtropical climate with average temperatures between 15–18uC. The outbreak occurred during the dry season, which generally lasts four months. The region presents accentuated anthropogenic disturbances, with several small plantations (sugar cane, coffee and corn) and livestock areas. Debris and garbage could be observed in the BV-affected properties and PdRs, and their excrements were also observed. In some cases, portions of forest marked the limit of the backyards, corrals and pastures, and wild animals including mice, rats, little spotted cats, capybaras, red-legged siriemas, opossums, marmosets and cattle egrets were frequently sighted around the properties. Figure 1. Location and phytoecological characteristics of Mariana county BV outbreak area. The outbreak region is located in a biome transition area, between the Mata Atlaˆntica (Atlantic rainforest) and the Cerrado (Savanna), with accentuated anthropogenic disturbance. doi:10.1371/journal.pone.0007428.g001 Figure 1. Location and phytoecological characteristics of Mariana county BV outbreak area. The outbreak region is located in a biome transition area, between the Mata Atlaˆntica (Atlantic rainforest) and the Cerrado (Savanna), with accentuated anthropogenic disturbance. doi:10.1371/journal.pone.0007428.g001 October 2009 \| Volume 4 \| Issue 10 \| e7428 PLoS ONE \| www.plosone.org 2 BV: VACV Circulation in PdRs Figure 2. Immunofluorescence assay of infected BSC-40 cells revealed that MARV-M, MARV-H and MARV-B are OPV. BSC-40 cells were infected with MARV isolates at a MOI of 3. After 24 h, an anti- OPV antibody revealed the presence of the B5R protein which was punctually distributed, especially in cytoplasmic membranes and the Golgi complex, similarly to the pattern observed in VACV-WR-infected cells. The DAPI staining highlights the DNA contained in viral factories (white arrows). doi:10.1371/journal.pone.0007428.g002 Exanthematic infections were notified in several small properties localized within the analyzed area. No outbreak had ever been declared in this region previously. Typical VACV lesions were observed on cattle’s teats and on milkers’ hands and arms. Although serological and molecular tests confirmed human and bovine infection by VACV, other farm animals, such as dogs, pigs, cats and horses were OPV-seronegative based on plaque reduction neutralizing tests (PRNT). Viral biological and phylogenetic analysis In order to investigate whether MARV was, in fact, the etiological agent of the local BV outbreak, viruses were also isolated from a human vesicle and from a bovine scab, and used for biological and molecular comparisons. The murine isolate was designated MARV-M; the bovine, MARV-B; and the human, MARV-H. Immunofluorescence microscopy from BSC-40 cells infected with MARV isolates (Figure 2) revealed the presence of the OPV B5R protein which was punctually distributed, especially in the cytoplasmic membranes and the Golgi complex, similarly to the pattern observed in VACV-WR infected cells. The DAPI staining highlighted the DNA contained in viral factories (white arrows). The inoculation of MARV isolates in the chorioallantoic membrane (CAM) of embryonated chicken eggs caused the appearance of white pocks, indicative of VACV infection, as seen in Figure 3-A. No difference was observed between MARV isolates and VACV-WR in the immunofluorescence and CAM inoculation assays. Figure 2. Immunofluorescence assay of infected BSC-40 cells revealed that MARV-M, MARV-H and MARV-B are OPV. BSC-40 cells were infected with MARV isolates at a MOI of 3. After 24 h, an anti- OPV antibody revealed the presence of the B5R protein which was punctually distributed, especially in cytoplasmic membranes and the Golgi complex, similarly to the pattern observed in VACV-WR-infected cells. The DAPI staining highlights the DNA contained in viral factories (white arrows). doi:10.1371/journal.pone.0007428.g002 respectively. Amplicons were directly sequenced in both orienta- tions. The alignment of the tk, vgf and ha sequences obtained from MARV-M, MARV-B and MARV-H displayed perfect homology; therefore, all isolates were considered identical (MARV) according to phylogenetic parameters. When compared to nucleotide sequences available in the GenBank databases using the BLASTN program, the tk and vgf genes from MARV were highly similar to homologous genes from other VACV strains. Optimal alignment showed similarity rates of up to 99.5% between MARV and VACV–WR genes and minimal differences from nucleic acid substitutions. The coding region of the ha gene was analyzed by alignment with similar sequences from other OPV isolates deposited in GenBank. The MARV ha inferred amino acid sequence contained a signature deletion (Figure 4-A) also present in the sequences of other Brazilian VACV strains, such as Arac¸atuba virus (ARAV), Cantagalo virus (CTGV), Serro virus (SV2), GuaraniP2 virus (GP2V), Muriae virus (MURV) and others, but absent in GuaraniP1 virus (GP1V), Belo Horizonte virus (VBH), BeAn58058 virus (BAV) and SPAn232 virus (SAV). Viral biological and phylogenetic analysis Although the deletion is also present in the VACV-Instituto respectively. Amplicons were directly sequenced in both orienta- tions. The alignment of the tk, vgf and ha sequences obtained from MARV-M, MARV-B and MARV-H displayed perfect homology; therefore, all isolates were considered identical (MARV) according to phylogenetic parameters. When compared to nucleotide sequences available in the GenBank databases using the BLASTN program, the tk and vgf genes from MARV were highly similar to homologous genes from other VACV strains. Optimal alignment showed similarity rates of up to 99.5% between MARV and VACV–WR genes and minimal differences from nucleic acid substitutions. The coding region of the ha gene was analyzed by alignment with similar sequences from other OPV isolates deposited in GenBank. The MARV ha inferred amino acid sequence contained a signature deletion (Figure 4-A) also present in the sequences of other Brazilian VACV strains, such as Arac¸atuba virus (ARAV), Cantagalo virus (CTGV), Serro virus (SV2), GuaraniP2 virus (GP2V), Muriae virus (MURV) and others, but absent in GuaraniP1 virus (GP1V), Belo Horizonte virus (VBH), BeAn58058 virus (BAV) and SPAn232 virus (SAV). Although the deletion is also present in the VACV-Instituto One-step growth curve assays in BSC-40 cells showed that MARV-B, MARV-H and MARV-M presented a similar replica- tion profile, but statistically distinct from that observed in VACV- WR infected cells (p,0.04) (Figure 3-C). While MARV isolates presented a growth rate of approximately 1.5 log, VACV-WR titers increased 2.0 log after 24 hours of infection. Additionally, plaque phenotype comparisons showed that MARV isolates induced the formation of small plaques in BSC-40 cells (average diameter: 0.6 mm), different from the large plaques formed by VACV-WR (average diameter: 1.4 mm) (Figure 3-B). One-step growth curve assays in BSC-40 cells showed that MARV-B, MARV-H and MARV-M presented a similar replica- tion profile, but statistically distinct from that observed in VACV- WR infected cells (p,0.04) (Figure 3-C). While MARV isolates presented a growth rate of approximately 1.5 log, VACV-WR titers increased 2.0 log after 24 hours of infection. Additionally, plaque phenotype comparisons showed that MARV isolates induced the formation of small plaques in BSC-40 cells (average diameter: 0.6 mm), different from the large plaques formed by VACV-WR (average diameter: 1.4 mm) (Figure 3-B). Viral biological and phylogenetic analysis Based on the ha nucleotide sequences from MARV and other OPV, we construct- ed an evolutionary tree employing the MEGA 3.1 program, which clustered MARV with several VACV isolates (Figure 4-B). Epidemiological and phylogenetic investigations of OPV have been targeting the ha nucleotide and amino acid sequences to identify viral species and estimate their evolutionary relationships [16,29,30]. Recently, rat-to-elephant and rat-to-human CPXV transmission were described [29,30], on the basis of the perfect homology of the ha gene open reading frame (ORF) of CPXV isolates from distinct hosts. Therefore, based on this model, we decided to analyze the ha gene and the vgf and tk conserved genes in parallel with biological assays to characterize and compare the murine, bovine and human MARV isolates. The analysis of tk, vgf and ha sequences from MARV isolates displayed perfect homology not only in their respective ORFs but also in the entire nucleotide sequences analyzed. The genetic similarity observed among these samples was corroborated by results obtained in one-step curve and plaque phenotype assays, indicating that the three isolates were in fact from a single VACV strain that had circulated among different hosts during the BV outbreak in Mariana County. Viral biological and phylogenetic analysis OPV timidine kinase (tk), virus growth factor (vgf) and hemmaglutinin (ha) genes from MARV isolates were submitted to PCR amplification, generating 528-, 381- and 960-bp fragments, PLoS ONE \| www.plosone.org October 2009 \| Volume 4 \| Issue 10 \| e7428 3 BV: VACV Circulation in PdRs Figure 3. MARV-M, MARV-B and MARV-H induced the formation of white pocks in CAM and presented a similar profile in plaque phenotype and one-step growth curve assays. (A) CEF monolayers infected with MARV isolates were harvested after 72 h and inoculated onto the CAM of embryonated chicken eggs. Typical VACV white pocks were observed in inoculated CAM after 72 hours. (B) MARV isolates presented small plaques in BSC-40 cells (average diameter: 0.6 mm), distinct from the prototypical VACV-WR plaque phenotype (average diameter: 1.4 mm). (C) One-step growth curve assays in BSC-40 cells showed that MARV-B, MARV-H and MARV-M present a similar replication profile, which was statically distinct from that observed for VACV-WR (p,0.04). doi:10.1371/journal.pone.0007428.g003 Figure 3. MARV-M, MARV-B and MARV-H induced the formation of white pocks in CAM and presented a similar profile in plaque phenotype and one-step growth curve assays. (A) CEF monolayers infected with MARV isolates were harvested after 72 h and inoculated onto the CAM of embryonated chicken eggs. Typical VACV white pocks were observed in inoculated CAM after 72 hours. (B) MARV isolates presented small plaques in BSC-40 cells (average diameter: 0.6 mm), distinct from the prototypical VACV-WR plaque phenotype (average diameter: 1.4 mm). (C) One-step growth curve assays in BSC-40 cells showed that MARV-B, MARV-H and MARV-M present a similar replication profile, which was statically distinct from that observed for VACV-WR (p,0.04). doi:10.1371/journal.pone.0007428.g003 Oswaldo Cruz (IOC) strain, other vaccine strains, including Lister- Butantan and Malbran, the vaccine strains used in Brazil during the WHO campaign, lacked this signature [1]. Based on the ha nucleotide sequences from MARV and other OPV, we construct- ed an evolutionary tree employing the MEGA 3.1 program, which clustered MARV with several VACV isolates (Figure 4-B). present work is the first to clearly establish a link between a human-, a bovine- and a murine-VACV isolates that are temporally and spatially related. Oswaldo Cruz (IOC) strain, other vaccine strains, including Lister- Butantan and Malbran, the vaccine strains used in Brazil during the WHO campaign, lacked this signature [1]. PLoS ONE \| www.plosone.org Discussion All VACV strains from group 1 presented a deletion of 6 amino acids at position 251, but not all isolates that present such a deletion were clustered in this group. VACV-IOC, for example, despite presenting the deletion, was grouped with VACV-Ankara and VACV-Lister, both vaccine strains. Therefore, our data suggest that MARV shares the same probable origin as many Brazilian VACV strains isolated during BV outbreaks, and it is distinct from the vaccine strains used in Brazil during the smallpox eradication campaign, such as LST-BTT, Malbran and IOC [1]. models [31]. Our analysis of the ha gene revealed this phylogenetic segregation pattern, and MARV clustered with the first group. All VACV strains from group 1 presented a deletion of 6 amino acids at position 251, but not all isolates that present such a deletion were clustered in this group. VACV-IOC, for example, despite presenting the deletion, was grouped with VACV-Ankara and VACV-Lister, both vaccine strains. Therefore, our data suggest that MARV shares the same probable origin as many Brazilian VACV strains isolated during BV outbreaks, and it is distinct from the vaccine strains used in Brazil during the smallpox eradication campaign, such as LST-BTT, Malbran and IOC [1]. wolves, foxes and opossums have been seen in peridomestic buildings. Therefore, if these VACV strains are indeed primarily circulating among wild mammals, the isolation of MARV from a peridomestic mouse could represent a possible link between wildlife and BV-affected properties, as PdRs usually wander into the farms and their surroundings, maintaining contact with humans, cattle and wild animals alike [31]. Based on our data and on previously published works [16,18,19,27], including epidemiological studies of other OPV [12,28,29], we developed the hypothetical VACV transmission cycle presented in Figure 5. PdRs could be infected by wild animals in the farms’ surroundings after territorial fights [32], ingestion or aspiration of contaminated feces [27], consumption of contaminat- ed carcasses [12] or consumption of food containing the saliva of an infected animal [12]. VACV and CPXV shedding and transmission have been consistently demonstrated in mice and rats [27,28], which can partially explain the circulation and spread of VACV among PdRs. Some infected PdRs eventually return to the farms, introducing VACV into bovine and human populations. Discussion The present study introduces a new element in the complex and poorly understood VACV transmission chain: the peridomestic rodent. The search for VACV-infected rodents in BV-affected areas seemed like a rational strategy, since (i) such animals have been described as CPXV reservoirs in Europe, promoting viral transmission to humans, cats and zoo animals [6]; (ii) rats and mice are frequently sighted in BV-affected farms and are in constant contact with wildlife, humans and farm animals [20,21]; and (iii) laboratory studies showed that PdRs can shed and transmit OPV by direct contact with contaminated excrement [27,28]. Although some VACV strains had consistently been isolated and re-isolated from Brazilian forests using mice as sentinel animals [22], we describe for the first time the isolation of a VACV strain from a Mus musculus mouse captured in the context of a BV outbreak. Previous phylogenetic analysis of conserved and variable OPV genes showed that some rodent/forest VACV isolates cluster with specific zoonotic/bovine VACV isolates [13,14]. However, the Trindade et al. [13] and Drumond et al. [14] demonstrated that there are two genetically distinct VACV groups circulating in Brazil. The first is composed of ARAV, CTGV, GP2V, SV2, PSTV and other strains, while group 2 includes BAV, SAV, GP1V and VBH strains. Interestingly, this genetic polymorphism is reflected in the VACV strains’ virulence profiles in Balb/c mouse PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org PLoS ONE \| www.p October 2009 \| Volume 4 \| Issue 10 \| e7428 PLoS ONE \| www.plosone.org 4 BV: VACV Circulation in PdRs Figure 4. MARV is grouped phylogenetically with several Brazilian VACV strains isolated during BV outbreaks, but is segregated from VACV vaccine strains. (A) Amino acid inferred sequence of the MARV hemagglutinin (HA) gene and comparison to the homologous sequence from several OPV. The box with an asterisk indicates the deletion region conserved in the sequences of MARV, several Brazilian field VACV samples and the IOC vaccine strain. (B) Phylogenetic tree constructed based on the nucleotide sequence of the OPV ha gene. The ha tree shows MARV grouping with VACV field samples. Despite the presence of the 6 aa deletion, the IOC strain grouped with other vaccine strains. Discussion Nucleotide sequences were obtained from GenBank (VACV-ARAV:AY523994; VACV-LOR:DQ810281; VACV-RIA:DQ810280; VACV-CTGV:AF229247; VACV-PSTV:DQ070848; VACV- MURV:DQ247770; VACV-GP2V:DQ206437; VACV-Acambis clone3:AY313848; VACV-DUKE:DQ439815; VACV-MALBRAN:AY146624; VACV- SV2:EF063677; Rabbitpox:AF37511; VACV-LST:AY678276; VACV-SPAn232:DQ222922; VACV-BeAn58058:DQ206442; VACV-GP1V:DQ206436; VACV-WR: AY243312; VACV-Ankara:U944848; VACV-VBH:DQ206435; VACV-Lister-Butantan:EF175985; VACV-IOC:AF225248; VACV- Bfl3906:AF375077; VACV-Bfl81:AF375078; HSPV:DQ792504; MPXV-ZAIRE:DQ011155; MPXV-mpv3945:AF375098; MPXV-Sierra Leone:AY741551; CPXV-Germany: DQ437593; CPXV-cowHA12:AY902253; CPXV-GRI90:AF375087; VARV:DQ437589. Bootstrap confidence intervals are shown on branches (1,000 replicates). doi:10.1371/journal.pone.0007428.g004 Figure 4. MARV is grouped phylogenetically with several Brazilian VACV strains isolated during BV outbreaks, but is segregated from VACV vaccine strains. (A) Amino acid inferred sequence of the MARV hemagglutinin (HA) gene and comparison to the homologous sequence from several OPV. The box with an asterisk indicates the deletion region conserved in the sequences of MARV, several Brazilian field VACV samples and the IOC vaccine strain. (B) Phylogenetic tree constructed based on the nucleotide sequence of the OPV ha gene. The ha tree shows MARV grouping with VACV field samples. Despite the presence of the 6 aa deletion, the IOC strain grouped with other vaccine strains. Nucleotide sequences were obtained from GenBank (VACV-ARAV:AY523994; VACV-LOR:DQ810281; VACV-RIA:DQ810280; VACV-CTGV:AF229247; VACV-PSTV:DQ070848; VACV- MURV:DQ247770; VACV-GP2V:DQ206437; VACV-Acambis clone3:AY313848; VACV-DUKE:DQ439815; VACV-MALBRAN:AY146624; VACV- SV2:EF063677; Rabbitpox:AF37511; VACV-LST:AY678276; VACV-SPAn232:DQ222922; VACV-BeAn58058:DQ206442; VACV-GP1V:DQ206436; VACV-WR: AY243312; VACV-Ankara:U944848; VACV-VBH:DQ206435; VACV-Lister-Butantan:EF175985; VACV-IOC:AF225248; VACV- Bfl3906:AF375077; VACV-Bfl81:AF375078; HSPV:DQ792504; MPXV-ZAIRE:DQ011155; MPXV-mpv3945:AF375098; MPXV-Sierra Leone:AY741551; CPXV-Germany: DQ437593; CPXV-cowHA12:AY902253; CPXV-GRI90:AF375087; VARV:DQ437589. Bootstrap confidence intervals are shown on branches (1,000 replicates). doi:10.1371/journal.pone.0007428.g004 Figure 4. MARV is grouped phylogenetically with several Brazilian VACV strains isolated during BV outbreaks, but is segregated from VACV vaccine strains. (A) Amino acid inferred sequence of the MARV hemagglutinin (HA) gene and comparison to the homologous sequence from several OPV. The box with an asterisk indicates the deletion region conserved in the sequences of MARV, several Brazilian field VACV samples and the IOC vaccine strain. (B) Phylogenetic tree constructed based on the nucleotide sequence of the OPV ha gene. The ha tree shows MARV grouping with VACV field samples. Despite the presence of the 6 aa deletion, the IOC strain grouped with other vaccine strains. Nucleotide sequences were obtained from GenBank (VACV-ARAV:AY523994; VACV-LOR:DQ810281; VACV-RIA:DQ810280; VACV-CTGV:AF229247; VACV-PSTV:DQ070848; VACV- MURV:DQ247770; VACV-GP2V:DQ206437; VACV-Acambis clone3:AY313848; VACV-DUKE:DQ439815; VACV-MALBRAN:AY146624; VACV- SV2:EF063677; Rabbitpox:AF37511; VACV-LST:AY678276; VACV-SPAn232:DQ222922; VACV-BeAn58058:DQ206442; VACV-GP1V:DQ206436; VACV-WR: AY243312; VACV-Ankara:U944848; VACV-VBH:DQ206435; VACV-Lister-Butantan:EF175985; VACV-IOC:AF225248; VACV- Bfl3906:AF375077; VACV-Bfl81:AF375078; HSPV:DQ792504; MPXV-ZAIRE:DQ011155; MPXV-mpv3945:AF375098; MPXV-Sierra Leone:AY741551; CPXV-Germany: DQ437593; CPXV-cowHA12:AY902253; CPXV-GRI90:AF375087; VARV:DQ437589. Bootstrap confidence intervals are shown on branches (1,000 replicates). doi:10.1371/journal.pone.0007428.g004 models [31]. Our analysis of the ha gene revealed this phylogenetic segregation pattern, and MARV clustered with the first group. PLoS ONE \| www.plosone.org Discussion On the other hand, peridomestic mice and rats could be infected with VACV after coming into contact with bovine/human scab fragments, contaminated milk (Abraha˜o & Oliveira et al., unpublished data) or fomites (bulk tanks, cloth contaminated with blood and tissue from bovine lesions and materials associated with milking procedures). Subsequently, infected PdRs could spread VACV to wild animals when they are predated upon [1], via their p g The occurrence of VACV circulation among wild animals was supported by viral isolations in the 1960s and 1970s [22–24] and by recent serological studies that showed a high prevalence of anti- OPV immunoglobulins from mammalians in Brazilian wildlife, including rodents, macaques, grey-foxes and coatis [25,26]. As mentioned, some BV outbreaks were temporally and spatially distant from other outbreaks, in areas with little or no pecuary activity. In these specific cases, it is believed that wildlife was the original source of VACV circulation. Viruses could then reach rural environments when their wild hosts eventually leave their wild habitat and venture to the boundaries of farms in search of food (mainly in plantations) or after the deforestation of their habitat. Nonetheless, the presence of wild animals is usually restricted to the properties’ borders, with their presence in places like corrals and the main house being quite atypical [31]. Only PLoS ONE \| www.plosone.org October 2009 \| Volume 4 \| Issue 10 \| e7428 5 BV: VACV Circulation in PdRs Figure 5. A hypothetical model of the VACV transmission cycle. Peridomestic rodents could promote the transmission of VACV between wild animals and cattle or humans, since they circulate both in farm buildings and their surroundings. This diagram was proposed based on published epidemiological and laboratory data on VACV and the behavioral characteristics of Brazilian rodents and wild animals. Solid lines indicate experimentally determined data, and dashed lines represent hypothetical propositions still under investigation. doi:10.1371/journal.pone.0007428.g005 Figure 5. A hypothetical model of the VACV transmission cycle. Peridomestic rodents could promote the transmission of VACV between wild animals and cattle or humans, since they circulate both in farm buildings and their surroundings. This diagram was proposed based on published epidemiological and laboratory data on VACV and the behavioral characteristics of Brazilian rodents and wild animals. Solid lines indicate experimentally determined data, and dashed lines represent hypothetical propositions still under investigation. doi:10.1371/journal.pone.0007428.g005 (Amphotericin B, Crista´lia, Sa˜o Paulo, Brazil), 500 U/ml penicillin and 50 mg/ml gentamicin (Schering-Plough, Brazil). Discussion The VACV strain Western Reserve (VACV-WR), kindly provided by Dr C. Jungwirth (Universitat Wurzburg, Germany), and the VACV- MARV isolates were grown in BSC-40 cells and purified in a sucrose gradient as previously described [34]. feces and carcasses or during fights. Although less probable, the possibility of direct VACV transmission between wild animals and humans/cattle cannot be discarded. Additional studies are required to elucidate the complex VACV transmission dynamics, especially when considering the virus’s large host range. We believe that the presence of VACV-infected PdRs in affected properties could represent both the cause and/or the consequence of BV outbreaks. Monitoring of PdRs could be used to predict BV outbreaks or to indicate the risk of VACV introduction into wild environments. feces and carcasses or during fights. Although less probable, the possibility of direct VACV transmission between wild animals and humans/cattle cannot be discarded. Additional studies are required to elucidate the complex VACV transmission dynamics, especially when considering the virus’s large host range. We believe that the presence of VACV-infected PdRs in affected properties could represent both the cause and/or the consequence of BV outbreaks. Monitoring of PdRs could be used to predict BV outbreaks or to indicate the risk of VACV introduction into wild environments. Sample preparation and viral isolation Scabs and murine organs were macerated in phosphate buffered saline (PBS) (0.1 g sample/0.9 ml PBS) using a homogenizer (Politron, Switzerland) and clarified by centrifugation at 20006g for 3 minutes. Vesicular liquid swabs were added to 200 mL of PBS and centrifuged at 20006g for 3 min. Viral isolation was performed by inoculation of 200 mL of processed samples onto CEF monolayers, followed by 72 hours of incubation. Monolayers presenting cytopathic effects were harvested and inoculated onto the chorioallantoic membrane (CAM) of embryonated chicken eggs and incubated at 37uC for 72 hours to analyze the appearance of typical VACV white pocks [35]. Viral infectivity assays BSC-40 cells were grown to a density of 56105 cells per well on a 6-well culture dish and then infected with MARV isolates and VACV-WR. Infections were carried out at a MOI of 10 for 3, 6, 12, 24, 36 and 48 h. The infected monolayers were then harvested and titrated in BSC-40 cells for one-step growth curve assays. MARV isolates and VACV-WR plaque phenotype assays were carried out in parallel, in BSC-40 cells at 0.01 MOI and incubated for 48 h. Statistical analysis using the two-tailed Student’s T-test was performed with ABI prism 3.0 software. Immunofluorescence microscopy BSC-40 cells were grown to a density of 4.56104 cells on sterile 13 mm coverslips and infected with MARV isolates or VACV- WR at a multiplicity of infection (MOI) of 3. After 24 hours, the cells were fixed with 4% paraformaldehyde solution and incubated with an anti-OPV B5R antibody, followed by incubation with secondary rhodamine-conjugated anti-mouse antibody. After incubation with antibodies, cells were stained with DAPI for 10 minutes. Images of fluorescently-labeled cells were obtained with an Olympus BX51 immunofluorescence microscope. Clinical sample collection and rodent capture Samples of vesicles and crusts (dried scabs) from cattle teats and milkers’ hands were collected during a BV outbreak in Mariana County, Brazil (2005), using 1 ml insulin syringes and 0.45 mm613 mm needles, cotton swabs or a pair of tweezers. Serum samples were collected from milkers, cows and other domestic animals, such as dogs, cats, pigs and horses and used in neutralization assays [33]. A written consent was provided by the inhabitants of the analyzed rural properties before participating in this study. During the same outbreak, a total of 20 slide-door traps for rodents were assembled using a mixture of cod liver oil, peanut butter, banana and bacon as bait. Traps were positioned at several points in and around the farms, including in barns, silos, corrals, food warehouses, grain plantations and the boundaries of adjacent forests. The captured rodents were anesthetized for blood collection and subsequently sacrificed by cervical dislocation. The brain, liver, lungs, heart, gonads, intestine, trachea, peritoneum, stomach and spleen were collected and stored until viral isolation experiments could be conducted. The rodent captures were performed with the written permission of the Brazilian Environmental Surveillance Institute (IBAMA), and all experiments were approved by the Ethical and Animals Use Committee at the Universidade Federal de Minas Gerais, Brazil. References Zona da Mata region, Minas Gerais. Arquivo brasileiro de medicina veterinaria e zootecnia 57: 423–429. 1. Fenner F, Henderson DA, Arita I, Jezek A, Ladnyi ID (1988) Smallpox and its eradication. Geneva: World Health Organization Press. 49 p. 1. Fenner F, Henderson DA, Arita I, Jezek A, Ladnyi ID (1988) Smallpox and its eradication. Geneva: World Health Organization Press. 49 p. 2. International Committee on Taxonomy of Viruses(ICTV) (2009) Available: http://www.ncbi.nlm.nih.gov/ICTVdb/. Accessed: 2009 May 14. 20. Silva-Fernandes AT (2008) Bovine Vaccinia of the Rio de Janeiro State: epidemiology and caractherization of Vaccinia virus samples. PhD dissertation, Universidade Federal do Norte Fluminense. 3. Damon I (2007) Poxviridae and their replication. Fields Virology. New York: Raven Press. pp 2079–2081. 21. Madureira M, Study of the Bovine Vaccine Outbreaks in Minas Gerais State (2009) PhD dissertation, Escola de Veterina´ria, Universidade Federal de Minas Gerais. 4. Parrino J, Graham BS (2006) Smallpox vaccines: Past, present, and future. J Allergy Clin Immunol 118: 1320–1326. 5. Heymann DL, Aylward RB (2006) Mass vaccination: when and why. Curr Top Microbiol Immunol 304: 1–16. 22. Lopes de S, Lacerda JP, Fonseca IE, Castro DP, Forattini OP, et al. (1965) Cotia Virus: a new agent isolated from sentinel mice in Sa˜o Paulo, Brazil. Am J Trop Med Hyg 14: 156–157. 6. Haenssle HA, Kiessling J, Kempf VA, Fuchs T, Neumann C, et al. (2006) Orthopoxvirus infection transmitted by a domestic cat. J Am Acad Dermatol 54: S1–4. 23. da Fonseca FG, Trindade GS, Silva RL, Bonjardim CA, Ferreira PC, et al. (2002) Characterization of a vaccinia-like virus isolated in a Brazilian forest. J Gen Virol 83: 223–228. 7. Reynolds MG, Yorita KL, Kuehnert MJ, Davidson WB, Huhn GD, et al. (2006) Clinical manifestations of human monkeypox influenced by route of infection. J Infect Dis 194: 773–780. 24. Fonseca FG, Lanna MC, Campos MA, Kitajima EW, Peres JN, et al. (1998) Morphological and molecular characterization of the poxvirus BeAn 58058. Arch Virol 143: 1171–1186. 8. Damaso CR, Esposito JJ, Condit RC, Moussatche N (2000) An emergent poxvirus from humans and cattle in Rio de Janeiro State: Cantagalo virus may derive from Brazilian smallpox vaccine. Virology 277: 439–449. 25. Guedes MIM, Formiga M, Trindade GS, Leite JA, Drumond BP, et al. (2005) Occurrence of Orthopoxvirus Infection in Native Mamal Species in Brazil. Brazilian Society of Virology Congress. . 9. Trindade GS, Lobato ZI, Drumond BP, Leite JA, Trigueiro RC, et al. References (2006) Short report: Isolation of two vaccinia virus strains from a single bovine vaccinia outbreak in rural area from Brazil: Implications on the emergence of zoonotic orthopoxviruses. Am J Trop Med Hyg 75: 486–490. 26. Simonetti BR, Abreu DC, Simonetti JP, Gonc¸alves MCR, Silva MEV, et al. (2007) Animal infections by vaccinia-like viruses in the state of Rio de Janeiro: Northwestern Region. Virus Rev Res 12: 1–12. 10. Singh RK, Hosamani M, Balamurugan V, Bhanuprakash V, Rasool TJ, et al. (2007) Buffalopox: an emerging and re-emerging zoonosis. Anim Health Res Rev 8: 105–114. 27. Ferreira JM, Abrahao JS, Drumond BP, Oliveira FM, Alves PA, et al. (2008) Vaccinia virus: shedding and horizontal transmission in a murine model. J Gen Virol 89: 2986–2991. 11. Hutson CL, Olson VA, Carroll DS, Abel JA, Hughes CM, et al. (2009) A prairie dog animal model of systemic orthopoxvirus disease using West African and Congo Basin strains of monkeypox virus. J Gen Virol 90: 323–333. 28. Maiboroda AD (1982) Experimental infection of Norvegian rats (Rattus norvegicus) with ratpox virus. Acta Virol 26: 288–291. 29. Kurth A, Wibbelt G, Gerber HP, Petschaelis A, Pauli G, et al. (2008) Rat-to- elephant-to-human transmission of cowpox virus. Emerg Infect Dis 14: 670–671. g yp J 12. Fenner F, Wittek R, Dumbell KR (1989) The orthopoxviruses. New York, N.Y: Academic Press. 13. Trindade GS, Emerson GL, Carroll DS, Kroon EG, Damon IK (2007) Brazilian vaccinia viruses and their origins. Emerg Infect Dis 13: 965–972. 30. Ninove L, Domart Y, Vervel C, Voinot C, Salez N, et al. (2009) Cowpox virus transmission from pet rats to humans, France. Emerg Infect Dis 15: 781–784. 14. Drumond BP, Leite JA, da Fonseca FG, Bonjardim CA, Ferreira PC, et al. (2008) Brazilian Vaccinia virus strains are genetically divergent and differ from the Lister vaccine strain. Microbes Infect 10: 185–197. 31. Ferreira JM, Drumond BP, Guedes MI, Pascoal-Xavier MA, Almeida-Leite CM, et al. (2008) Virulence in murine model shows the existence of two distinct populations of Brazilian Vaccinia virus strains. PLoS ONE 3: 3038–3043. populations of Brazilian Vaccinia virus strains. PLoS ONE 3: 3038 15. Leite JA, Drumond BP, de Souza Trindade G, Bonjardim CA, Ferreira PC, et al. (2007) Brazilian Vaccinia virus strains show genetic polymorphism at the ati gene. Virus Genes 35: 531–539. 32. Washington State Departament of Ecology (WSDE) (2008) Available: http:// www.ecy.wa.gov/. Accessed 2009 may 14. 33. Acknowledgments We thank Joa˜o Rodrigues dos Santos, Angela Sana Lopes, Ilda Gamma, and colleagues from Laborato´rio de Vı´rus (ICB-UFMG /Escola de Veterina´ria) for their excellent technical support, and Dr. Bernard Moss (National Institute of Allergy and Infectious Diseases, USA) for anti-B5R antibodies. Amplification of tk, vgf and ha genes and phylogenetic analyses Amplification of tk, vgf and ha genes and phylogenetic analyses y Primers based on the tk and vgf nucleotide sequence of VACV– WR were produced as described by Fonseca et al. [24]. The ha coding sequence was amplified using primers EACP1 and EACP2, as described by Ropp et al. [36]. The MARV PCR-amplified tk, vgf and ha fragments were directly sequenced in both orientations and in triplicate (Mega-BACE sequencer, GE Healthcare, Buckinghamshire, UK). The sequences were aligned with previously published OPV sequences from GenBank using the ClustalW method, and were manually aligned using MEGA software version 3.1 (Arizona State University, Phoenix, AZ, USA). The Modeltest software was used determine which model of evolution was most appropriate for our analysis [37]. A phylogenetic tree based on the ha gene sequence was constructed using the maximum parsimony method with 1,000 bootstrap replicates implemented by MEGA 3.1. The MARV tk Author Contributions Conceived and designed the experiments: JSA MICG GST FGDF BEFM ZIPL ATSF PCPF CAB EGK. Performed the experiments: JSA MICG GST FGDF RKC BEFM ZIPL ATSF GOR LSL. Analyzed the data: JSA GST FGDF EGK. Contributed reagents/materials/analysis tools: PCPF CAB EGK. Wrote the paper: JSA GST FGDF EGK. p J GST FGDF RKC BEFM ZIPL ATSF GOR LSL. Analyzed the data: JSA GST FGDF EGK. Contributed reagents/materials/analysis tools: PCPF CAB EGK. Wrote the paper: JSA GST FGDF EGK. GST FGDF EGK. Contributed reagents/materials/analysis tools: PCPF CAB EGK. Wrote the paper: JSA GST FGDF EGK. Cells and viruses Chicken embryo fibroblasts (CEF) and BSC-40 cells were grown at 37uC in Eagle’s minimum essential medium (MEM; GibcoBRL, Invitrogen, Carlsbad, California, USA) supplemented with 5% fetal calf serum (FCS, Cultilab, Brazil), 25 mg/ml fungizone PLoS ONE \| www.plosone.org October 2009 \| Volume 4 \| Issue 10 \| e7428 October 2009 \| Volume 4 \| Issue 10 \| e7428 6 BV: VACV Circulation in PdRs (GQ226042), vgf (GQ226041) and ha (GQ226040) sequences were deposited in GenBank. (GQ226042), vgf (GQ226041) and ha (GQ226040) sequences were deposited in GenBank. References Abdalrhman I, Gurt I, Katz E (2006) Protection induced in mice against a lethal orthopox virus by the Lister strain of vaccinia virus and modified vaccinia virus Ankara (MVA). Vaccine 24: 4152–4160. 16. de Souza Trindade G, da Fonseca FG, Marques JT, Nogueira ML, Mendes LC, et al. (2003) Aracatuba virus: a vaccinialike virus associated with infection in humans and cattle. Emerg Infect Dis 9: 155–160. ( ) 34. Joklik WK (1962) The purification of four strains of poxvirus. Virology 18: 9–18. 34. Joklik WK (1962) The purification of four str g 17. Silva-Fernandes AT, Travassos CE, Ferreira JM, Abrahao JS, Rocha ES, et al. (2009) Natural human infections with Vaccinia virus during bovine vaccinia outbreaks. J Clin Virol 44: 308–313. g 17. Silva-Fernandes AT, Travassos CE, Ferreira JM, Abrahao JS, Ro 35. Overman JR, Tamm I (1957) Multiplication of vaccinia virus in the chorioallantoic membrane in vitro. Virology 3: 173–84. 36. Ropp SL, Jin Q, Knight JC, Massung RF, Esposito JJ (1995) PCR strategy for identification and differentiation of small pox and other orthopoxviruses. J Clin Microbiol 33: 2069–2076. J 18. Leite JA, Drumond BP, Trindade GS, Lobato ZI, da Fonseca FG, et al. (2005) J 18. Leite JA, Drumond BP, Trindade GS, Lobato ZI, da Fonseca FG, et al. (2005) Passatempo virus, a vaccinia virus strain, Brazil. Emerg Infect Dis 11: 1935–1938. Passatempo virus, a vaccinia virus strain, Brazil. Emerg Infect Dis 11: 1935–1938. 37. Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817–818. 19. Lobato ZIP, Trindade GS, Frois MCM, Ribeiro EBT, Dias GRC, et al. (2005) Outbreak of exantemal disease caused by Vaccinia virus in human and cattle in 19. Lobato ZIP, Trindade GS, Frois MCM, Ribeiro EBT, Dias GRC, et al. (2005) Outbreak of exantemal disease caused by Vaccinia virus in human and cattle in , , , , , (2 ) Outbreak of exantemal disease caused by Vaccinia virus in human and cattle in PLoS ONE \| www.plosone.org October 2009 \| Volume 4 \| Issue 10 \| e7428 7 PLoS ONE \| www.plosone.org
https://openalex.org/W3159810962	https://link.springer.com/content/pdf/10.1007/s00590-021-02990-6.pdf	English	null	The role of radiotherapy in adult soft tissues sarcoma of the extremities	European journal of orthopaedic surgery & traumatology	2,021	cc-by	10,301	Abstract Local management of adult soft tissue sarcoma of the extremities has evolved over the past decades. Until the 1970s, radical surgery (amputations) was the standard therapeutic procedure resulting in significant physical and psychological morbidity for the patients. In the present era, limb sparing surgery combined with radiotherapy represents the current standard of care for high grade and > 5 cm STSs. This approach guarantees high local control rate and function preservation. The aim of this paper is to summarize the current evidence for RT in STSs of the extremities. Outcomes, technical details (techniques, tim- ing, dose, volumes of treatment) and the emerging role of RT in the management of oligometastatic disease will be analysed. Finally, results of the recent clinical trials testing new scenarios in RT of STSs will be described. Keywords Soft tissue sarcoma · Radiotherapy · Extremities · Limbs · Review European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 https://doi.org/10.1007/s00590-021-02990-6 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 https://doi.org/10.1007/s00590-021-02990-6 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 https://doi.org/10.1007/s00590-021-02990-6 GENERAL REVIEW GENERAL REVIEW The role of radiotherapy in adult soft tissues sarcoma of the extremities Silvia Cammelli1,2 · Annalisa Cortesi3 · Milly Buwenge1,2 · Alice Zamagni1,2 · Martina Ferioli1,2 · Giulia Ghigi3 · Antonino Romeo3 · Alessio G. Morganti1,2 Received: 27 February 2021 / Accepted: 18 April 2021 / Published online: 6 May 2021 © The Author(s) 2021 * Silvia Cammelli silvia.cammelli2@unibo.it 1 Radiation Oncology, IRCCS Azienda Ospedaliero- Universitaria di Bologna, Bologna, Italy 2 Department of Experimental, Diagnostic and Specialty Medicine—DIMES, Alma Mater Studiorum University of Bologna, Bologna, Italy 3 Radiotherapy Unit, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola, Italy Role of radiotherapy on overall survival From the available literature, RT impact on survival is unclear. The two prospective randomized trials by Yang et al. [5] and Pisters et al. [6] did not demonstrate an impact of adjuvant RT on OS. Thereafter, several studies have investigated the role of RT on tumour LC, suggesting a positive effect [5, 6]. According to these evidences, the addition of RT to con- servative surgery is currently recommended in international guidelines [1], 2]. The combined modality is associated with a local recurrence rates lower than 15% [5–7]. Two subsequent retrospective studies from the surveil- lance, epidemiology, and end results (SEER) database [9, 10] have shown different conclusions. Two randomized studies compared conservative sur- gery alone versus surgery combined with adjuvant RT [5, 6]. Yang et al. reported results from 141 patients affected by STSs, 99 high-grade (G2-G3), and 50 low grade (G1). Patients were randomized to receive adjuvant external beam RT (EBRT) or not, after conservative surgery. With a median follow-up of 9.6 years, a statistically significant improvement in LC was recorded for high grade sarcomas treated with adjuvant EBRT vs surgery alone: 10-year LC were 100% vs. 78%, respectively (p = 0.003). A trend was recorded in low grade sarcomas in favour of patients treated with EBRT vs surgery alone: 10-year LC were 95% vs. 68%, respectively (p = 0.067). These data were confirmed in a recent update:[7]: after a median follow-up of 17.9 years, local recurrence rate was 25% following limb sparing sur- gery alone compared to 1.4% in those treated with adjuvant EBRT (p = 0.0001). f Kachare et al. [9] analysed 2606 high grade extremi- ties sarcoma patients divided in two cohorts, created using propensity score matching between irradiated and non- irradiated groups. In the final analysis, RT was associated with survival advantage in both univariate and multivariate analysis. Kohsy et al. [10] included 6960 patients in their analysis: in high-grade tumours, 3-year OS in patients who received RT and those who did not was 73% versus 63%, respectively (p < 0.001), supporting the benefit of RT on survival. i Although these data are encouraging and are in favour of a wide use of RT, they need to be interpreted with cau- tion. Role of radiotherapy on overall survival The retrospective study designs and the fact that SEER registry does not provide data that could have an impact on outcomes, such as the use of chemotherapy or information on RT technique, might have influenced the results. l Major literature data about overall survival in extremities STSs are described in Table 1. (p ) Pisters et al. randomized 164 patients to receive conserv- ative surgery + adjuvant brachytherapy (BRT) vs surgery alone. With a median follow-up of 76 months, patients with high-grade sarcomas treated with BRT presented statistically higher 5-year LC rates than patients with low grade lesions: 89% versus 66%, respectively (p = 0.0025). BRT had no impact on LC in patients with low-grade lesions (p = 0.49). Data from the largest retrospective study conducted by Jebsen et al. [8] (1093 adult patients) indicate a benefit from adjuvant RT regardless of the state of margins. The ben- efit was more pronounced in deep-seated and high-grade Pisters et al. randomized 164 patients to receive conserv- ative surgery + adjuvant brachytherapy (BRT) vs surgery alone. With a median follow-up of 76 months, patients with high-grade sarcomas treated with BRT presented statistically higher 5-year LC rates than patients with low grade lesions: 89% versus 66%, respectively (p = 0.0025). BRT had no impact on LC in patients with low-grade lesions (p = 0.49). Introduction Extremity and trunk STSs show about 60% of disease- specific survival at 10 years; local recurrence rates range from 20 to 30% and 30–50% of cases develop metastases (most frequently in the lungs). The incidence of distant metastases at diagnosis is about 10% and is more likely in patients with large, deep and high-grade lesions. Soft tissue sarcomas (STSs) are a rare and heterogeneous group of tumours originating from connective tissue, repre- senting 1% of all adult and 15% of paediatric malignancies [1]. Adult STSs originate mostly from the extremities (43%), followed by visceral (19%), retroperitoneum (15%), trunk (10%) and head and neck (9%) [1].f Radiotherapy (RT) plays an increasing role in the multi- modal treatment of soft tissue sarcomas. Technological evo- lutions like intensity-modulated, image-guided and stereo- tactic radiotherapy have considerably increased the potential of radiotherapy in the last 20 years, leading to significant improvements in patient management. In fact, they have allowed either dose escalation (with consequent improved efficacy) or reduction in toxicity (with consequent improved functional outcome). More than 50 different histological and molecular sub- types of STS have been described by the World Health Organization (WHO) among which the most common sub- types in adults are undifferentiated pleomorphic sarcoma, liposarcoma and leiomyosarcoma [2]. Prognosis depends on histological subtypes, grading, size, tumour location, pres- ence of distant metastases and other factors [2]. The primary aim of this paper is to summarize the state of the art about the role of RT on extremity STSs of adult. Furthermore, the impact of RT on local control (LC), overall survival (OS), outcomes in oligometastatic presentation, and future direction about RT application in clinical practice will be analysed. RT in bone sarcomas [3], sarcomas typical of paedi- atric and adolescent age (like rhabdomyosarcoma and Ewing sarcoma), sarcoma originating from visceral organs and head and neck, as well as sarcoma-like lesion (like (0121 3456789) 3 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 1584 dermatofibrosarcoma protuberans and desmoid fibromato- sis) will not be described in this review. tumours. In this group of patients, the risk of local recur- rence without RT was more than three times greater than that with RT. The 5-year local control rate was 28% without RT and 62% after RT for intralesional margin, 74% versus 81% for marginal margin and 87% vs. 93% after wide margin. Role of radiotherapy on local tumour control Major literature data about local control in extremities STSs are described in Table 1. Surgery is the mainstay of treatment for resectable STSs. Until the 1970s, the standard surgical procedure was amputations. Based on these evidence, international guidelines recom- mend addition of RT to limb sparing surgery in patients with high grade, large (> 5 cm), deep lesion. For low grade lesions, RT is usually reserved for patients with positive margins or local recurrence without prior RT. In selected cases, it can be useful in a preoperative setting even in Grade 1 lesion to help the surgeon to have adequate margins. In 1982, the prospective randomized trial conducted by Rosemberg et al. [4] compared patients to receive either amputation or limb sparing surgery plus adjuvant RT. Twenty-seven patients were randomized to receive limb- sparing resection and RT, 16 patients received amputation (randomization was 2:1). The authors found no statistically significant differences in OS (p = 0.99) and LC (p = 0.06) rates: 5-year OS and LC for amputation and limb-sparing surgery plus RT were 88% versus 83% and 100% versus 85%, respectively. 1 3 Role of combined chemo‑radiation therapy There is no consensus on the current role of combined radia- tion and chemotherapy for adult extremities STSs. Study results are conflicting about this topic, and consequently, it cannot be considered the standard treatment. Data from the largest retrospective study conducted by Jebsen et al. [8] (1093 adult patients) indicate a benefit from adjuvant RT regardless of the state of margins. The ben- efit was more pronounced in deep-seated and high-grade A meta-analysis published in 2008 found a statistically significant benefit in terms of both relapse-free survival and 1 3 1 3 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 1585 al of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 Table 1 Summary of outcome from major studies EBRT: external beam radiotherapy, LSS: limb sparing surgery References Study design Setting No. of patients Median F/U (years) 5 year LC (% 5 year DFS (%) 5 year OS (%) Yang et al. [5] Randomized LSS alone 71 17.9 78 (10 year) High grade 68 (10 year) Low grade Not reported 74 (10 year) LSS + post-op EBRT 70 100 (10 year) 95 (10 year) 75 (10 year) O'Sullivan et al. [17] Randomized Pre-op EBRT 94 3.3 p = 0.791 Not reported p = 0.791 Post-op EBRT 96 Jebsen et al. [8] Retrospective LSS alone 598 5 28 (Intralesional margins) 74 (Marginal margins) 87 (Marginal margins) Not reported Not reported LSS + EBRT (pre or post) 381 62 (Intralesional margins) 81 (Marginal margins) 93 (marginal margins) Pisters et al. [6] Prospective randomized LSS alone 164 6.3 69 81 Not reported LSS + BRT 82 84 Sampath et al. [19] Retrospective Pre-op EBRT 293 5.3 93 Not reported 65 Post-op EBRT 528 87 60 Wang et al. [27] Prospective phase II Pre-op EBRT + LSS 79 3.6 94 (2 year) 61.5 (2 year) 80.6 (2 year) Koshy et al. [10] Retrospective LSS alone 3.689 Not reported Not reported Not reported 73 (3 year) LSS + EBRT (pre or post) 3.271 63 (3 year) Alektiar et al. [21] Retrospective LSS + EBRT (pre or post) 41 3 84 Not reported 64 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 1586 OS in the adjuvant setting [11]. A randomized trial pub- lished afterwards [12] did not confirm these results show- ing no benefit from adjuvant EBRT with doxorubicin and ifosfamide in both relapse free survival and OS. Role of combined chemo‑radiation therapy was associated with greater incidence of acute wound com- plications compared to post-operative RT, 35% versus 17%, respectively (p = 0.01). On the other hand, late side effects like fibrosis, edema and joint stiffness were more common in patients receiving post-operative RT. The revised data at prolonged follow-up (5 years) confirmed these results [18]. The literature data about neoadjuvant setting are con- troversial as well. Only one randomized trial comparing pre-operative chemotherapy and surgery with or without radiotherapy versus surgery with or without RT alone was done and did not demonstrate any advantage from adding systemic treatment [13]. Another study found a significant benefit in overall survival for neoadjuvant chemotherapy plus hyperthermia [14].lf i Sampath et al. published the largest retrospective com- parison between pre-operative and post-operative RT [19] conducted on 821 patients. The primary aim was to assess the impact of RT sequencing with surgery on OS and LC. With a median follow-up of 5.2 years, better outcomes on local and distant control were found in the pre-operative setting. Five-year local failure free survival was 93% and 87% in the pre-operative and in the post-operative group, respectively (p < 0.05). Five-year overall survival was 65% and 60% in the pre-operative and post-operative group, respectively (p < 0.01). Despite the retrospective nature of this study, the authors suggested the hypothesis that pre-op RT may provide patients with the opportunity to improve long-term survival. As result of these conflicting data, differences in clinical practice regarding use of adjuvant and/or neoadjuvant chem- otherapy worldwide are relevant. It can be proposed as an option to high risk cases (high grade, deep, > 5 cm tumour) with sensitive histological types. The decision should be shared in a multidisciplinary setting taking into account the potential increased toxicity of the combined treatment. About this topic, literature data [15] reported high rate of toxicity (5% grade 5, 83% grade 4) in patients received three cycles of neoadjuvant chemotherapy (modified mesna, doxo- rubicin, ifosfamide, and dacarbazine [MAID]), interdigitated preoperative radiation therapy (RT; 44 Gy administered in split courses), and three cycles of postoperative CT (modi- fied MAID). In the study published by Palassini et al. [16], the combined radio-chemotherapy (epirubicin plus ifosfa- mide) treatment was found to be feasible, safe and with a limited increase in toxicities compared to patients receiving preoperative CHT alone. Role of combined chemo‑radiation therapy Based on the available literature data, it is possible to affirm that both treatments, pre-operative and post-operative, lead to similar results in terms of LC and OS and that the main differences are in terms of side effects. ff As already discussed, the most frequents pre-operative EBRT related side effect are wound complications (fre- quency 19.5–35%) [6]. The internal side of the thigh, tumour size > 10 cm, the tumour proximity to skin surface are risks factor for the development of this unwanted side effect. [7, 8]. Major literature data about EBRT related acute and late toxicity in extremities STSs are described in Table 2. In clinical practice, the indication for each treatment must be evaluated in multidisciplinary committee, consid- ering the optimal sequence between surgery and RT, tak- ing into account various factors for each patient (tumour size, anatomical position of the disease, histological sub- type, patient's comorbidity, risk factors). For both treat- ment settings, the evolution of RT techniques has led to an improvement in the planning of RT and a consequent reduc- tion in short- and long-term toxicity. From commonly used 3D-CRT techniques, in the recent era, there has been an evo- lution towards intensity-modulated radiation therapy (IMRT) and volumetric modulated arc therapy (VMAT), techniques that involve the use of variable intensity multiple beams: they allows a more conformal dose distribution to the target volumes (Fig. 1), a reduction in locoregional failure [20], an excellent local control in patients with high risk features and a greater sparing of neighbouring healthy tissues [21]. External beam radiation therapy (EBRT) The standard of radiation treatment after limb sparing sur- gery was originally the adjuvant setting [4, 5]. However, the indications for preoperative RT are increasing due to the various advantages compared to postoperative RT [17, 18]. Several recently published studies have evaluated the benefits and risks of neoadjuvant and adjuvant RT treatment [17, 18]. O’Sullivan et al. [17] from the Canadian Sarcoma Group, conducted a phase III randomized study to compare both approaches [17]. One hundred and ninety patients were enrolled, 94 in pre-operative arm and 96 in post-operative. The primary end point was the rate of wound complica- tions within 120 days of surgery. After a median follow-up of 3.3 years, they did not observe any statistically signifi- cant difference in LC and progression free survival rates between the two groups. However, important differences with regard to side effects were recorded: preoperative RT Preoperative EBRT The main advantages of preoperative EBRT are that the total radiation dose is lower than in the post-operative setting 3 1 3 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 1587 Table 2 Summary of side effects from major studies EBRT: external beam radiotherapy; LSS: limb sparing surgery References Study design Setting No. of patients Median F/U (years) Wound com- plications (%) Late toxicity grade ≥ 2 (%) O'Sullivan et al. [17] Randomized Pre-op EBRT 94 3.3 35 Not reported Post-op EBRT 96 17 Davis [18] Randomized Pre-op EBRT 73 Not reported Not reported 31.5 Fibrosis 15.1 Edema 17.8 Joint stiffness Post-op EBRT 56 48.2 23.2 23.2 Wang et al. [22] Prospective phase II Pre-op EBRT + LSS 79 3.6 36.6 10.5 Beane et al. [7] Randomized LSS alone 71 17.9 20 4 Bone frac- ture 12 Edema LSS + post- opEBRT 70 27 10 25 Alektiar et al. [21] Retrospective LSS + EBRT (pre or post 41 3 19.5 4.8 Bone fracture 12 Edema 17.1 Joint stiffness Table 2 Summary of side effects from major studies In case of positive surgical margins, the possibility of per- forming an adjuvant boost using EBRT or BRT (14–20 Gy is described in the international guidelines [1]. However, the results of two retrospective trials [25, 26] showed that post- operative RT boost did not clearly provide any advantage in preventing local relapse in patients with positive surgi- cal margins. Furthermore, the advantage of adding postop- erative RT boost has not yet been evaluated in randomized clinical trial. The topic is highly debatable and risk of local failure vs. potential toxicity should be very carefully evalu- ated individually for each patient.i (50 Gy vs. 60–66 Gy) [17] and treatment volumes are fre- quently smaller with consequently lower late toxicity and improved long-term functional outcomes [22]. Furthermore, the definition of the target volumes is easy, as the disease is clearly visible in magnetic resonance imaging (MRI) [23]. Moreover, there is an increase in radiobiological efficacy due to the better oxygenation and vascularization of the tumour. Preoperative EBRT Finally, from a theoretical point of view, surgical resection could become easier due to the possible shrinkage and cel- lular modification of the pseudocapsule diseases).i i The patients who can benefit most from neoadjuvant EBRT are those who present deep disease, greater than 5 cm, high grade and where surgery is complex due to the proximity of STS to neurovascular bundle or bone [23]. A consensus on the definition of gross target volume (GTV) and clinical target volume (CTV) was obtained by the Radiation Therapy Oncology Group (RTOG) [27] and by Haas and his European, American and Canadian colleagues [23]. GTV is defined on T1 contrast-weighted MRI images; fusion of CT and MRI images is recommended for treat- ment planning. CTV is defined as GTV + microscopic clini- cal extension: currently, it is obtained with an expansion of 3 to 4 cm longitudinal margins and 1.5 cm radial margins to GTV. CTV extension can be limited to the anatomical com- partment limits. Planning target volume (PTV) corresponds to an isotropic expansion of 5–10 mm of the CTV (Fig. 2); it varies according to the immobilization systems, the tech- niques and the image-guided RT used in each center [28]. Preoperative RT also has clear advantages both for sur- gery and for local control in patients affected by myxoid liposarcoma, in consideration of the high rate of shrinkage/ size reduction that the tumour can achieve [24]. Main disadvantage of neoadjuvant EBRT is high rate of surgical wound complications. The randomized trial by O’Sullivan et al. (3) reported 35% of wound complications in the preoperative group versus 17% in the postoperative group. Another possible disadvantage of preoperative RT is potential risk for patients unresponsive to radiation treat- ment to progress during RT, making them unable to receive definitive surgery. Postoperative EBRT i Surgery should be performed 4–6 weeks after the end of RT, to reduce the risk of wound complications and limit acute reactions [23]. However, it is not advisable to wait too long due to the risk of development of late fibrosis that could hinder surgery. Postoperative RT allows a definitive pathologic assessment. Moreover, it is associated with a lower rate of scar and post- operative wound healing complications. It is especially indi- cated in patients with significant comorbidities, at risk of wound complications. Currently, the standard dose for neoadjuvant EBRT is 50 Gy in 25 fractions. The overall RT period is 5 weeks. 1 3 1588 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 1588 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 Fig. 1 example of isodose distribution for a patient with soft tissue sarcoma of the thigh treated with volumetric modulated arc therapy (VMAT) Fig. 1 example of isodose distribution for a patient with soft tissue sarcoma of the thigh treated with volumetric modulated arc therapy (VMAT) bution for a patient with soft tissue sarcoma of the thigh treated with volumetric modulated arc therapy (VMAT) Fig. 1 example of isodose distribution for a patient with soft tissue sarcoma of the thigh treated with volumetric m On the other hand have to be considered several disad- vantages of post-operative RT: larger target volumes need to be irradiated than neoadjuvant RT and higher total dose are requested. Thus, postoperative RT is associated with a higher rates of late toxicity than preoperative RT. Side effects such as fibrosis, joint weakness, bone fracture, oedema are fre- quently permanent and can consequently lead to a reduction in patient's quality of life [17]. the surgical bed with appropriately safe margins, followed by a boost to the tumour bed of 10–20 Gy if surgical margins are not adequate, resulting in a total dose of 60–70 Gy. CTV must include tumour bed, all surgically manipu- lated tissues, all visible metal clips, the entire surgical scar and extent of the operative field and drain sites, with a 3.5 to 4 cm of longitudinal and 1.5 cm of radial margins, with the exception of osseous planes or fascia that act as natural barriers [29]. 1 3 Postoperative EBRT reported a study [32] involving 130 patients, 25 treated with BRT with a mean dose of 16 Gy (range 10–20 Gy) + EBRT with a dose of 50 Gy (range 40–70 Gy), while 61 patients were treated with exclusive EBRT with a median dose of 59 Gy (range, 50–74). Five- year OS, MFS and LC for patients treated with BT + EBRT vs EBRT alone were 82% vs. 72% (p = 0.93), 90% vs. 78% (p = 0.15) and 90% vs 83%, respectively (p = 0.25). In the univariate analysis, improved 5-year LC was found for high- grade tumours when treated with BRT + EBRT instead of Andrews et al. reported a study [32] involving 130 patients, 25 treated with BRT with a mean dose of 16 Gy (range 10–20 Gy) + EBRT with a dose of 50 Gy (range 40–70 Gy), while 61 patients were treated with exclusive EBRT with a median dose of 59 Gy (range, 50–74). Five- year OS, MFS and LC for patients treated with BT + EBRT vs EBRT alone were 82% vs. 72% (p = 0.93), 90% vs. 78% (p = 0.15) and 90% vs 83%, respectively (p = 0.25). In the univariate analysis, improved 5-year LC was found for high- grade tumours when treated with BRT + EBRT instead of Postoperative EBRT It is recommended to keep a drainage area radio protected to reduce distal oedema and late severe Currently, the standard dose for adjuvant EBRT is 50 Gy with standard fractionation to a larger volume encompassing 1 3 1 3 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 1589 Fig. 2 Example of target delineation for a patient with soft tissue sarcoma of the thigh. Yellow line represents gross tumour volume (GTV). Red line represents planning target volume (PTV) Fig. 2 Example of target delineation for a patient with soft tissue sarcoma of the thigh. Yellow line represents gross tumour volume (GTV). Red line represents planning target volume (PTV) complications [23]. The boost target volume should be cre- ated according to the original tumour extension. To define it accurately, it is necessary to provide a pre-operative CT or MRI data set. In post-operative setting, positioning of metal surgical clips during excision by surgeon is crucial to permit radiation oncologist to define the target volume in the proper manner [23] For the delineation of PTV, an isotropic expansion of the CTV of 5–10 mm is given. been drawn up [30] and previous American Brachytherapy Society guideline [31] has been updated. Guidelines reported use of BRT as RT-boost in cases at high risk of recurrence. been drawn up [30] and previous American Brachytherapy Society guideline [31] has been updated. Guidelines reported use of BRT as RT-boost in cases at high risk of recurrence. Andrews et al. reported a study [32] involving 130 patients, 25 treated with BRT with a mean dose of 16 Gy (range 10–20 Gy) + EBRT with a dose of 50 Gy (range 40–70 Gy), while 61 patients were treated with exclusive EBRT with a median dose of 59 Gy (range, 50–74). Five- year OS, MFS and LC for patients treated with BT + EBRT vs EBRT alone were 82% vs. 72% (p = 0.93), 90% vs. 78% (p = 0.15) and 90% vs 83%, respectively (p = 0.25). In the univariate analysis, improved 5-year LC was found for high- grade tumours when treated with BRT + EBRT instead of Andrews et al. Intraoperative radiation therapy (IORT) IORT consists of the delivery of a high dose of radiation in a single fraction during surgical procedure to the tumour bed in order to obtain an enhanced biological effect reducing toxicity, being able to move radiosensitive structures at risk out of the radiation field. i Proton therapy is up to date the standard of care for the skull base, spinal and paraspinal sarcomas. Moreover, it is often indicated in case of bone sarcomas, pediatric diseases, retro- peritoneal or large volume sarcomas, second tumours or for re-irradiation in case of relapses. Guttmann et al. [42] conducted a prospective study on 23 patients with locally recurrent or new primary STS in previ- ously irradiated fields who underwent proton re-irradiation. Treatment was generally well tolerated with low high-grade toxicity rates. Therefore, they concluded that proton re-irradi- ation for STS may be considered a possible and safe treatment option. Guttmann et al. [42] conducted a prospective study on 23 patients with locally recurrent or new primary STS in previ- ously irradiated fields who underwent proton re-irradiation. i Updated recommendations on the use of intraoperative RT (IORT) have recently been published [36], in the light of an updated systematic review of literature. Experts recommend IORT as a possible alternative to EBRT boost after pre-operative RT with R1 or R2 resection. Furthermore, it can be used as part of the post-operative RT treatment. Post-operative RT, as already mentioned, is usually divided in two part: a dose of 50 Gy prescribed to an extended PTV, followed by a RT dose of 10–20 Gy to a limited PTV (based on surgical margin status). The latter ‘‘boost” phase can be replaced by a preceded IORT boost. In both cases, the main advantage is the smaller irradiated volume with consequent potential lower side effects. i Treatment was generally well tolerated with low high-grade toxicity rates. Therefore, they concluded that proton re-irradi- ation for STS may be considered a possible and safe treatment option. Gary et al. from Loma Linda University are running a phase II single arm trial (NCT01819831) investigating the role of preoperative proton therapy (total dose 50 Gy, 25 daily frac- tions) followed by limb sparing surgery. The primary end point of the study is late radiation toxicities > grade 2 at 2 years. Hadron therapy A large series on adjuvant EBRT plus BRT boost in STS was recently performed [34]: 107 patients affected by high grade primary or recurrent STS underwent adjuvant BRT 20 Gy + EBRT 46 Gy ± chemotherapy. Five-year LC and OS rates were 80.9% and 87.4%, respectively. Hadron therapy is a form of EBRT using beams of energetic carbon ions, protons, or other heavier positive ions. The most common type of particle therapy is proton therapy. The chief advantage of proton therapy is that the RT dose is delivered over a narrow range of depth, which results in minimal entry, exit, or scattered radiation dose to healthy nearby tissues. Indeed, particle beams exhibit a Bragg peak [40] in energy loss through the body, delivering their maximum radiation dose at or near the tumour and minimizing damage to surrounding normal tissues [41]. BRT as a monotherapy can be considered in low-risk diseases, in case of small high-grade STS with negative margins or for re-irradiation. In fact, BRT should also be considered for patients with STS relapse who have previ- ously undergone limb-sparing surgery and EBRT, and where complications increase after re-treatment of previously irra- diated tissues [35]. Hadron therapy does not represent the standard of RT in extremity STS because modern technologies of conventional EBRT are satisfactory. However, some patients affected by proximal thigh or trunk sarcomas of big dimension could ben- efit from proton therapy for better healthy tissues sparing. Brachytherapy After a recent systematic literature review and clinical expe- riences, American guidelines on the use of BRT in STS have 1 3 1 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 1590 EBRT alone, 100% vs 74%, respectively (p = 0.09). These data suggested that the combination of BRT to EBRT may offer a better LC than EBRT alone for large deep-seated high-grade STS. (like major nerves or skin) can be spared from the field of irradiation. Furthermore, since there are no problems of intra- and inter-fraction movements, margins can be reduced to a minimum, allowing to deliver high dose to a much smaller irradiated volume compared to an EBRT boost [39]. In the early 2000s, with the introduction of technologi- cal advancement in EBRT, BRT started to be replaced by EBRT techniques such as IMRT. Between 1995 and 2006, a large retrospective study comparing adjuvant BRT to IMRT was conducted at Memorial Sloan-Kettering Cancer Center by Alektiar et al. [33]. One hundred and thirty-four adult patients affected by high grade extremities STS were enrolled: 71 received BRT, 63 received EBRT using IMRT technique, both in adjuvant setting. LC was significantly bet- ter in the IMRT group compared to the BRT one. Sometimes IORT is used in case of recurrent extremity STS. Tinkle et al. [38] reported a retrospective analysis of 26 patients with locally recurrent STS who underwent IORT (median dose 15 Gy, range 10–18 Gy) after salvage limb-spar- ing resection, reporting 58%, 81% and 50% 5-year LC, ampu- tation-free, and OS, respectively, with acceptable morbidity. Role of radiotherapy in oligometastatic disease cord compression. Recent technological advances in RT offer the opportunity to change the role of this modal- ity in the strategic approach to oligometastatic STS. The selection of patients who can benefit is complex, and it depends on several factors and should always require a multidisciplinary approach. Unfortunately, natural evolution of STSs is characterized by a high risk of distant metastasis (DM). Approximately 50% of STS patients develop DM [43] even in case of opti- mal primary treatment. The risk is higher for those patients with large tumour and/or with high grade histology. The most common site of DM is the lung (70–80%) followed by bone, liver and brain. One technical possibility for DM-directed RT is stereo- tactic body RT (SBRT). It is characterized by highly focused radiation to small volume in a single or few large dose frac- tions. This hypofractionated regimen is more biologically effective than conventionally fractionated RT. Furthermore, it can help to overcame radiation resistance, typical of radi- oresistant tumour such as STS. The approach to metastatic STS depends on several factor, such as number and site of metastases, histologi- cal subtype of primary tumour, interval between primary tumour diagnosis and development of DM, performance status and presence of comorbidities. Even if chemother- apy remains the standard of care for metastatic sarcoma patients, lack of durable responses and limited effect from systemic therapy highlights the importance of local treat- ment in the management of metastatic STS. The study with the largest number of patients was pub- lished by Lindsay and colleagues [45]. They treated 117 metastatic soft tissue and bone sarcoma lung nodules in 44 patients. With a median follow-up of 14 months, 2-year OS and pulmonary LC were 82% and 95%, respectively; only six out of 177 pulmonary nodules showed progression. Twenty- five percent of patients reported radiation-associated com- plications graded as ≤ 3 except one patient who developed an oesophageal stricture. Increasing literature evidence suggests, in selected sub- group of patients affected by oligometastatic disease, a benefit of locally ablative treatments (often in combination with systemic agents) resulting in longer life expectancy and better quality of life [44]. An additional advantage in the use of local ablative therapy could be its role in delaying initiation or change of systemic therapy. The larg- est retrospective study about this topic was published by Falk et al. Intraoperative radiation therapy (IORT) f Several studies in literature have shown how the combina- tion of limb sparing surgery, IORT and EBRT proved very high 5-year LC rates, 82–97% [37], 38, furthermore associ- ated with high limb preservation rates and good functional outcomes [37, 38]. The good functional results are probably due to the fact that radiosensitive structures or organs at risk 1 3 1 3 1591 European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 Role of radiotherapy in oligometastatic disease [44], reporting improved OS for patients who received local ablative treatment (surgery, RT, radiofre- quency ablation) of lung, liver or other metastases site compared to patients who do not received any local treat- ment (p = 0.0001). Other literature series also reveal favourable outcomes and confirm low toxicity profile supporting SBRT as a valid alternative to surgery, not only for lung (Table 3) but also for other sites such as bone [46] and brain [47] † These studies included patients with both soft tissue and bone sarcomas ‡ Local pulmonary control rate ents with both soft tissue and bone sarcomas Future directions Extensive pathological treatment response was observed in 91% of patients; with a median follow-up of 25 months, rate of grade 2 or higher toxicity was 14%, lower compared to the 37% of the historical Canadian study [17]. Based on these evidence, authors propose the use of 36 Gy (2 Gy/fraction) as an alternative approach of pre-operative RT for myxoid liposarcoma. This represents the first attempt to histology-stratified RT approach for STS.i Results published by Kosela-Paterczyk et al. [48] are encouraging and suggest a possible use of this approach in clinical practice. They treated 272 patients with pre- operative RT for five consecutive days in 5 Gy per fraction, immediately followed by surgery. LC was comparable to previously published studies (81%); early toxicity was simi- lar to the Canadian study by O’Sullivan (32.4% vs. 35%), but only 7% of patients required surgery for treatment of the complications (vs. 16% in the Canadian study); late toxicity was 16% (vs. 32% in the Canadian study [17]).i The high interest on this topic is confirmed by several trials currently underway in this area. At least six are now registered at www.clinicaltrials.gov and are currently accru- ing patients. One of them, the running trial NCT03989596 conducted by Maria Sklodowska-Curie Institute—Oncology Center, is investigating hypofractionated pre-operative RT schedule (10 fractions × 3.25 Gy) combined hyperthermia in marginally resectable STSs followed by a RT boost 4 × 4 Gy within one week in case of unresectability.if Finally, another recent field of investigation is about combined approach of RT with new systemic therapy like target agents or checkpoint inhibitors. Encouraging pre- liminary data regarding efficacy of combined treatment of RT with, Sorafenib [52], Pazopanib [53] and Sunitinib [54] have been recently published, although some of them are accompanied by unexpected high toxicity. Lewin and colleagues [54] studied a combination of RT (28 frac- tions,1.8 Gy/die) with sunitinib: 44% of patients presented grade 3 hepatotoxicity rate. However, tumour necrosis was higher in patients treated with combined treatment com- pared to the group with RT alone (75% vs. 40%) even if higher local failure rate was apparent in patients receiv- ing sunitinib. Several clinical trials are currently on going about this topic and results, to validate role of combined treatment with new agent, are awaited. Future directions Based on these preliminary data, although they have to be con- firmed by long-term analysis and further trials, this could be considered as a possible new treatment option.i To reach the first objective, the introduction of IMRT and image-guided radiation therapy has allowed to better con- form irradiated volume and to use smaller safety margins resulting in reduced side effect. f The recent phase II RTOG 0630 trial [22] demonstrated reduced grade 2 late toxicity rates (11%) compared to 37% reported in the historical randomized NCIC Trial. Eighty- six patients were treated in a preoperative setting (50 Gy in 25 fractions) with reduced margin around the macroscopic lesion compared to that reported in International guidelines [23].i Another aspect often debated among sarcoma scientific community is the possibility to treat different histological subtypes by specific treatment approaches, also includ- ing tailored RT treatment schedule. The rationale is based on the knowledge that the big group of these rare solid tumours of STS includes many histological subtypes with many differences in biological and clinical behaviour. For example, myxoid liposarcoma has been reported in several studies as characterized by higher radiation sensitivity. It shows greater reduction in size and greater pathological response to RT compared to other histological subtypes [50].i Another field of research, aimed at saving resources, involves shortening radiation schedule using hypofrac- tionated RT regimen. Hypofractionation represents a kind of RT fractionation in which the total dose is delivered in fewer fractions with an increased fraction dose. This treat- ment may lead to additional biological effects compared to conventionally fractionated RT. The main advantages of hypofractionation are the decreased overall treatment time which is more convenient for both patients and physicians, the increased compliance and the best cost-effectiveness ratio of the treatment. Furthermore, such an approach may provide an additional radiobiological benefit when treating radioresistant tumours such as sarcomas. On the basis of this finding, the recent international prospective phase II DOREMY trial [51] was conducted in order to assess whether a dose reduction in preoperative RT for myxoid liposarcoma would result in comparable oncological outcome to the present standard of care with less morbidity. Seventy-nine patients who received a pre- operative RT dose of 36 Gy in once-daily 2 Gy fractions were enrolled. Future directions The current local approach for extremities STSs, based on limb sparing surgery and RT, has permitted to reach sat- isfactory results in terms of local recurrence rate (lower than 15%) [5, 6, 8]. Furthermore, advancement in RT field leads to two main objectives: reducing treatment associated acute and late side effects and increasing the efficacy of RT. Historically, surgery of metastatic site has been consid- ered the treatment of choice. Instead, RT has been mainly used for palliation and to reduce risk of bone fracture and Table 3 Published series about SBRT for sarcoma lung metastases 5 year rate † These studies included patients with both soft tissue and bone sarcomas Authors Year No. of patients No. of lesions Total dose/ No. of fractions Median F/U (months) 2 years L.C (%) 2 years O.S (%) Toxicity Gr ≥ 3 (%) Dhakal et al. (pre- ferred)[55] 2012 14 74 50 Gy/5 fr 11 88 69 None Frakulli et al.[56]† 2015 24 68 30–60 Gy/3–8 fr 17 86 66 None Navarria et al. [57] 2015 28 51 48 Gy/4 fr (pre- ferred) 21 96 56 None Soyfer et al. [58] 2017 22 53 60 Gy/3 fr 95 100 50* n.r Lindsay et al.[45]† 2018 44 117 50 Gy/10 fr (pre- ferred) 14 95‡ 82 2 Baumann et al.[59]† 2020 56 44 50 Gy/4–5 fr 16 90 46 None Table 3 Published series about SBRT for sarcoma lung metastases y † These studies included patients with both soft tissue and bone sarcomas European Journal of Orthopaedic Surgery & Traumatology (2021) 31:1583–1596 1592 To reach the first objective, the introduction of IMRT and image-guided radiation therapy has allowed to better con- form irradiated volume and to use smaller safety margins resulting in reduced side effect. response in 14 out of 87 patients (16%) in the NBTXR3 group and in 7 out of 89 patients (8%) who underwent RT alone, (p = 0.044), and a higher rate of R0 resections (77% vs. 64%, p = 0.042) in the NBTXR3 group. They did not record significant differences in severe side effects. 1 3 Future directions Another field of research is studying effects of intra- tumoural injections of radio-enhancing substances in order to increase the radiobiological effects of radiation with minimized systemic side effects. Bonvalot et al. [49] recently published encouraging results for intratumoural injection of nanoparticles of hafnium oxide (NBTXR3) [49]. They conducted a phase II-III randomized trial com- paring standard pre-operative RT (50 Gy) versus standard pre-operative RT preceded by NBTXR3 injection into the primary tumour site. They reported pathological complete Conclusions Montemurro M, Pantaleo MA, Piana R, Picci P, Piperno-Neu- mann S, Pousa AL, Reichardt P, Robinson MH, Rutkowski P, Safwat AA, Schöffski P, Sleijfer S, Stacchiotti S, Sundby Hall K, Conservative surgery combined with RT represents the standard of care for local treatment of high grade STSs of limbs. RT can be administered both pre- or post-oper- atively. Post-operative modality increases late side effects like fibrosis, oedema and joint stiffness. Pre-operative setting is associated with higher rate of wound complications, but literature evidence suggests that advanced techniques can decrease these complications. f Unk M, Van Coevorden F, Van Der Graaf W, Whelan J, Wardel- mann E, Zaikova O, Blay JY (2018) Soft tissue and visceral sarcomas: ESMO-EURACAN Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. https://doi.org/ 10.1093/annonc/mdy096 3. Mavrogenis AF, Angelini A, Vottis C, Palmerini E, Rimondi E, Rossi G, Papagelopoulos PJ, Ruggieri P (2015) State-of-the- art approach for bone sarcomas. Eur J Orthop Surg Traumatol 25:5–15 4. Rosenberg SA, Tepper J, Glatstein E, Costa J, Baker A, Brennan M, DeMoss EV, Seipp C, Sindelar WF, Sugarbaker P, Wesley R (1982) The treatment of soft-tissue sarcomas of the extremities. 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https://openalex.org/W4308569313	https://ejnpn.springeropen.com/counter/pdf/10.1186/s41983-022-00563-w	English	null	SARS-CoV-2 and monkeypox: what is common and what is not in a present pandemic versus a potential one—a neuropsychiatric narrative review	The Egyptian Journal of Neurology, Psychiatry and Neurosurgery /The Egyptian Journal of Neurology, Psychiatry and Neurosurgery	2,022	cc-by	8,232	© 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/. Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 https://doi.org/10.1186/s41983-022-00563-w Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 https://doi.org/10.1186/s41983-022-00563-w The Egyptian Journal of Neurology, Psychiatry and Neurosurgery The Egyptian Journal of Neurology, Psychiatry and Neurosurgery Abstract Pandemic represents challenging medical emergency as it is usually associated with high rates of mortalities and morbidities. Along the last 2 and half years the world has faced the emergence of severe acute respiratory syndrome corona virus 2 pandemic that caught medical agencies and health authorities by surprise and costed more than half billion morbidities and 6 million mortalities. Unfortunately, the way developed countries contained the novel corona virus was unsatisfactory in means of early quarantines as well as obtaining and distributing an effective vaccine. This failure in management might have been responsible for the emergence of a new potential pandemic caused by monkeypox virus. Along the current review article, a detailed comparison is presented between corona virus and monkeypox virus based on virological characteristics, role of corona virus in monkeypox spread, pathogenesis, neuropsychiatric manifestations, and treatment and management. It is obvious that both viruses have a major role in causing various neuropsychiatric manifestations. Neurological manifestations are either bound directly to the virus spread to central and peripheral nervous system or secondary to triggering an immune reaction. Psychiatric ones are mostly related to stigmatization, isolation as well as changes that takes place in neurotransmitters and their metabo- lites within the nervous system. Dealing properly with monkeypox virus spread through previously learned lessons from corona virus might protect the world from a new pandemic. Keywords: SARS-CoV-2 virus, Coronavirus, Pandemic, Monkeypox virus, Neuropsychiatric manifestations, Virology, Mortality and morbidity, Quarantine, Stigma Meanwhile an endemic is a prevalent infectious disease within a specific population confined to geographical area [2]. SARS‑CoV‑2 and monkeypox: what is common and what is not in a present pandemic versus a potential one—a neuropsychiatric narrative review Tamer Roushdy* Introduction Pandemic is an infectious disease that originates in a confined area (single country or region) under the term epidemic state then spreads on a wide scale to involve different countries, regions, and even continents affecting a large proportion of world’s population [1]. An example of an endemic disease is malaria that is present mainly in sub-Saharan Africa besides broad geo- graphical zones along the equator yet nearly absent in the rest of the world due to absence of its transmitting vector [3]. Unlike malaria, cholera is another infectious disease that is endemic in sub-Saharan Africa with an outbreak epidemic that can turn to become pandemic if not prop- erly managed. Historically cholera had seven pandemic *Correspondence: Tamer.roushdy@med.asu.edu.eg Neurology Department, Faculty of Medicine, Ain Shams University, 38 Abbasia, Cairo 11591, Egypt Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 Page 2 of 11 attacks the latest was in 1961 with emergence of a new strain [4].f systems with a wide range of autoimmune conditions and diverse neuropsychiatric manifestations that are either a result of direct viral effect or secondary to excessive inflammatory response or crossed immunological reac- tion [8]. Epidemics usually occur secondary to different reasons as reduced general hygiene or low immune system in a community. An example of an epidemic is the cholera epidemic outbreak of Yemen that occurred as a result of the Yemeni war [5]. Meanwhile the world is facing another unfamiliar out- break of a previously considered endemic which is monk- eypox (MPX) that has crossed borders in a behavior that is uncommon for endemic diseases [9, 10]. So, it could be concluded that pandemics originate from uncontrolled epidemics. Since the year 2020 the world is facing a pandemic that originated from a central market in Wuhan city in China that sells food besides ani- mals and other products. It is caused by a novel corona virus and was termed after its commonest presentation being severe acute respiratory syndrome of corona virus 2 (SARS-CoV-2) [6]. According to World Health Organization (WHO), monkeypox is now considered a health emergency of global concern. MPX also can cause some neuropsychiat- ric manifestations. Although such manifestations are not fully understood. Yet, it shares similar general viremia related symptoms as well as psychiatric stigmatization that is originating out of fear from an unknown patho- gen, its effect and to what extent it is contagious [11–13]. SARS-CoV-2 belongs to the same corona viruses that emerged in the beginning of the twenty first century which were termed severe acute respiratory syndrome of corona virus (SARS-CoV) and middle east respiratory syndrome of corona virus (MERS-CoV). SARS-CoV-2 is highly infectious if compared to its earlier ancestors since its lipid envelope where the protein spikes are emerging has some changes in its fusion sites that facilitate better attachment to cells and augment entrance [7]. f Along the current review a comparative presentation between SARS-CoV-2 and MPX regarding their cross- relation, features and neuropsychiatric manifestations as well as ways of management and control is presented (Table 1). Main text Moreover, it is continuously mutating and this further facilitates its ability to escape acquired immunity from previous attacks within the same individual and being a novel virus to humans there is already no innate immu- nity against it. SARS‑CoV‑2 and MPX virology SARS-CoV-2 belongs to the orthocoronavirinae subfam- ily that is derived from the Coronaviridae family. It is a single stranded RNA virus that is enveloped in a bilayer of lipid structured envelope with spikes on its surface [14]. For such reasons, SARS-CoV-2 pandemic is still run- ning for more than 2 years affecting different body Table 1 Similarities and differences between SARS-CoV-2 and MPX Table 1 Similarities and differences between SARS-CoV-2 and MPX SARS-CoV-2 MPX Virological classification Orthocoronavirinae subfamily, Coronaviridae family Chordopoxvirinae subfamily, Poxviridae family Nucleic acid structure Single stranded RNA Double stranded DNA Forms Alpha, Beta, Gamma, Delta, Omicron with its different clades Nigerian clade and democratic republic of Congo clade Abundant form worldwide Omicron Nigerian clade Way of transmission Droplet, direct contact, aerosol Long-term close direct contact with infected animals, con- tact with infected body fluids, human-to-human transmis- sion among homosexual men Nature of spread Tides and waves Constant rise Source of body entrance Attaching its surface spikes to ACE2 receptors on endothe- lial cells Macropinocytosis Commonest neuropsychi- atric manifestations Strokes, encephalitis, peripheral neuropathy, autoimmune neuropathy, fatigue, lack of concentration, inattention, memory lapses, generalized weakness, sleep disturbance, depression, anxiety, post-traumatic stress, chronic head- ache Headache, myalgia, photophobia, pain and fatigue, seizures, encephalitis, anxiety, depression up to suicide Manifestations in pediatrics Less aggressive yet rarely MIS-C can develop More aggressive Long-term close direct contact with infected animals, con- tact with infected body fluids, human-to-human transmis- sion among homosexual men Constant rise Macropinocytosis Headache, myalgia, photophobia, pain and fatigue, seizures, encephalitis, anxiety, depression up to suicide Manifestations in pediatrics Less aggressive yet rarely MIS-C can develop More aggressive More aggressive The way through which SARS-CoV-2 gain entrance to the human body is through attaching its spikes to the endothelial cells angiotensin converting enzyme 2 recep- tors (ACE-2) that are abundant along different organs beginning from the nasal cells, respiratory system, renal system, cardiac, vascular system and nervous system [6]. As for MPX, it belongs to the chordopoxvirinae sub- family that is derived from the Poxviridae family. Unlike SARS-CoV-2, monkeypox is a double stranded 197 kb DNA virus with a lipoprotein envelope and it enters cells through macropinocytosis. Being a DNA virus makes it The way through which SARS-CoV-2 gain entrance to the human body is through attaching its spikes to the endothelial cells angiotensin converting enzyme 2 recep- tors (ACE-2) that are abundant along different organs beginning from the nasal cells, respiratory system, renal system, cardiac, vascular system and nervous system [6]. As for MPX, it belongs to the chordopoxvirinae sub- family that is derived from the Poxviridae family. Unlike SARS-CoV-2, monkeypox is a double stranded 197 kb DNA virus with a lipoprotein envelope and it enters cells through macropinocytosis. Being a DNA virus makes it More aggressive SARS-CoV-2: severe acute respiratory syndrome corona virus 2, MPX: monkeypox virus, RNA: ribonucleic acid, DNA: deoxyribonucleic acid, ACE2: angiotensin converting enzyme 2 receptors, MIS-C: multisystem inflammatory syndrome in children Page 3 of 11 Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 SARS-CoV-2 is considered one of the largest known RNA viruses reaching 29.9 kb. This makes it difficult to be transmitted through air-borne transmission, yet it could be transmitted through droplet transmission as well as direct contact with infected surfaces or through aerosol transmission [15]. stable when compared to SARS-CoV-2 as DNA viruses have the ability to proof-read their genome while repli- cating with a less chance to have mutated forms although not fully impossible [18]. Along its original endemic site being west and central Africa, MPX virus has two clades, one in Nigeria with less mortality rates being 1/100 cases and the other in Democratic Republic of Congo with mortality reaching 1/10 cases while Cameron act as a border zone with the two clades endemic in it [18]. RNA viruses are liable to mutation secondary to errors that develop in their genetic codes while replicating. Such mutation can result in new strains making it diffi- cult to control a pandemic caused by RNA viruses [16]. SARS-CoV-2 has mutated from its original alpha form to beta, gamma, delta, and omicron with its different clades within the last 2 years [17]. Along its current 2022 cross-border outbreak, MPX had a 15 single nucleotide polymorphism mutation which is an uncommon and a relatively rapid mutation for a DNA virus [19]. Mutations in SARS-CoV-2 are associated with rapid spread and such spread up till now has infected more than half billion mankind with more than 6 million deaths. Monkeypox spread through close long-term contact with an infected animal as well as through body fluids, until the current 2022 global outbreak with a total of more than 60,000 cases worldwide with only few hun- dred cases in endemic countries there was no direct wide spread human-to-human transmission [20, 21] (Fig. 1, Table 2). Whether such direct spread among humans who have never traveled to the endemic areas along Nigeria where the running clade worldwide is present is related to SARS-CoV-2 is a question of concern. Is MPX spread related to SARS‑CoV‑2? Infection with SARS-CoV-2 is associated with reduc- tion in total leucocytic count and lymphopenia in Fig. 1 World map showing countries with monkeypox cases based on numbers that is obtained from centers of disease control and prevention by 28th of September 2022. countries in yellow represent that numbers of cases detected in single digit (units), red represent detected cases in tens, green represent detected cases in hundreds, blue represent detected cases in thousands, and black represent detected cases in tens of thousands Fig. 1 World map showing countries with monkeypox cases based on numbers that is obtained from centers of disease control and prevention by 28th of September 2022. Is MPX spread related to SARS‑CoV‑2? countries in yellow represent that numbers of cases detected in single digit (units), red represent detected cases in tens, green represent detected cases in hundreds, blue represent detected cases in thousands, and black represent detected cases in tens of thousands Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 Page 4 of 11 Table 2 Countries with reported cases of spreading monkeypox based on centers for disease control and prevention from 17th of August to 28th of September 2022 Table 2 Countries with reported cases of spreading monkeypox based on centers for disease control and prevention from 17th of August to 28th of September 2022 August to 28th of September 2022 11th Aug 16th Aug 22nd Aug 27th Aug 6th Sept 10th Sept 15th Sept 20th Sept 28th Sep Countries/total 31,803 36,591 41,360 44,507 53,031 56,613 56,935 62,411 66,554 Andorra 4 4 4 4 4 4 4 4 4 Argentina 37 49 72 72 170 170 170 265 326 Aruba 0 0 0 0 2 2 2 3 3 Australia 58 70 89 89 125 125 125 132 135 Austria 175 200 217 231 271 278 278 300 309 Bahamas 1 1 1 2 2 2 2 2 2 Barbados 1 1 1 1 1 1 1 1 1 Belgium 546 546 624 671 706 726 726 744 757 Benin 3 3 3 3 3 3 3 3 3 Bermuda 1 1 1 1 1 1 1 1 1 Bolivia 5 11 31 42 79 89 89 129 175 Bosnia and Herzegovina 1 3 3 3 3 3 3 3 5 Brazil 2131 2584 3359 3788 5037 5525 5525 6649 7445 Bulgaria 4 4 4 4 4 5 5 6 6 Cameroon 7 7 7 7 7 7 7 7 8 Canada 957 1059 1112 1173 1251 1317 1317 1363 1389 Central African Republic 8 8 8 8 8 8 8 8 8 Chile 91 141 189 207 381 450 450 728 842 Colombia 55 84 129 273 582 938 938 1260 1653 Costa Rica 3 3 3 3 3 3 3 4 4 Croatia 12 17 17 22 26 27 27 27 29 Curaçao 0 0 1 1 1 1 1 1 3 Cuba 0 0 0 1 2 2 2 2 3 Cyprus 3 3 4 4 5 5 5 5 5 Czechia 29 35 36 41 48 58 58 62 66 Democratic Republic of Congo 163 163 163 163 195 195 195 195 195 Denmark 126 151 163 169 175 181 181 183 184 Dominican Republic 4 5 6 7 7 7 7 31 31 Ecuador 10 17 19 35 53 53 53 68 120 Egypt 0 0 0 0 0 1 1 1 1 El Salvador 0 0 0 0 1 1 1 2 5 Estonia 9 9 9 10 10 10 10 11 11 Finland 22 22 22 22 22 30 30 33 33 France 2423 2673 2889 2889 3558 3646 3646 3898 3970 Georgia 1 1 2 2 2 2 2 2 2 Germany 2982 3142 3266 3329 3493 3518 3519 3563 3607 Ghana 35 35 47 47 76 76 76 84 84 Gibraltar 5 6 6 6 6 6 6 6 6 Greece 41 49 50 52 58 66 66 69 74 Greenland 0 2 2 2 2 2 2 2 2 Guadeloupe 1 1 1 1 1 1 1 1 1 Guatemala 2 3 3 4 8 11 11 12 20 Guyana 0 0 0 0 2 2 2 2 2 Honduras 0 3 3 3 4 4 4 4 6 Hong Kong 0 0 0 0 0 1 1 1 1 Hungary 51 57 62 64 70 71 71 75 77 Iceland 11 11 12 12 12 12 12 12 14 Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 Page 5 of 11 Table 2 (continued) 11th Aug 16th Aug 22nd Aug 27th Aug 6th Sept 10th Sept 15th Sept 20th Sept 28th Sept India 9 9 9 9 10 10 10 12 12 Indonesia 0 0 0 1 1 1 1 1 1 Iran 0 0 1 1 1 1 1 1 1 Ireland 101 101 113 128 144 160 160 173 178 Israel 166 189 194 213 239 241 241 247 250 Italy 599 644 689 714 760 787 787 828 846 Jamaica 3 4 4 4 5 9 9 12 14 Japan 3 4 4 4 4 4 4 4 4 Jordan 3 4 4 4 4 4 4 4 4 Latvia 3 3 4 4 4 4 4 4 5 Lebanon 6 6 6 6 6 8 8 11 11 Liberia 2 2 2 2 2 2 2 3 3 Lithuania 3 5 5 5 5 5 5 5 5 Luxembourg 34 43 45 47 53 53 53 55 55 Malta 30 30 31 31 31 33 33 33 33 Martinique 1 1 1 1 1 1 1 1 1 Mexico 91 147 252 252 504 788 804 1050 1367 Moldova 1 1 2 2 2 2 2 2 2 Monaco 0 1 1 3 3 3 3 3 3 Montenegro 1 1 1 2 2 2 2 2 2 Morocco 1 1 1 1 3 3 3 3 3 Netherlands 959 1025 1087 1087 1166 1195 1199 1209 1221 New Caledonia 1 1 1 1 1 1 1 1 1 New Zealand 3 4 4 4 4 5 5 5 9 Nigeria 157 157 157 157 220 220 220 277 277 Norway 66 72 76 76 82 82 82 89 92 Panama 2 2 4 7 10 12 12 12 14 Paraguay 0 0 0 0 1 1 1 1 1 Peru 547 775 937 1188 1531 1724 1724 2054 2423 Philippines 1 1 1 4 4 4 4 4 4 Poland 85 95 104 121 130 145 145 160 182 Portugal 710 770 810 810 871 871 871 898 917 Qatar 3 3 3 3 3 3 3 4 5 Republic of Congo 3 3 3 3 3 3 3 5 5 Romania 28 32 33 35 36 36 36 37 40 Russia 1 1 1 1 1 1 1 2 2 Saint Martin 1 1 1 1 1 1 1 1 1 Saudi Arabia 5 5 6 6 8 8 8 8 8 Serbia 23 23 23 31 31 31 31 31 40 Singapore 15 15 15 15 16 16 16 19 19 Slovakia 8 10 10 12 12 14 14 14 14 Slovenia 40 43 43 43 43 45 45 46 47 South Africa 3 3 4 4 5 5 5 5 5 South Korea 1 1 1 1 1 2 2 2 2 South Sudan 0 0 0 0 0 2 2 2 2 Spain 5162 5719 5792 6284 6543 6749 6749 6947 7122 Sudan 1 1 1 2 2 2 2 6 7 Sweden 123 129 139 150 161 165 165 179 186 Switzerland 347 387 392 416 476 480 480 494 513 Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 Page 6 of 11 Table 2 (continued) 11th Aug 16th Aug 22nd Aug 27th Aug 6th Sept 10th Sept 15th Sept 20th Sept 28th Sept Taiwan 3 3 3 3 3 3 3 3 3 Thailand 4 4 5 5 7 7 7 8 8 Turkey 1 1 1 1 1 1 1 1 1 Ukraine 0 0 0 0 0 0 0 1 3 United Arab Emirates 16 16 16 16 16 16 16 16 16 United Kingdom 2914 3017 3081 3207 3413 3484 3484 3552 3585 United States 9492 11,889 14,594 15,908 19,961 21,504 21,805 23,892 25,340 Uruguay 2 2 2 2 4 5 5 5 6 Venezuela 1 1 1 1 3 3 3 3 5 many cases. Pathogenesis of SARS‑CoV‑2 and MPX neuropsychiatric manifestations SARS-CoV-2 pandemic that is in the form of tides and waves which differs from MPX more linear spread (Fig. 2) have aided different health authorities and organizations to study its pathogenesis along different body systems with nervous system one of them. Another link between SARS-CoV-2 and MPX out- break is absence of immunity in young generations against smallpox virus since stopping its vaccination campaigns after its eradication in 1980. Smallpox belongs to the same family of monkeypox and stop- ping the administration of its vaccine aided in rela- tive increase in monkeypox cases in its endemic areas together with reduction in immunity of local residents on top of spreading SARS-CoV-2 that is associated as well in increasing numbers of monkeypox cases [23]. Besides gaining entrance to cells through ACE-2 receptors which causes direct invasion, the common- est pathophysiologic mechanisms responsible for central and to an extent peripheral neurological mani- festations of SARS-CoV-2 are attributed to exposure of the immune system to a novel virus with absence of innate immunity previous experience, which is fol- lowed by over activity and overexpression of cytokine and chemokine responses that reaches up to triggering autoimmune reactions [25].l The world failure to equally distribute vaccines against SARS-CoV-2 among developing and devel- oped countries plays a role in increasing the liability for catching SARS-CoV-2 that further reduces the patient’s immunity and increase his liability to have a co-infection with monkeypox. This failure in vaccine distribution is causing backfire on developed world with the cross-border spread of an increasing number of MPX cases [23, 24]. Such overexpression of inflammatory and immune markers is reported along different studies that proved the presence of elevated values of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) besides pleo- cytosis in examined cerebrospinal fluid (CSF) of SARS- CoV-2 victims [26]. Central nervous system invasion of SARS-CoV-2 is through two routes, first is through hematological spread, where the virus reaches the endothelial cells lining the blood capillaries of the blood brain barrier (BBB) and invades such cells through attaching its spikes to ACE-2 receptors that are abundant within the endothelial cell surfaces and this causes break down in the BBB which in turns causes direct inflammation to the brain and brain edema that could compress vital centers along the brain- stem with further affection of respiratory and circulatory systems. Is MPX spread related to SARS‑CoV‑2? Such low circulatory immune cells count can make infected cases with SARS-CoV-2 more liable for co-infection with MPX and this in turn may be associated with change in form and course of infection in one or both diseases as well as response to vaccina- tion [22]. Pathogenesis of SARS‑CoV‑2 and MPX neuropsychiatric manifestations Pathogenesis of SARS‑CoV‑2 and MPX neuropsychiatric manifestations Second route is retrospectively either from the endothelial cells lining the olfactory bulb and along the olfactory pathway to the CNS or from chemo and mecha- noreceptors of the respiratory system to the brainstem cardiorespiratory system [27–29]. Another possible explanation for the outbreak of endemic Nigerian clade of MPX through many coun- tries worldwide with most of them within the western world is the reduction in restrictions related to travel and lockdown after reaching more than 70% vaccina- tion against SARS-CoV-2 in the developed world and the mass gathering that reached up to 80,000 attend- ants most of them from the gay and bisexual commu- nity along Maspalomas festival in the Canary Island between 5 and 15th May 2022. Such gathering may have played a role in the spread of viruses like MPX in case of the presence of one or more carrier within such gathering [23]. Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 Page 7 of 11 0 10000 20000 30000 40000 50000 60000 70000 Rising Cases of MPX within one month of monitoring Fig. 2 Rising cases of monkeypox within 1 month of monitoring Rising Cases of MPX within one month of monitoring Rising Cases of MPX within one month of monitoring Pathogenesis behind peripheral nervous system dis- orders associated with SARS-CoV-2 is either secondary to direct viral infection and that is why some literature reported cases were having a para-infectious presentation or secondary to triggering the immune system through molecular mimicry to attack the myelin sheath with post- infectious presentation [30–32]. tryptophan to kynurenine is associated with reduction in serotonin that can explains psychiatric manifestations accompanying infection with SARS-CoV-2 as depression and anxiety [35]. As for neurological manifestations pathogeneses of MPX up till now are mainly attributed to inflammatory viremia effects of the virus causing fever that in turns affect muscles leading to myalgia. Raised body tempera- ture is associated with tachycardia, tachypnea, hypercap- nia up to hypoxia which affects all body systems and can cause headache, encephalopathy up to seizures in vulner- able cases [36]. Psychiatric manifestations presenting with SARS- CoV-2 is attributed to the lockdown and curfew that was declared along the initial waves of the pandemic which deprived medically chronic patients of their usual check- ups with their treating physicians besides the media effect of presenting mortalities with daily counts that had direct mental traumatizing effect. Pathogenesis of SARS‑CoV‑2 and MPX neuropsychiatric manifestations Social distancing is also a main trigger to frustration, aggression, mood dis- orders, insomnia and psychosis [33, 34]. Psychiatric pathogeneses of MPX are to a great extent stigma related especially that most available data are speaking on homosexuality role in transmission of MPX. And since it is a relatively recently human-to-human transmitted disease, its stigma originates from lacking full medical knowledge about it that resembles what epi- lepsy had in the ancient civilization thoughts [37]. Stigma from being infected with fear of transmitting infection also played a role in overexpression of psychiat- ric manifestations besides the changes that develop along neurotransmission pathway and its metabolites triggered indirectly by stress and directly by the immune response effect to the virus on such neurotransmitters [35]. Neuropsychiatric aspects of SARS‑CoV‑2 and MPX Unlike its name, SARS-CoV-2 does not just cause influ- enza like manifestations. Along the last 2 and half years since its declaration as a pandemic by WHO in March 2020, SARS-CoV-2 affected the nervous system in a plenty of ways. f Kucukkarapinar and colleagues succeeded in linking psychiatric manifestations that takes place post SARS- CoV-2 exposure to the kynurenine pathway and trypto- phan metabolism. Infection with SARS-CoV-2 alter the tryptophan pathway with increase in kynurenic acid, kynurenine, and quinolinic acid and decrease in tryp- tophan which in turn increases oxidative stress, and impairs glutamate action with a net affection on cogni- tive functions. Meanwhile increase in conversion of Such affection is either central or peripheral, some- times preceded the respiratory manifestations, and in other occasions is either conjoint with it or appears few weeks following diagnosis [8]. Page 8 of 11 Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 These different phases of neurological affection spot- light on the diverse pathophysiology of neurological complications of SARS-CoV-2 [8, 38]. termed long Covid. Neurological and psychiatric symp- toms are the commonest in long Covid. Such symptoms range between fatigue, lack of concentration, inattention, memory lapses, generalized weakness, sleep disturbance, depressive or anxiety symptoms, post-traumatic stress, chronic headaches, autonomic dysfunctions. Psychiatric manifestations may reach up to delusions [45, 46]. Neurovascular complications of SARS-CoV-2 whether in the form of ischemic strokes or hemorrhagic ones and whether arterial or venous are very common and are reported with mild, moderate, or severe forms of SARS- CoV-2 [39, 40]. Pathogenesis of SARS‑CoV‑2 and MPX neuropsychiatric manifestations Long Covid symptoms are much like the myalgic encephalomyelitis/chronic fatigue syndrome symptoms [47]. Stroke in some cases is the only manifestation of SARS- CoV-2. Its pathophysiology was described by Roushdy and Hamid which is related to uncontrolled rise in blood pressure secondary to down regulation in ACE-2 recep- tors that are used by SARS-CoV-2 spikes to gain entrance to endothelial cells, reduction in platelet count, distur- bance in coagulation parameters whether procoagulants or anticoagulants as well as cytokine storm and elevated inflammatory biomarkers that can affect vessel wall integrity and induce vasculitis [6]. Autopsy of the brain of SARS-CoV-2 victims showed widespread of macrophages and inflammatory infiltrates as well as microglia within the brain and disruption of the blood brain barrier; such findings highlight the possibility of developing neurodegenerative conditions in the future as Parkinson’s and Alzheimer’s diseases [8, 48] As for young children who catch SARS-CoV-2, the usual symptoms are mild and do not extend beyond the general viremia signs. Yet, few develop the rare multisys- tem-inflammatory syndrome in children (MIS-C) which is associated with excessive endothelial activation and this might be accompanied by neurological manifesta- tions that range between mild symptoms as headache and anosmia, and can extend up to meningitis, seizures, encephalopathy, cerebellar ataxia, proximal myopathy and bulbar palsy. Such neurological manifestations are secondary to immune system overactivation with auto- immune reactions [49]. Besides the usual symptoms accompanying any viremia being myalgia, bony aches, fatigue, anorexia and fever, it is noticed that SARS-CoV-2 is associated with anosmia in many cases. Anosmia is believed to be secondary to viral invasion of olfactory nerve endings within the cribriform plate that is lined by endothelium cells abundant with ACE-2 [41]. Besides dysfunction of the olfactory nerve, other cra- nial nerves are occasionally affected following SARS- CoV-2 infection and secondary to long-term treatment with steroids in patients who are usually immunocom- promised. Such patients might catch rhino-orbito-cere- bral mucormycosis with infiltration of the orbital cavity, or nasal sinuses with affection of oculomotor, abducent, trochlear and optic nerves with or without cavernous sinus thrombosis [42, 43]. Unlike SARS-CoV-2, MPX virus has few reported neu- ropsychiatric manifestations, yet such statement might be misleading in the context that MPX cases are still in the range of thousands if compared to millions studied cases in SARS-CoV-2. Pathogenesis of SARS‑CoV‑2 and MPX neuropsychiatric manifestations Reported neuropsychiatric manifestations of the endemic MPX are headache, myalgia, photophobia, pain and fatigue, seizures, encephalitis, anxiety, and depres- sion that can mount up to suicide [50–52]. Encephalitis is also reported with SARS-CoV-2 that is explained as direct invasion of the virus through the nasal cavity and backflow through the olfactory nerve to the brain. Yet evidence supporting this explanation is weak as analysis of cerebrospinal fluid of patients along plenty of cases failed to detect the viral RNA through reverse tran- scription polymerase chain reaction (PCR). Yet encepha- litis with SARS-CoV-2 is suggested to be a result of direct immune reaction releasing cytokines, chemokines and inflammatory biomarkers within the central nervous sys- tem [8, 38]. Fatigue, myalgia, and headache can be attributed to the viremia that is common with any viral infection [53]. As for psychiatric manifestations that ranges from anxi- ety up to major depression with suicidality it could be explained on basis of fear of stigma as most historical patients were hospitalized in quarantine hospitals [54]. Encephalitis was reported in cases along endemic regions of west and central Africa and also was previ- ously reported in 2 pediatric cases both were diagnosed by MPX, and only one of them was subjected to cerebro- spinal fluid analysis that showed antibodies (IgM) against the virus yet no evidence of the virus itself which could be explained on immunological basis rather than direct invasion of the brain by the virus [55, 56]. Peripheral nervous system is also involved in acute cases. Many reports spoke about peripheral neuropathy and autoimmune reactions with autoantibodies against the peripheral nervous system causing Guillain–Barre spectrum syndromes few weeks after a negative PCR [44]. Many of the cases that recovered the acute phase of illness suffer a diverse natured symptom; such cases are Along the current cross-border outbreak besides three deaths in Nigeria, two in Central African Republic, and Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 Page 9 of 11 non-replicating virus vector vaccine, and protein subunit vaccine [58]. one in Ghana, there are 2 reported deaths in Spain, one in Brazil and one in India. The two deaths in Spain are secondary to encephalitis and meningoencephalitis. MPX does not have a definite treatment yet. Brincido- fovir and tecovirimat are under investigation for possibil- ity of being effective based on previous partial success on sporadic cases in the United Kingdom. Preventive measures and management Again, vaccines are the only way to guard against wide spread of MPX. Historically smallpox vaccine used to cause cross-immune protection against other viruses belonging to poxviridae family including MPX. The WHO is recommending to administer new forms of smallpox vaccine for those in medical field who deal with cases of MPX or as a prophylactic treatment within 4 days of contact with a case [61]. Being viruses, the general recommendation by differ- ent health authorities is just symptomatic treatment for symptoms as the use of analgesics and antipyretics to guard against constitutional symptoms as fever, headache and body aches is applied. As for current National Institutes of Health (NIH) guidelines for SARS-CoV-2 it is divided into two phases. The first one is targeting the virus itself while in the phase of early infection and replication and the second one is targeting the dysregulated immune system [57]. guidelines for SARS CoV 2 it is divided into two phases. The first one is targeting the virus itself while in the phase of early infection and replication and the second one is targeting the dysregulated immune system [57]. Pathogenesis of SARS‑CoV‑2 and MPX neuropsychiatric manifestations Such medications are still not recommended as a general treatment for all cases, but are left for those immunocompromised, or severely ill [59, 60]. Despite SARS-CoV-2 manifestations in children are mild yet manifestations of MPX in children may be severe [56]. Conclusion Aggressiveness of treatment is also based on the ill- ness status whether mild, moderate or severe as well as critically ill. For those who are not hospitalized, dexa- methasone or any kind of systemic corticosteroids is not recommended. Novel SARS-CoV-2 took the world by surprise when it emerged as an epidemic and within few months became a pandemic. SARS-CoV-2 has plenty of neuropsychiatric manifestations that play a great deal in its morbidities. As for MPX, it is an already known virus yet still surpris- ing the unknown mode through which it crossed borders from its endemic areas in west and central Africa to other continents. As for those patients who are not hospitalized but are at risk of passing into severe form of SARS-CoV-2 infec- tion as those who are immunocompromised or diabet- ics with uncontrolled diabetes, antiviral medications are to be administered as ritonavir-boosted nirmatrelvir or remdesivir and in case of unavailability then bebt- elovimab or molnupiravir could be administered [57]. Mutations that have been already detected in MPX may have played a role in escaping its endemic region to other regions as well as suspicion of SARS-CoV-2 role in caus- ing mass immunity reduction facilitating the spread of MPX as an opportunistic virus. Hospitalized patients who do not require oxygen sup- plementation are managed as non-hospitalized but with a prophylactic dose of heparin. Meanwhile, hospitalized patients who are on oxygen support are supplied with dexamethasone besides remdesivir and full-dose hepa- rin in case of elevated d-dimer and not pregnant. As for pregnant patients, prophylactic dose of heparin is recommended. As SARS-CoV-2, monkeypox has neuropsychiat- ric challenges. The world health authorities still have a chance to control and manage MPX and prevent a poten- tial pandemic through lessons learned from SARS-CoV-2. Ethics approval and consent to participate Not applicable. 18. Kumar N, Acharya A, Gendelman HE, Byrareddy SN. The 2022 out- break and the pathobiology of the monkeypox virus. J Autoimmun. 2022;131:102855. https://doi.org/10.1016/j.jaut.2022.102855. 18. Kumar N, Acharya A, Gendelman HE, Byrareddy SN. The 2022 out- break and the pathobiology of the monkeypox virus. J Autoimmun. 2022;131:102855. https://doi.org/10.1016/j.jaut.2022.102855. Competing interests Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. 20. Sklenovská N, Van Ranst M. Emergence of monkeypox as the most important orthopoxvirus infection in humans. Front Public Health. 2018;6:241. https://doi.org/10.3389/fpubh.2018.00241. Received: 16 August 2022 Accepted: 10 October 2022 Received: 16 August 2022 Accepted: 10 October 2022 p g p 21. Harris E. What to know about monkeypox. JAMA. 2022;327(23):2278–9. https://doi.org/10.1001/jama.2022.9499. 21. Harris E. What to know about monkeypox. JAMA. 2022;327(23):2278–9. https://doi.org/10.1001/jama.2022.9499. 22. Lai CC, Wang CY, Hsueh PR. Co-infections among patients with COVID- 19: the need for combination therapy with non-anti-SARS-CoV-2 agents? J Microbiol Immunol Infect. 2020;53(4):505–12. https://doi.org/ 10.1016/j.jmii.2020.05.013. Acknowledgements Not applicable. 14. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species severe acute respiratory syndrome- related coronavirus: classifying 2019-nCoV and naming it SARS- CoV-2. Nat Microbiol. 2020;5(4):536–44. https://doi.org/10.1038/ s41564-020-0695-z. g No funds were received to fulfill this work. No funds were received to fulfill this work. Availability of data and materials 16. Novella IS, Presloid JB, Taylor RT. RNA replication errors and the evolu- tion of virus pathogenicity and virulence. Curr Opin Virol. 2014;9:143–7. https://doi.org/10.1016/j.coviro.2014.09.017. 16. Novella IS, Presloid JB, Taylor RT. RNA replication errors and the evolu- tion of virus pathogenicity and virulence. Curr Opin Virol. 2014;9:143–7. https://doi.org/10.1016/j.coviro.2014.09.017. The corresponding author takes full responsibility for the data, has full access to all of the data, and has the right to publish any and all data separate and apart from any sponsor. 17. Ding K, Jiang W, Xiong C, Lei M. Turning point: a new global COVID-19 wave or a signal of the beginning of the end of the global COVID-19 pandemic? Immun Inflamm Dis. 2022;10(4):606. https://doi.org/10. 1002/iid3.606. Consent for publication Not applicable. 19. Isidro J, Borges V, Pinto M, Sobral D, Santos JD, Nunes A, et al. Phy- logenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. Nat Med. 2022. https:// doi.org/10.1038/s41591-022-01907-y. 19. Isidro J, Borges V, Pinto M, Sobral D, Santos JD, Nunes A, et al. Phy- logenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. Nat Med. 2022. https:// doi.org/10.1038/s41591-022-01907-y. Limitations and future directionsh The current review has some limitations. First, it is an initial narrative review as data regarding monkeypox manifestations are still minimal. Future in-depth reviews including systematic ones will for sure add to the current review. Intravenous tocilizumab is kept for hospitalized patients with rapid oxygen demands or systemic inflam- mation. Dexamethasone, remdesivir and intravenous tocilizumab are administered to critically ill patients on high-flow oxygen, non-invasive oxygen, or mechanical ventilation. Second, although presenting a link between two viruses one of them was never faced by the world and another that did not spread to such extent worldwide before although known since the middle of the twenti- eth century yet such presentation ought to be laboratory checked to confirm the role of lowered immunity caused by one virus in facilitating the spread of another.h As for preventive measures directed against SARS- CoV-2, it is recommended to keep a distance not less than 2 m on dealing with a patient, and an infected patient should wear a face covering, with frequent hand washing of caregiver as well as the patient. Vaccines are the most reliable preventive ways against SARS-CoV-2 infection. There are four main types of vaccines: whole virus vaccine, RNA or mRNA vaccine, Third, indirect role of improper distribution of corona virus vaccine along developing countries in monkeypox spread needs in-depth research and accordingly proper Page 10 of 11 Roushdy Egypt J Neurol Psychiatry Neurosurg (2022) 58:127 future guidelines on medical services and preventive medicine distribution ought to be implemented by the world health authorities. 9. El Eid R, Allaw F, Haddad SF, Kanj SS. Human monkeypox: a review of the literature. PLoS Pathog. 2022;18(9):e1010768. https://doi.org/10. 1371/journal.ppat.1010768. 9. El Eid R, Allaw F, Haddad SF, Kanj SS. Human monkeypox: a review of the literature. PLoS Pathog. 2022;18(9):e1010768. https://doi.org/10. 1371/journal.ppat.1010768. 10. Bunge EM, Hoet B, Chen L, Lienert F, Weidenthaler H, Baer LR, et al. The changing epidemiology of human monkeypox—A potential threat? A systematic review. PLoS Negl Trop Dis. 2022;16(2):e0010141. https:// doi.org/10.1371/journal.pntd.0010141. Abbreviations 11. World Health Organization. Monkeypox factsheets. www.who.int/ news-room/fact-sheets/detail/monkeypox. 11. World Health Organization. Monkeypox factsheets. www.who.int/ news-room/fact-sheets/detail/monkeypox. SARS-CoV-2: Severe acute respiratory syndrome corona virus 2; SARS-CoV: Severe acute respiratory syndrome corona virus; MERS-CoV: Middle east respiratory syndrome corona virus; MPX: Monkeypox virus; WHO: World Health Organization; ACE-2: Angiotensin converting enzyme 2; PCR: Polymerase chain reaction; IL: Interleukin; TNF-α: Tumor necrosis factor-alpha; MIS-C: Multisystem inflammatory syndrome in children; NIH: National Institutes of Health. 12. McEntire CRS, Song KW, McInnis RP, Rhee JY, Young M, Williams E, et al. Neurologic manifestations of the World Health Organization’s list of pandemic and epidemic diseases. Front Neurol. 2021;22(12):634827. https://doi.org/10.3389/fneur.2021.634827. g 13. Deshmukh P, Vora A, Tiwaskar M, Joshi S. Monkeypox: what do we know so far? A short narrative review of literature. J Assoc Physicians India. 2022;70(7):11–2. https://doi.org/10.5005/japi-11001-0071. 13. Deshmukh P, Vora A, Tiwaskar M, Joshi S. Monkeypox: what do we know so far? A short narrative review of literature. J Assoc Physicians India. 2022;70(7):11–2. https://doi.org/10.5005/japi-11001-0071. Author contributions TR: conceptualization, collection of the scientific data, writing—original draft preparation, writing and editing, figure preparation. The author read and approved the manuscript. 15. Chan JF, Yuan S, Kok KH, To KK, Chu H, Yang J, et al. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet. 2020;395(10223):514–23. https://doi.org/10.1016/S0140-6736(20) 30154-9. 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https://openalex.org/W4206539395	https://essopenarchive.org/doi/pdf/10.1002/essoar.10510004.1	English	null	Transforming to Open Science in the NASA Science Mission Directorate	null	2,022	cc-by	1,066	Transforming to Open Science in the NASA Science Mission Directorate Steven Crawford1 1NASA Headquarters November 24 2022 November 24, 2022 November 24, 2022 Abstract For over 30 years, NASA Science Mission Directorate has been a world leader in making the data from its ﬂagship missions available for anyone to use. This data represents a signiﬁcant public investment and NASA has a mandate to maximize the accessibility of this information for the public good. Along with the data collected by the Missions, NASA has also supported the curation of data that enables ground breaking research in each of our diﬀerent disciplines. These novel technologies for data access and curation from the diﬀerent divisions have enabled groundbreaking science. As technology advances and data volumes grow, SMD is working towards even further enhancing the accessibility of the information that NASA produces. This includes new policies to assure that NASA data is FAIR (Findable, Accessible, Interoperable, and Reusable) in the future, leverage techniques and technologies developed by the diﬀerent divisions for accessing information across the SMD, enabling new technologies like machine learning and cloud processing along with high performance computing, providing support for citation of data through digital object identiﬁers, creating the ability to search across SMD to enable cross divisional science, and making sure SMD data are accessible to everyone. With these developments, NASA SMD aims to help the community to Transform to Open Science and accelerate the process of scientiﬁc discovery. In the process, this will enable SMD science to be even more inclusive, reproducible, and transparent. 1 1 Transform to Open Science Steve Crawford Kevin Murphy, Katie Baynes, Chelle Gentemann, Yvonne Ivey, Manil Maskey, Kaylin Bugbee, Yaitza Luna-Cruz, Elena Steponaitis, Emily Cassidy, Christian Reyes, Frances Adele 17 December 2021 OPEN-SOURCE SCIENCE Building on the concepts from Open-Source Software, open source science conducts science openly to expand access and uses transformative technology to accelerate science. Fiscal year OSS Total ($M) FY21 $8 FY22 $21 FY23 $20 FY24 $20 FY25 $20 FY26 $20 FY27 $20 • Transform to OPen Science (TOPS) • Investments in open-source science infrastructure • Cross-divisional AI capabilities and • Digital Transformation activities • ROSES elements for open source software • Open scientific cloud environments • Data analysis platform prototypes Prototype common data catalog by FY22Q4 and expand Astrophysics Data System $130M in Divisional investments in Open-Source Science that are aligned with this program. SPD-41: Scientific Information Policy SPD-41: The Science Information Policy is the first SMD-wide policy on data, software and information. Learn more at the Science Information Policy Website. SPD-41: The Science Information Policy is the first SMD-wide policy on data, software and information. Learn more at the Science Information Policy Website. ●Consolidation of existing policies and laws applicable to SMD. ●Consolidation of existing policies and laws applicable to SMD. ●Consolidation of existing policies and laws applicable to SMD. ●Applies to all SMD-funded activities related to producing scientific information. The policy excludes restricted information such as ITAR, export control, CUI. ●Applies to all SMD-funded activities related to producing scientific information. The policy excludes restricted information such as ITAR, export control, CUI. ●A new Request for Information (RFI) solicits feedback on SPD-41, including support needed for successful implementation and proposed additions: go.nasa.gov/RFISPD41. Responses are due Feb 11, 2021. ●A new Request for Information (RFI) solicits feedback on SPD-41, including support needed for successful implementation and proposed additions: go.nasa.gov/RFISPD41. Responses are due Feb 11, 2021. ●Questions can be sent to HQ-SMD-SPD41@mail.nasa.gov. ●Questions can be sent to HQ-SMD-SPD41@mail.nasa.gov. nd P Data ● Mission data will be shared with no period of exclusive access ● Data shall be FAIR (Findable, Accessible, Interoperable, and Reusable) ● Mission software is developed openly ● Research software released as open- source software with a permissive license ● Mission software is developed openly ● Research software released as open- source software with a permissive license ● Research software released as open- source software with a permissive license ● Data shall be FAIR (Findable, Accessible, Interoperable, and Reusable) Increasing Access to SMD Publications SA has a mandate to ensure access to and able preservation of peer-reviewed publications t arise from NASA-funded research. NASA SMD supports publishing as Open Access and encourages using preprint servers. SMD is funding ADS to expand its holdings in Heliophysics and Planetary Science. NASA STI is working on an agreement with CHORUS and improving the PubSpace interface. We will be developing further guidance and services to make it automatic to preserve and make your publications accessible. 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https://openalex.org/W4224289920	https://www.auctoresonline.org/uploads/articles/1646042385Stress_fractures_in_Sports.pdf	English	null	Stress Fractures in Sports	Surgical case reports and images	2,022	cc-by	4,356	Stress Fractures in Sports Philip Catalá-Lehnen* Department of Orthopedics Surgery, Germany. ponding Author: Philip Catalá-Lehnen, Department of Orthopedics Surgery, Germany. Corresponding Author: Philip Catalá-Lehnen, Department of Orthopedics Surgery, Germany. Corresponding Author: Philip Catalá-Lehnen, Department of Orthopedics Surgery, Germany. Received date: September 28, 2021; Accepted date: October 25, 2021; published date; January 05, 2022 Copyright: © 2022, Philip Catalá-Lehnen, 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. Copyright: © 2022, Philip Catalá-Lehnen, This is an open access article distributed under the Creative Comm unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. , This is an open access article distributed under the Creative Commons Attribution License, which permits ion in any medium, provided the original work is properly cited. Abstract Foot injuries make up a not insignificant proportion of sports injuries, but the kind of frequency varies depending on the sport. According to the 2018 Sports Report of the Verwaltungs-Berufsgenossenschaft (VBG), basketball accounted for 7.4 % of foot injuries Keywords: stress fractures; sports; foot injuries Keywords: stress fractures; sports; foot injuries Introduction fractures mostly involve the second and third metatarsals [6]. Irrespective of whether an acute or stress fracture is present, it is essential to identify as soon as the cause and contributory risk factors as quickly as possible factors. Subsequent treatment should take these into account. Foot injuries make up a not insignificant proportion of sports injuries, but the kind of frequency varies depending on the sport. According to the 2018 Sports Report of the Verwaltungs-Berufsgenossenschaft (VBG), basketball accounted for 7.4 % of foot injuries, ice hockey for 5.6 %, football for 10.3 % and handball for 4.3 % [20]. Metatarsal fractures are the most common entity [3]. The first metatarsal is the least commonly fractured, accounting for approximately 5 % of metatarsal fractures, and the fifth metatarsal the most commonly fractured, accounting for approximately 56 %. Fractures are evenly spread among the other metatarsals [6]. Stress fractures may occur as well as traumatic fractures. They account for 38 % of stress fractures of the lower extremity Stress J. Surgical Case Reports and Images Copy rights@ Philip Catalá-Lehnen. Stress Fractures in Sports Open Access Brief Report Journal of Surgical Case Reports and Images Philip Catalá-Lehnen * AUCTORES Globalize your Research J. Surgical Case Reports and Images Copy rights@ Philip Catalá-Lehnen. Stress Fractures in Sports Open Access Brief Report Journal of Surgical Case Reports and Images Philip Catalá-Lehnen * AUCTORES Globalize your Research Symptoms Whereas in acute fractures sudden onset of pain is typically a cardinal symptom, in metatarsal stress fractures it generally develops gradually. Reduced loading capacity is associated with fractures irrespective of aetiology. Adequate early diagnosis and identification of risk factors are particularly detraining important in competitive sports to avoid long periods without training or competition. Fig. 1: 20-year-old patient, 2nd Handball Bundesliga, recurrent fracture sustained during the game, fifth metatarsal: (a) initial MRI with visible fracture gap and marginal fluid collection, laboratory tests showed no vitamin D deficiency, decision made for conservative treatment with physiotherapy, shock wave therapy (ESWT) and carbon inserts; (b) follow-up X-ray after 9 weeks, clear callus formation with surroun-ding oedema, plantar cortical gap still present; (c) X-ray on completion of conservative treatment after 13 weeks, clear callus formation in the fracture area with some small areas of still unconsolidated cortex. Fig. 1: 20-year-old patient, 2nd Handball Bundesliga, recurrent fracture sustained during the game, fifth metatarsal: (a) initial MRI with visible fracture gap and marginal fluid collection, laboratory tests showed no vitamin D deficiency, decision made for conservative treatment with physiotherapy, shock wave therapy (ESWT) and carbon inserts; (b) follow-up X-ray after 9 weeks, clear callus formation with surroun-ding oedema, plantar cortical gap still present; (c) X-ray on completion of conservative treatment after 13 weeks, clear callus formation in the fracture area with some small areas of still unconsolidated cortex. Auctores Publishing LLC – Volume 5(1)-098 www.auctoresonline.org ISSN: 2690-1897 Page 1 of 6 Copy rights@ Philip Catalá-Lehnen. J. Surgical Case Reports and Images hyperparathyroidism (sHPT). Prolonged sHPT is associated with impaired bone turnover [23, 17] and can also lead to loss of mineral salts [26, 17]. Guidelines reco-mmend measurement of 25-hydroxy vitamin D serum levels in all patients with sHPT [22]. hyperparathyroidism (sHPT). Prolonged sHPT is associated with impaired bone turnover [23, 17] and can also lead to loss of mineral salts [26, 17]. Guidelines reco-mmend measurement of 25-hydroxy vitamin D serum levels in all patients with sHPT [22]. Diagnosis Following medical history and physical examination, it is advisable to perform diagnostic ultrasound of the foot to gain a more better understanding of the fracture. In the hands of an experienced investigator, diagnostic ultrasound is just as sensitive and specific as diagnostic X-ray [4]. However, if a fracture is suspected, this should be followed by diagnostic X-ray of the foot in three planes [3]. In fractures of the base of metatarsals 1 – 4, computer tomography should also be perfor-med, as such fractures may be associated with Lisfranc dislocation fractures. In the case of stress fractures, MRI is the gold standard, as X-ray images are often falsely negative [36]. Osteopenia Osteopenia can occur alone or as a result of -vitamin D deficiency [17]. Diagnosis mostly involves dual-energy X-ray absorptiometry (DXA). A new procedure will allow bone density to be determined without the need for exposure to radiation based on calcium homeostasis in the skeleton [9]. Risk factors It is crucial to identify any predisposing factors. For example, stress fractures are more common in ballet dancers due to repeated maximum plantar flexion [36, 27]. Athletes in sports with high running or jumping loads are also at risk. As retiring from the sport is not an option for competitive athletes, it is essential to identify other possible causes. These may include changes in loading, such as increased training volumes, changes in surface, new shoes, special shoe inserts or biomechanical compensation mechanisms following prior injury [7, 21]. Systemic causes should also be considered. These include changes in hormone balance, eating disorders and malabsorption syndrome [1, 2]. The female athlete triad is a common example [33, 1]. It is therefore important to check intake of protein, calcium and vitamins D and K2, as well as use of alcohol, opioids and tobacco [28, 10]. Nonsteroidal anti-inflammatory drugs (NSAIDs) in particular can have a negative impact on fracture healing. Use of NSAIDs has caused a significant decrease in trabecular bone mass in ectopic ossification areas due to the decreased number and activity of osteoblasts. This is caused by interference with the bone morphogenetic protein-7 (BMP-7) signalling pathway. Although trauma is the direct cause in acute fractures, the other above factors can predispose to fracture and should therefore also be considered even if the immediate cause is apparent. [32] Biomechanical compensation mechanisms If the above mentioned risk factors have been ruled out or recurrent metatarsal injuries occur, structural compensation mechanisms should also be considered [8]. These mechanisms can result in an imbalance in the loading and unloading of bones and soft tissue structures, resulting in fracture [21]. From a biomechanical perspective, chan-ges in movement vectors and the resulting force vectors lead to incorrect loading e.g overloading [31]. A picture of the movement pattern and the resulting loading vectors can be gained using gait/running analysis, which allows the kinematics and kinetics of the lower extremity to be observed [5]. Use can also be made of dynamic procedures such as foot pressure measurement and gait analysis [7, 5, 11]. These allow observation of, for example, running and foot roll behaviour. Based on the findings, it is possible to counteract incorrect biomechanical and compensation loading with the choice of individual shoe inserts and foot muscle training Diet Diet can have both a positive and negative effect on fracture healing. First, it is essential to avoid any toxins. Smoking and heavy drinking are associated with loss of bone mass (osteo-penia) and increased risk of fracture [28]. The available data on moderate drinking is still inconclusive [38]. In general, a fully nutritious diet is essential in any injury to ensure an adequate supply of energy and protein. In this case, 2 – 2.5 g protein / kg body weight are recommended. It is especially important to prevent micronutrient deficiencies. Vitamins D and K2, magnesium and boron are parti-cularly important in promoting bone healing. Auctores Publishing LLC – Volume 5(1)-098 www.auctoresonline.org ISSN: 2690-1897 Conservative or surgical treatment gradual restoration of weight bearing as the bone heals [15]. Stress fractures of the fourth and fifth metatarsal in particular are more prone to non-union [36]. In a case series, 11 athletes with stress fractures of the base of the fourth metatarsal were therefore treated with plate fixation and autologous bone grafting from the calcaneus. On average, all athletes were able to return to sports after 12 weeks [30]. Shaft fractures of metatarsals 2 – 5 with no or slight dislocation can be treated conservatively. Dislocations of up to 3 mm and plantar malalignment up to 10° are amenable to conservative treatment [3]. In such cases, immobilisation in a short walker with full weight bearing adjusted according to pain level is sufficient. This treatment when combined with a change of shoe to one with a rigid sole after 4 – 6 weeks has the advantage of a better functional outcome compared with immobilisation in a lower leg plaster cast [4]. Provision with a carbon sole is also a primary consideration. Fractures of the first metatarsal with dislo-cation should be surgically stabilised, as this bone plays a central role in carrying body weight and in the foot arch [4]. Fractures of more than one meta-tarsal, even with slight dislocation, require surgery [3]. In subcapital and metatarsal head fractures, axial deviations up to 10o are similarly amenable to conservative treatment. Procedures to promote bone healing Non-union may occur with both acute and stress fractures. There are various treatment procedures that may be used before surgical revision needs to be considered. It is essential not to delay their use until it is too late but to initiate application as early as possible in the healing process. Shock wave therapy Shock waves ensure increased production of growth factors, nitrogen oxides and free radicals, which trigger the healing process. This can be exploited to treat stress fractures [15]. Shock wave therapy induces angiogenesis, and mesenchymal stem cells differentiate to osteoblasts. The periosteum is also stimulated, which plays a major role in callus formation [15]. There are a number of case series that report positive outcomes for stress fractures. These involved the use of focused medium to high energy shock waves. Conservative treatment consists of unloading the forefoot by means of a rigid sole with full weight bearing adjusted according to the pain level. For fractures with greater dislocation and extra-articular fractures, intramedullary wires are the fracture-fixation procedure of choice [3]. Surgery should also be indicated for shortened metatarsals or rotation defects of the toes [12]. The periosteum is also stimulated, which plays a major role in callus formation [15]. There are a number of case series that report positive outcomes for stress fractures. These involved the use of focused medium to high energy shock waves. In most cases the same doses were used as for non-union. The best outcomes were achieved with 2000 impulses at 0.2 mJ/mm2 in 2 sessions. For non-union, there are individual rando-mised, controlled studies as well as case series. The healing rates with shock wave therapy do not differ from those following surgery. In the studies 4000 impulses were applied at an energy flow density of 0.09 – 0.7 mJ/mm2 in each of generally 3 or 4 sessions. Auctores Publishing LLC – Volume 5(1)-098 www.auctoresonline.org ISSN: 2690-1897 Vitamin D deficiency Vitamin D plays an important role in calcium and phosphate homeostasis. Particularly in combination with calcium it helps maintain a balanced bone metabolism [18, 19]. It is therefore essential to rule out vitamin D deficiency (< 25 nmol/L) [9] and any possibility of resulting secondary Auctores Publishing LLC – Volume 5(1)-098 www.auctoresonline.org ISSN: 2690-1897 Page 2 of 6 Copy rights@ Philip Catalá-Lehnen. J. Surgical Case Reports and Images Fig.2: Diagnosis and conservative treatment of metatarsal fractures Treatment Fi 2 Di i d i f l f T Pulsating electromagnetic field therapy (PEMF) studies indicate greater cost effectiveness compared with surgical revision [14]. studies indicate greater cost effectiveness compared with surgical revision [14]. PEMF is another procedure being advocated for non-union and delayed healing. Studies have demonstrated good healing of Jones fractures with non-union, particularly when the devices have been used for more than 9 hours a day [24]. Fracture healing was more quickly achieved following surgery in the treatment group than in the group receiving placebo magnetic field therapy [34]. The evidence of the results, however, is problematic because a control group was part of the study design in only one of the studies. Special features of stress fractures Stress fractures commonly involve the neck of the second and third metatarsal and the shaft of the fifth metatarsal. A very pronounced foot arch increases the risk of stress fractures of the fifth metatarsal due to increased loading [12]. It can take 3 – 6 months for the fracture to heal. As with traumatic fractures, treatment consists of initial unloading with Auctores Publishing LLC – Volume 5(1)-098 www.auctoresonline.org ISSN: 2690-1897 Page 3 of 6 J. Surgical Case Reports and Images Copy rights@ Philip Catalá-Lehnen. patient, 2nd Handball Bundesliga with recurrent fracture of the fifth metatarsal four months after the initial injury: MRI after (a) bone marrow oedema in the fifth metatarsal and (b) proximal fracture gap. Follow-up X-ray 2 months after (c) recurrent onservative treatment with physiotherapy, shock wave therapy and provision with carbon inserts, and (d) consolidation of the fracture. omagnetic field therapy (PEMF) studies indicate greater cost effectiveness compared with surgical revision Fig. 3: 20-year-old patient, 2nd Handball Bundesliga with recurrent fracture of the fifth metatarsal four months after the initial injury: MRI after repeat injury shows (a) bone marrow oedema in the fifth metatarsal and (b) proximal fracture gap. Follow-up X-ray 2 months after (c) recurrent fracture following conservative treatment with physiotherapy, shock wave therapy and provision with carbon inserts, and (d) consolidation of the fracture. Fig. 3: 20-year-old patient, 2nd Handball Bundesliga with recurrent fracture of the fifth metatarsal four months after the initial injury: MRI after repeat injury shows (a) bone marrow oedema in the fifth metatarsal and (b) proximal fracture gap. Follow-up X-ray 2 months after (c) recurrent fracture following conservative treatment with physiotherapy, shock wave therapy and provision with carbon inserts, and (d) consolidation of the fracture. Platelet rich plasma (PRP) Animal studies have shown positive effects of PRP on fracture healing [13]. In view of these results a beneficial effect is also conceivable in humans. Clinical experience shows that additional treatment with PRP does lead to a positive course in a protracted course of fracture healing. Overall, the evidence for PRP promoting fracture healing is scarce [29]. On the other hand, injection of PRP has been successful in alleviating pain and in associated biomechanical compensation mechanisms as well as protective guarding and movement avoidance [25, 37]. However, further evidence-based studies are required for a more evidence-based and conclusive assessment [16]. Ultrasound Studies point to improved healing in non-union, but in some there was no control group [35, 14]. Good healing rates were achieved, and individual Auctores Publishing LLC – Volume 5(1)-098 www.auctoresonline.org ISSN: 2690-1897 Auctores Publishing LLC – Volume 5(1)-098 www.auctoresonline.org ISSN: 2690-1897 Page 4 of 6 Copy rights@ Philip Catalá-Lehnen. J. Surgical Case Reports and Images J. Surgical Case Reports and Images Fig. 4: 23-year-old patient, 2nd Handball Bundesliga with proximal fracture of the third metatarsal: (a) CT shows proxi- mal fracture of the third metatarsal with joint involvement; (b) also evidence of bone marrow oedema throughout the third metatarsal on MRI. Fig. 4: 23-year-old patient, 2nd Handball Bundesliga with proximal fracture of the third metatarsal: (a) metatarsal with joint involvement; (b) also evidence of bone marrow oedema throughout th Fig. 4: 23-year-old patient, 2nd Handball Bundesliga with proximal fracture of the third metatarsal: (a) CT shows proxi- mal fracture of the third metatarsal with joint involvement; (b) also evidence of bone marrow oedema throughout the third metatarsal on MRI. Summary 7. Geoffrey J Dowling, George S Murley, Shannon E Munteanu, Melinda M Franettovich Smith, Bradley S Neal, Ian B Griffiths, Christian J Barton, and Natalie J Collins. (2014) Dynamic foot function as a risk factor for lower limb overuse injury: a systematic review. Journal of foot and ankle research, 7(1):53. The first important step in the treatment of meta--tarsal stress fractures is to figure out the cause and identify any risk factors. It can help prevent secondary injury. This should be followed by multimodal treatment comprising modified weight bearing and optimised bone healing. The pain experienced by the patient plays a central role particularly with regard to modi-fying weight bearing. However, due to their negative effect on fracture healing, administration of NSAIDs should be avoided. Pain can be reduced, and bone healing promoted by diet, shock wave therapy and, when applicable, supplementary electromagnetic field therapy. 8. Katherine M Edenfield, Charlie Michaudet, Guy W Nicolette, and Peter J Carek. (2018) Foot and ankle conditions: Midfoot and forefoot conditions. FP essentials, 465: 30-34. 9. Anton Eisenhauer, M Müller, Alexander Heuser, Ana Kolevica, C-C Glüer, M Both, C Laue, Uv Hehn, S Kloth, R Shroff, et al. (2019) Calcium isotope ratios in blood and urine: A new biomarker for the diagnosis of osteoporosis. Bone reports, 10:100200. References 1. 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(2017) Stress fractures of the footand ankle. Injury, 48(8):1722-1726. 25. H Namazi and A Mehbudi. (2016) Investigating the effect of intra-articular prp injection onpain and function improvement in patients with distal radius fracture. Orthopaedics & Traumatology: Surgery & Research, 102(1):47-52. 37. Yung-Tsan Wu, Kao-Chih Hsu, Tsung-Ying Li. (2018) Effects of platelet-rich plasma on pain and muscle strength in patients with knee osteoarthritis. American journal of physical medicine & rehabilitation, 97 (4):248-254. 26. HJ Oh, B-H Yoon, Y-C Ha, D-C Suh, S-M Lee, K-H Koo, and Y-K Lee. (2020) The change of bone mineral density and bone metabolism after gastrectomy for gastric cancer: a meta- analysis. Osteoporosis International, 31(2):267-275. 38. Shuai Yuan, Karl Michaelsson, Zihao Wan, and Susanna C Larsson. (2019) Associations of smoking and alcohol and coffee intake with fracture and bone mineral density: a mendelian randomization study. Calcified tissue international, 105(6):582-588. 38. Shuai Yuan, Karl Michaelsson, Zihao Wan, and Susanna C Larsson. References (2019) Associations of smoking and alcohol and coffee intake with fracture and bone mineral density: a mendelian randomization study. Calcified tissue international, 105(6):582-588. 27. Martin J O’Malley, William G Hamilton, John Munyak, and Michael J DeFranco. (1996)Stress fractures at the base of the Ready to submit your research? Choose Auctores and benefit from:  fast, convenient online submission  rigorous peer review by experienced research in your field  rapid publication on acceptance  authors retain copyrights  unique DOI for all articles  immediate, unrestricted online access At Auctores, research is always in progress. Learn more https://auctoresonline.org/journals/journal-of-surgical-case- reports-and-images Ready to submit your research? Choose Auctores and benefit from:  fast, convenient online submission  rigorous peer review by experienced research in your field  rapid publication on acceptance  authors retain copyrights  unique DOI for all articles  immediate, unrestricted online access At Auctores, research is always in progress. Learn more https://auctoresonline.org/journals/journal-of-surgical-case- reports-and-images Ready to submit your research? Choose Auctores and benefit from: This work is licensed under Creative Commons Attribution 4.0 License To Submit Your Article Click Here: Submit Manuscript DOI: 10.31579/2690-1897/098 This work is licensed under Creative Commons Attribution 4.0 License To Submit Your Article Click Here: Submit Manuscript DOI: 10.31579/2690-1897/098 Auctores Publishing LLC – Volume 5(1)-098 www.auctoresonline.org ISSN: 2690-1897 Auctores Publishing LLC – Volume 5(1)-098 www.auctoresonline.org ISSN: 2690-1897 Page 6 of 6
https://openalex.org/W2170135958	https://www.frontiersin.org/articles/10.3389/fcvm.2014.00017/pdf	English	null	The Importance of Patency in Patients with Critical Limb Ischemia Undergoing Endovascular Revascularization for Infrapopliteal Arterial Disease	Frontiers in cardiovascular medicine	2,015	cc-by	7,063	bli h d 0 J 20 MINI REVIEWS IN MEDICINE published: 07 January 2015 doi: 10.3389/fcvm.2014.00017 Frederic Baumann1, Christoph Ozdoba2, Ernst Gröchenig 3 and Nic 1 Miami Cardiac and Vascular Institute, Baptist Hospital, Miami, FL, USA 2 Clinical and Interventional Neuroradiology, University Hospital Bern, Bern, Switzerland 3 Clinical and Interventional Angiology, Kantonsspital Aarau, Aarau, Switzerland 4 University of Applied Sciences Furtwangen, Villingen-Schwenningen, Germany 1 Miami Cardiac and Vascular Institute, Baptist Hospital, Miami, FL, USA 2 Clinical and Interventional Neuroradiology, University Hospital Bern, Bern, Switzerland 3 Clinical and Interventional Angiology, Kantonsspital Aarau, Aarau, Switzerland 4 University of Applied Sciences Furtwangen, Villingen-Schwenningen, Germany Critical limb ischemia (CLI) represents the most severe form of peripheral arterial disease (PAD) and frequently occurs in medically frail patients. CLI patients frequently exhibit multi- segmental PAD commonly including the tibial arterial segment. Endovascular therapy has been established as ﬁrst-line revascularization strategy for most CLI patients. Restenosis was reported to occur in up to more than two-thirds of CLI patients undergoing angio- plasty of complex tibial arterial obstructions. Nevertheless, favorable clinical outcomes were observed for infrapopliteal angioplasty when compared with bypass surgery, despite higher patency rates for the latter. Based on these observations, infrapopliteal patency was considered to be only of secondary importance upon clinical outcomes in CLI patients. In contrast to these earlier observations, however, recent ﬁndings from two randomized clinical trials indicate that infrapopliteal patency does impact on clinical outcomes in CLI patients. The purpose of the present manuscript is to provide a critical reappraisal of the present literature on the clinical importance of tibial arterial patency in CLI patients undergo- ing endovascular revascularization and to discuss utility and limitations of currently available anti-restenosis technologies. METHODS INTRODUCTION Critical limb ischemia (CLI) represents a medically frail subgroup of patients presenting with the most severe form of peripheral arterial disease (PAD) (1–3). In these patients, revascularization is among the cornerstones of treatment and aims at the prevention of amputation and improvement of quality of life (1, 4, 5). CLI patients frequently present with multilevel PAD including com- plex obstructions of tibial arteries and, thus, frequently require challenging revascularization procedures. Endovascular therapy has been established as a ﬁrst-line revascularization strategy for most CLI patients (5, 6) since it was shown to provide com- parable clinical outcomes when compared with bypass surgery, despite higher patency rates for the latter (5, 7). Based on these observations, the tide over concept was established assuming that tibial arterial patency was mandatory only during the process of wound healing but not thereafter for the maintenance of skin integrity and resolution of CLI symptoms (7–9). More recent stud- ies, however, have stressed the importance of tibial patency upon clinical outcomes in CLI patients,thereby challenging the tide over concept (10–13). A comprehensive literature research was performed based on Pubmed database. All studies included in the meta-analysis by Romiti et al. (7) were acquired using Pubmed and analyzed there- after (researched April 2013). In addition, we reviewed literature for completed and ongoing randomized trials on drug-eluting stents (DESs) and drug-eluting balloon (DEB) versus bare metal stent (BMS) and/or plain-old balloon angioplasty (POBA) for tib- ial arterial revascularization. The latter literature research was based on Pubmed (www.pubmed.org, last accessed on March 13, 2014) and clinicaltrials.gov (last accessed on April 28, 2014) entries. INCIDENCE OF TIBIAL ARTERIAL RESTENOSIS AFTER ANGIOPLASTY Correspondence: Correspondence: Nicolas Diehm, Clinical and Interventional Angiology, Kantonsspital Aarau, Tellstrasse, 5001 Aarau, Schweiz, Switzerland e-mail: nicolas.a.diehm@gmail.com *Correspondence: Nicolas Diehm, Clinical and Interventional Angiology, Kantonsspital Aarau, Tellstrasse, 5001 Aarau, Schweiz, Switzerland e-mail: nicolas.a.diehm@gmail.com Keywords: critical limb ischemia, below the knee, endovascular revascularization, patency Edited by: Edited by: Marianne Brodmann, Medical University Graz, Austria Reviewed by: Erich Minar, Medical University of Vienna, Austria Heiko Uthoff, University Hospital Basel, Switzerland Martin Werner, Medical University of Vienna, Austria Marianne Brodmann, Medical University Graz, Austria Reviewed by: Erich Minar, Medical University of Vienna, Austria Heiko Uthoff, University Hospital Basel, Switzerland Martin Werner, Medical University of Vienna, Austria IMPACT OF TIBIAL ARTERIAL PATENCY ON CLINICAL OUTCOMES IN CLI Inadditiontotheselimitations,theimportanceof tibialpatency upon clinical outcomes was recently endorsed by various studies (10–13). It was shown that CLI patients require frequently tar- get lesion revascularization (TLR) to maintain favorable clinical results subsequent to tibial angioplasty. Within the aforemen- tioned study by Iida, TLR was necessary in 48% of patients with documented restenosis of the tibial target lesion in 73% at 12-month follow-up. In addition, the authors observed a pro- longed time of wound healing in patients with tibial resteno- sis when compared to patients without restenosis: 127 versus 66 days (P = 0.02). The high prevalence of tibial TLR was recently corroborated by Baumann et al. within a consecutive series of 128 CLI patients undergoing tibial angioplasty (13). That group aimed for a comparison analyzing the clinically driven need for TLR versus target extremity revascularization (TER) after tib- ial angioplasty. After 1 year, TLR was performed in 41.6% and TER in 17.2% of patients. While adding proof to the high preva- lence of TLR after tibial angioplasty, that observation more- over indicated that tibial restenosis is of greater clinical impact than progression of atherosclerotic disease as reﬂected by TER rates. Despite the high rates of tibial restenosis subsequent to POBA, endovascular revascularization is considered the ﬁrst-line treat- ment strategy for most CLI patients (5, 6). Until now, the BASIL trial was the ﬁrst and only trial randomly comparing endovascular therapy with open surgery in CLI patients undergoing infrain- guinal revascularization (5). In that trial, a total of 452 patients (224 angioplasty, 228 surgery) were analyzed for amputation-free survival. After 1 year, an amputation-free survival was obtained in 68 versus 71% and after 3 years 57 versus 53% comparing bypass surgery with angioplasty (P > 0.05). This observation was under- lined by a frequently cited meta-analysis by Romiti et al. (7). In that meta-analysis, the impact of tibial patency on clinical out- comes comparing endovascular versus surgical revascularization strategies in CLI was scrutinized. For that reason, a total of 30 studies including 2646 patients were analyzed (Table 1). Primary patency was 58.1, 51.3, and 48.6% after tibial angioplasty and 81.5, 76.8,and 72.3% after tibial bypass surgery at 12,24,and 36 months (P < 0.05). INCIDENCE OF TIBIAL ARTERIAL RESTENOSIS AFTER ANGIOPLASTY Restenosis remains the major drawback in CLI patients undergo- ing endovascular therapy of tibial arterial obstructions. Various studies reported tibial restenosis to occur in up to more than two- thirds of patients undergoing angioplasty of complex tibial arterial obstructions (10–12, 14, 15). Within a prospective study, Schmidt and colleagues evaluated the incidence of tibial arterial resteno- sis in CLI patients undergoing POBA. Restenosis was deﬁned as a lumen compromise ≥50% on serial angiography after 3 months (15). A total of 58 CLI patients (77 limbs) with a mean tibial lesion length of 184 mm were analyzed. In that cohort, binary The purpose of this review article is to provide a critical reap- praisal of the importance of tibial arterial patency on clinical outcomes in CLI patients. Moreover, we seek to analyze recently performed trials on endovascular revascularization for tibial PAD and to stress their applicability to everyday clinical practice. January 2015 \| Volume 1 \| Article 17 \| 1 www.frontiersin.org www.frontiersin.org Patency and infrapopliteal revascularization Baumann et al. restenosis was observed in 68.8% of limbs. Similar results were observed by Liistro and coworkers within the DEBATE-BTK trial (10) comparing POBA versus DEB. In that study, binary restenosis was assessed by angiography and deﬁned as a reduction of luminal diameter > 50%orbyduplexsonographydeﬁnedasapeaksystolic velocity index ≥2.5. After 1 year, tibial restenosis was observed in 74% in the POBA (74 limbs, mean lesion length: 131 mm) and 27% in the DEB group (74 limbs, mean lesion length: 129 mm [P < 0.001]). In addition, Iida and coworkers analyzed the inci- dence of tibial restenosis and its impact on clinical outcomes in CLI patients after POBA in a total of 63 patients (12). Restenosis was evaluated angiographically and deﬁned as a reduction of lumi- nal diameter ≥50%. After 3 months,tibial restenosis was observed in 74/102 (73%) of treated lesions. Of note, no detailed informa- tion on tibial lesion length was provided in that study, although tibial lesion length was shown to be indicative for the risk of tibial restenosis (16). functional clinical outcomes is limited due to substantial variabil- ity of clinical end point deﬁnitions or the lack of clinical outcome reports at all (20, 33). Clinical end point deﬁnitions included sub- jective relief,freedom from CLI,improvement of clinical classiﬁca- tion, and limb salvage (8, 32, 40, 41). Moreover, clinical outcomes were not reported throughout all studies but only in 26/30 (86.7%) studies. INCIDENCE OF TIBIAL ARTERIAL RESTENOSIS AFTER ANGIOPLASTY Third, systematic patency evaluation was performed in only 9/30 (30%) of studies included within the meta-analysis (17– 19, 24, 33, 37, 38, 44, 45). Remarkably, patency was assessed by duplex sonography in 8/30 (26.7%) studies and by angiography in only 1 (3.3%) study in a total of 60/2646 (2.3%) patients. Thus, the vast majority of patency evaluation was performed by duplex sonography, although its validity in tibial arteries is highly contro- versial (42). In addition,information on tibial patency was derived from the clinical need for repeated intervention in 4/30 (13.3%) studies. Thus, arterial patency rates may have been overestimated utilizing this surrogate deﬁnition. Fourth, Romiti et al. published 3-year outcomes, although only 16/30 (53.3%) studies reported follow-up results beyond 24 months. IMPACT OF TIBIAL ARTERIAL PATENCY ON CLINICAL OUTCOMES IN CLI Of interest, despite these signiﬁcant differences in patency rates, no signiﬁcant differences in clinical outcomes were observed: limb salvage rates were 86.0, 83.8, and 82.4% in patients treated by angioplasty and 88.5, 85.2, and 82.3% after tibial bypass surgery at same intervals. Based on these ﬁndings, the tide over concept was established assuming that increased perfusion was mandatoryforulcerhealinginCLIbutnotthereafterformaintain- ing skin integrity. Therefore, tibial arterial patency was considered to be of minor importance during mid- and long-term follow-up of CLI patients. Moreover, Rastan et al. were the ﬁrst to underline the impor- tance of tibial patency within a randomized setting (11). The Yukon BTK trial randomly assigned a total of 161 patients compar- ing DES (82 patients) to BMS (79 patients) for tibial angioplasty in CLI. Primary clinical end point in the Yukon BTK trial was an event-free survival deﬁned as freedom from target limb ampu- tation, target vessel revascularization, myocardial infarction, and death. After a follow-up period of 1016 days, an event-free sur- vival was attained in 65.8% in the DES group versus in 44.6% in the BMS group (P = 0.02). In line with clinical observations, pri- mary tibial patency at 1 year was 80.6 versus 55.6% (P = 0.004) when comparing DES with BMS (46). For several reasons, however, the validity of the methodological design and conclusion of that meta-analysis must be consid- ered as limited. First, the sample size in the majority of stud- ies included was small and ranged from 23 to 537 patients. Of note, 16/30 (53.3%) studies had included less than 50 patients. In addition, 9/30 (30%) studies included both CLI and patients with intermittent claudication. Thus, a substantial fraction of the patients (548/2646, 20.7%) did not suffer from limb-threatening ischemia. Accordingly, statistical power of both individual studies and the meta-analysis was limited. Second, a direct comparison of Thus, in consideration of these substantial limitations behind the tide over concept and given observations from more recent clinical trials (11–13), the ultimate importance of tibial arter- ial patency subsequent to endovascular therapy remains to be determined. January 2015 \| Volume 1 \| Article 17 \| 2 Frontiers in Cardiovascular Medicine \| Vascular Medicine Patency and infrapopliteal revascularization Baumann et al. Table 1 \| Summary of all studies analyzed within the meta-analysis of Romiti and coworkers. Ref. Patients (n) Limbs (n) s/e Patency evaluation (speciﬁcations) End points Reported fu (months) Mean fu (months Haider et al. IMPACT OF TIBIAL ARTERIAL PATENCY ON CLINICAL OUTCOMES IN CLI (17) 32 32 e DUS PP: 60% 24 n.i. Kudo et al. (18) 52 52 e DUS/ABI PP: 23.5%, SP: 46.1%, LS: 77.3% 36 14.7 Boyer et al. (19) 49 49 e DUS PP: 81%, SP: 88%, LS: 87% 36 21 Parsons et al. (20) 66 66 e ABI/pulse volume recordings PP: < 15% 12 n.i. Spinosa et al. (21) 37 37 e ABI/pulse volume recordings LS: 66% 12 7.8 No info on patency Wölﬂe et al. (22) s:125 130, IC: 3, CLI: 127 s/e CI/ABI (DUS after 1991) PPs: 46%, SPs: 49%, LSs: 63% 84 (s) n.i. e:84 89, IC: 5, CLI: 84 LSe: 63%, e: no patency information 72 (e) n.i. Marzelle et al. (23) 23 23 e Clinical PP: 34%, LS: 71% 12 8.6 Vraux et al. (24) 36 40 e DUS PP: 56%, SP: 72%, LS: 81% 12 10 Treiman et al. (25) 25, IC: 5, CLI: 20 25 e CI: ABI, DUS/angiography, (if ABI-impair > 0.1 or clinical deterioration) CI: 59% (32%, 20%) 12 (24, 36) 44 Brosi et al. (26) 29 38, IC: 13, CLI: 25 e ABI/clinical LS: 73% 12 5.9 Aulivola et al. (27) 79 90 e n.i. LS: 84.4% (52.5%) non-ESRD, LS: 80.2% (52.5%) ESRD (12, 36) 14.3 Sigala et al. (28) 50 50 e Clinical LS: 68.9% 24 15 Brillu et al. (29) 37 37 e Clinical LS: 87% 24 28 Brown et al. (30) 40 55 e CI CI: 44% 25.8 25.8 Bull et al. (31) 168, IC: 40, CLI: 128 168 e CI CI: 83% (single stenosis), CI: 76% (multilevel lesions), CI: 44% (lytic therapy), CI: 36% (segmental occlusion) 36 26.1 Danielsson et al. (32) 140 155, IC: 16, CLI: 139 e CI (improvement of subjective relief) CI: 66% (non-DM) CI: 32% (DM), LS: 66% (non-DM), LS: 90% (DM) 12 n.i. Favre et al. (33) 24, IC: 4, CLI: 20 25 e DUS PP: 46%, SP: 64% 24 15 Löfberg et al. (34) 82 86 e CP (according to SVS/ISCVS standards) CP: 36%, LS: 72% 36, 36 n.i. Ingle et al. (35) 67, IC: 6, CLI: 61 70 e CP (freedom from CLI) CP: 84%, LS: 94%, 36 n.i. Vraux et al. (24) 46 50 e Intention to treat CP PP: 46%, SP: 55%, CP: 63%, LS: 87% 12 15 Nydahl et al. IMPACT OF TIBIAL ARTERIAL PATENCY ON CLINICAL OUTCOMES IN CLI (36) 27, IC: 4, CLI: 24 28 e CP (symptomatic patency) CP: 56%, LS: 85%, survival: 81% 12 n.i. Table 1 \| Summary of all studies analyzed within the meta-analysis of Romiti and coworkers. January 2015 \| Volume 1 \| Article 17 \| 3 www.frontiersin.org www.frontiersin.org Patency and infrapopliteal revascularization Baumann et al. Table 1 \| Continued Ref. Patients (n) Limbs (n) s/e Patency evaluation (speciﬁcations) End points Reported fu (months) Mean fu (months) Tisi et al. (37) 57 57 e DUS: n = 26, Angiography: n = 3 (angiography if ABI-impair > 0.1 or clinical deterioration) PP: 27%, SP: 33%, LS: 88% 12 n.i. Söder et al. (38) 60 72 e Angiography PP: 48%, SP: 56%, LS 80% 18 10 Barton et al. (39) 43 n.i. e CI (asymptomatic) CI: 60% 36 28 Lazaris et al. (40) 24 24 e Intention to treat PP: 50%, LS: 92% 12 n.i. Sivananthan et al. (41) 38, IC: 18, CLI: 20 41 e CI: (improvement ≥1 Fontaine category) CI: 58% at last fu 21 Faglia et al. (8) 537, s: 117, e: 420 537 s/e CP (no recurrence of pain/ulcer) CP, PTA: 78%, Bypass: 77% 60 40 Bosiers et al. (42) 443 443 e DUS PP: 74.2%, LS: 96.6% 12, 12 n.i. Schwarten (43) 96 112 e n.i. LS: 83% 24 n.i. Ascher et al. (44) 30 32, IC: 12, CLI: 20 e DUS LS: 100%, PP: 85% 3 5.2 Table 1 \| Continued Table 2 \| Overview of randomized series comparing BMS with POBA for tibial revascularization in CLI patients. Ref. No. patients/ lesions Lesion length (mm) Follow-up Patency evaluation (number) Patency (%) Clinical end points (%) COMPLETED RANDOMIZEDTRIALS ON BMS FOR BTK Rand et al. (49) 51/95 24 6 months Angiography: 18 BMS: 79.7 LS BMSa: 42 BMS: 9 PTA 45.6 (P = 0.02) BMS: 92 PTA: 53 PTA: 9 PTA: 95 (P = ns) CT-Angio: 19 BMS: 8 PTA: 11 Randon et al. (50) 35/38 BMS: 22 12 months Clinical patency BMS: 66.0 LS BMSb: 16 PTA: 39 PTA: 79.5 (P = ni) BMS 92.7 PTA: 22 PTA: 90.0 (P = 0.76) Brodmann et al. IMPACT OF TIBIAL ARTERIAL PATENCY ON CLINICAL OUTCOMES IN CLI (48) 54/54 BMS: 28 12 months BMS: 35.3 CI BMSa: 21 PTA: 79 PTA: 41.8 (P = ns) BMS: 64.7 PTA: 33 PTA: 81.5 (P = ns) PLANNED OR ONGOING RANDOMIZEDTRIALS ON BMS FOR BTK ANGIOPLASTY XXSc 180 <150 12 Angiography – TLR BMS, bare metal stent; BTK, below the knee; No., number; PTA, percutaneous angioplasty; ns, not signiﬁcant; ni, no information; LS, limb salvage; CI, clinical improvement (improvement ≥1 category according to Rutherford classiﬁcation); P, P value; TLR, target lesion revascularization. aBalloon-expandable BMS. bIncluding balloon-expandable and self-expandable BMS. cSelf-expandable BMS. Table 2 \| Overview of randomized series comparing BMS with POBA for tibial revascularization in CLI patients. PTA: 33 PTA: 81.5 (P = ns) PLANNED OR ONGOING RANDOMIZEDTRIALS ON BMS FOR BTK ANGIOPLASTY XXSc 180 <150 12 Angiography – TLR BMS, bare metal stent; BTK, below the knee; No., number; PTA, percutaneous angioplasty; ns, not signiﬁcant; ni, no information; LS, limb salvage; CI, clinical improvement (improvement ≥1 category according to Rutherford classiﬁcation); P, P value; TLR, target lesion revascularization. aBalloon-expandable BMS. bIncluding balloon-expandable and self-expandable BMS. cSelf-expandable BMS. CURRENTLY AVAILABLE TECHNOLOGIES AIMED AT THE PREVENTION OF RESTENOSIS Mechanical scaffolding as provided by a stent may be an ideal solution to address elastic recoil, an important contributor to restenosis in tibial arteries (47). The application of tibial BMS was assessed in various studies (48–50). However, no substantial Given the excessive incidence of tibial arterial restenosis (10, 12, 14, 15) subsequent to POBA, various endovascular technologies have been assessed in the framework of clinical trials. January 2015 \| Volume 1 \| Article 17 \| 4 Frontiers in Cardiovascular Medicine \| Vascular Medicine Frontiers in Cardiovascular Medicine \| Vascular Medicine Patency and infrapopliteal revascularization Baumann et al. superior when compared with BMS/POBA for tibial angioplasty in respect to patency and the need for repeated TLR. In addi- tion, DES was shown to improve event-free survival rates when compared with BMS as shown within the aforementioned Yukon trial (11). beneﬁt of BMS application when compared with POBA was observed within three randomized trials (48–50) (Table 2). Based on these observations, it may be assumed that neointimal pro- liferation induced by BMS outweighs the potential beneﬁt of mechanical scaffolding in the prevention of restenosis induced by elastic recoil. Of note, however, tibial arterial lesion lengths of patients included in all randomized DES trials were ≤35 mm. Within a consecutive series, Baumann et al. analyzed tibial lesion mor- phology in 105 CLI patients undergoing tibial angioplasty (16). Thereby, a mean lesion length of 87 mm was observed for stenotic and 124 mm for occlusive tibial PAD. According to these mor- phological ﬁndings, only 11% of that study population would have qualiﬁed for participation within the above-mentioned ran- domized DES trials. Thus, currently available coronary DES is applicable to only a minority of CLI patients treated in everyday clinical practice. DRUG-ELUTING STENTS h b l d Given the above-outlined drawbacks of BMS in tibial interven- tions, great hope was attributed to DES technology. The principle of DES is to provide mechanical scaffolding but with a minimum of neointimal proliferation based on the antiproliferative coating. Four randomized trials compared DES versus POBA or BMS for tibial angioplasty (46, 51–53) (Table 3). Whilst the Yukon trial compared DES with BMS (46), the remaining randomized studies compared DES to POBA (51–53). Without exception, DES was Table 3 \| Overview of randomized trials comparing DES versus BMS or POBA for BTK angioplasty. Reference Devices Rutherford categories Renal insufﬁciency Inclusion criteria Patients (n) Follow-up (months) Final LL End point Results Yukon (46) DES° versus BMS (° Yukon, Translumina, Hechingen, Germany) 2–5 n.i. de novo lesions steno- sis > 70%, LL < 45 mm 161 12 31 ± 9 Restenosis (>50%) (a) DUS (PSVR > 2.4) Primary patency DES: 80.6% BMS: 55.6% (P = 0.004) Secondary patency (b) Angiography DES: 91.9% BMS: 71.4% (P:0.005) Destiny (52) DES° versus BMS (° Xience V stent) 4, 5 n.i. de novo steno- sis > 50%, LL < 40 mm 140 12 n.i. Restenosis > 50% by angiography Primary patency DES: 85% BMS: 54% (P = 0.0001) Falkowski (53) DES° versus BMS (° Cipher Cordis Europa N.V.) 3–5 n.i. de novo steno- sis > 60%, LL 5–30 mm 50 6 17.8 PE: Resteno- sis > 50% by angiography Primary patency DES: 16% BMS: 76% (P = 0.001) SE: TLR TLR DES: 12% BMS: 56% (P < 0.05) RANDOMIZEDTRIALS ON DES VERSUS POBA FOR BTK ANGIOPLASTY Achilles (51) DES° versus POBA (° Cipher Select, Cordis Cooperation, USA) 3–5 exclusion: creatinine > 2.5 mg/dl de novo and restenotic native steno- sis > 70%, LL < 120 mm 200 (99 versus 101) 12 27 ± 21 Restenosis by angiography Primary patency DES: 77.6% POBA: 58.1% n, number; n.i., no information; LL, lesion length; DUS, duplex ultrasound; PSVR, peak systolic velocity ratio; DES, drug-eluting stent; BMS, bare metal stent; TLR, target lesion revascularization; PE primary end point; SE secondary end point; POBA plain old balloon angioplasty Table 3 \| Overview of randomized trials comparing DES versus BMS or POBA for BTK angioplasty. Reference Devices Rutherford categories Renal insufﬁciency Inclusion criteria Patients (n) Follow-up (months) Final LL End point Results Yukon (46) DES° versus BMS (° Yukon, Translumina, Hechingen, Germany) 2–5 n.i. DRUG-ELUTING BALLOONS in the DEB group (P = 0.9). In the meantime, results from further randomized trials are awaited (Table 4). Of these, the Euro Canal trialwasterminatedearlyduetostrategicreorientationof thecom- pany. A second randomized trial performed was the InPact Deep trial, which was ﬁnished but upon completion the company with- drew the DEB of investigation from the market. This was based on the 12-month results with lacking efﬁcacy of DEB and moreover higher major amputation rates for DEB (8.8%) when compared to POBA (3.6%, P = 0.08). DEB technology was introduced with the intention of reducing neointimal proliferation. First, by limiting the mechanical irri- tation to the duration of balloon inﬂation, and second, by the applicationof anantiproliferativesubstanceduringtheendothelial injury phase induced by angioplasty. According to ﬁrst obser- vations, the application of DEB may reduce restenosis and the need for repeated revascularization when compared with POBA for tibial revascularization (10, 54). Within a non-randomized study setting, Schmidt et al. eval- uated the application of DEB for tibial revascularization in 104 patients (CLI: 82.6%, severe claudication: 17.4%, limbs: n = 109) (54). Binary restenosis was evaluated using angiography and deﬁned as a > 50% reduction of lumen diameter. After 3 months, binary restenosis after DEB was observed in 27.4%. Within a similarcohortof historiccontrolpatientsatthesamecenterunder- going tibialPOBA,restenosiswas reportedin 68.8% after3 months (54). According to this, DEB was shown to reduce tibial resteno- sis by around 60% when compared with POBA and, thus, great hope was placed on DEB technology aimed at improving tibial patency. The superiority of DEB over POBA in tibial arteries was furthermore shown by Liistro et al. who were the ﬁrst to report results from a randomized trial (10). Within the DEBATE-BTK trial, Liistro and coworkers analyzed 132 patients for tibial angio- plasty randomly assigned for 67 DEB and 65 POBA. Mean lesion length was 129 mm in patients treated with DEB and 131 mm treated with POBA (P = 0.9). Primary end point in that trial was binary restenosis deﬁned > 50% after 12 months by angiography (>90% of patients) and/or duplex sonography for the remain- ing. Secondary clinical end points were the incidence of TLR and amputation. Binary restenosis was 27% in the DEB and 74% in the POBA group (P < 0.001). In addition, the need for secondary TLR was lower in patients treated with DEB compared to those treated with POBA (18 versus 43%, P = 0.002). DRUG-ELUTING STENTS h b l d \| Overview of ongoing/not completed randomized trials on DEB versus BMS or POBA for BTK angioplasty. DRUG-ELUTING STENTS h b l d de novo lesions steno- sis > 70%, LL < 45 mm 161 12 31 ± 9 Restenosis (>50%) (a) DUS (PSVR > 2.4) Primary patency DES: 80.6% BMS: 55.6% (P = 0.004) Secondary patency (b) Angiography DES: 91.9% BMS: 71.4% (P:0.005) Destiny (52) DES° versus BMS (° Xience V stent) 4, 5 n.i. de novo steno- sis > 50%, LL < 40 mm 140 12 n.i. Restenosis > 50% by angiography Primary patency DES: 85% BMS: 54% (P = 0.0001) Falkowski (53) DES° versus BMS (° Cipher Cordis Europa N.V.) 3–5 n.i. de novo steno- sis > 60%, LL 5–30 mm 50 6 17.8 PE: Resteno- sis > 50% by angiography Primary patency DES: 16% BMS: 76% (P = 0.001) SE: TLR TLR Table 3 \| Overview of randomized trials comparing DES versus BMS or POBA for BTK angioplasty. ( ) RANDOMIZEDTRIALS ON DES VERSUS POBA FOR BTK ANGIOPLASTY Achilles (51) DES° versus POBA (° Cipher Select, Cordis Cooperation, USA) 3–5 exclusion: creatinine > 2.5 mg/dl de novo and restenotic native steno- sis > 70%, LL < 120 mm 200 (99 versus 101) 12 27 ± 21 Restenosis by angiography Primary patency DES: 77.6% POBA: 58.1% n, number; n.i., no information; LL, lesion length; DUS, duplex ultrasound; PSVR, peak systolic velocity ratio; DES, drug-eluting stent; BMS, bare metal stent; TLR, target lesion revascularization; PE, primary end point; SE, secondary end point; POBA, plain-old balloon angioplasty. RANDOMIZEDTRIALS ON DES VERSUS POBA FOR BTK ANGIOPLASTY n, number; n.i., no information; LL, lesion length; DUS, duplex ultrasound; PSVR, peak systolic velocity ratio; DES, drug-eluting stent; BMS, bare metal stent; TLR, target lesion revascularization; PE, primary end point; SE, secondary end point; POBA, plain-old balloon angioplasty. January 2015 \| Volume 1 \| Article 17 \| 5 www.frontiersin.org Patency and infrapopliteal revascularization Baumann et al. Table 4 \| Overview of ongoing/not completed randomized trials on DEB versus BMS or POBA for BTK angioplasty. DRUG-ELUTING BALLOONS Incontrasttoearlierobservations,patencyappearstoaffectclinical outcomes in CLI patients, and thus, remains the major drawback of tibial arterial angioplasty. DES and DEB were shown to improve tibial patency but both with speciﬁc limitations. Accordingly, cur- rently applied and evaluated DES for tibial revascularization do not address infrapopliteal lesion morphology. While DEB technol- ogy complies well with tibial lesion morphology in CLI patients, it may not address acute elastic recoil, an important contributor to tibial restenosis. Further studies assessing anti-restenosis concepts speciﬁcally dedicated to the unique requirements of complex tibial arterial obstructions are warranted. DRUG-ELUTING STENTS h b l d Study name Devices Rutherford categories Predeﬁned LL (mm) Patients (n) Follow-up (months) End points PLANNED/ONGOING RANDOMIZEDTRIALS ON DEB FOR BTK ANGIOPLASTY IDEAS-I DEB versus BMS 3–6 70–220 50 6 Restenosis (angiography) Piccolo DEB versus POBA 3–5 15–150 114 6 Late lumen loss (angiography) InPact Deepa DEB versus POBA 4–6 <100 450 12 Clinically driven TLR, restenosis (angiography) Euro Canalb DEB versus POBA 4–6 10–270 120 6 Late lumen loss (angiography) DEB, drug-eluting balloon; BMS, bare metal stent; POBA, plain-old balloon angioplasty; BTK, below the knee; LL, lesion length; n, number; TLR, target lesion revascularization; DUS, duplex ultrasound. aStudy terminated early based on safety concerns. bStudy terminated early based on strategic company decision, no safety concerns. Table 4 \| Overview of ongoing/not completed randomized trials on DEB versus BMS or POBA for BTK angioplasty. Study name Devices Rutherford categories Predeﬁned LL (mm) Patients (n) Follow-up (months) End points PLANNED/ONGOING RANDOMIZEDTRIALS ON DEB FOR BTK ANGIOPLASTY IDEAS-I DEB versus BMS 3–6 70–220 50 6 Restenosis (angiography) Piccolo DEB versus POBA 3–5 15–150 114 6 Late lumen loss (angiography) InPact Deepa DEB versus POBA 4–6 <100 450 12 Clinically driven TLR, restenosis (angiography) Euro Canalb DEB versus POBA 4–6 10–270 120 6 Late lumen loss (angiography) DEB, drug-eluting balloon; BMS, bare metal stent; POBA, plain-old balloon angioplasty; BTK, below the knee; LL, lesion length; n, number; TLR, target lesion revascularization; DUS, duplex ultrasound. aStudy terminated early based on safety concerns. bStudy terminated early based on strategic company decision, no safety concerns. DRUG-ELUTING BALLOONS DEB technology was introduced with the intention of reducing neointimal proliferation. First, by limiting the mechanical irri- tation to the duration of balloon inﬂation, and second, by the applicationof anantiproliferativesubstanceduringtheendothelial injury phase induced by angioplasty. According to ﬁrst obser- in the DEB group (P = 0.9). In the meantime, results from further randomized trials are awaited (Table 4). Of these, the Euro Canal trialwasterminatedearlyduetostrategicreorientationof thecom- pany. A second randomized trial performed was the InPact Deep trial, which was ﬁnished but upon completion the company with- drew the DEB of investigation from the market. This was based on Table 4 \| Overview of ongoing/not completed randomized trials on DEB versus BMS or POBA for BTK angioplasty. St d D i R th f d P d ﬁ d P ti t F ll E d mpleted randomized trials on DEB versus BMS or POBA for BTK angioplasty. REFERENCES Brosi P, Baumgartner I, Silvestro A, Do DD, Mahler F, Triller J, et al. Below-the- knee angioplasty in patients with end-stage renal disease. 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J Endovasc Ther (2014) 21:44–51. doi:10.1583/13-4486MR.1 Conﬂict of Interest Statement: Nicolas Diehm was the Principal Investigator for Switzerland of the Euro Canal trial and received consulting honoraria from Medrad. This trial, however, was terminated early due to strategical reorientation of the com- pany. Thereafter, Nicolas Diehm has no more ﬁnancial disclosures or conﬂicts of interest to report. The remaining authors have neither ﬁnancial disclosures nor a conﬂict of interest to state. 48. Brodmann M, Froehlich H, Dorr A, Gary T, Portugaller RH, Deutschmann H, et al. Percutaneous transluminal angioplasty versus primary stenting in infrapopliteal arteries in critical limb ischemia. Vasa (2011) 40:482–90. doi:10. 1024/0301-1526/a000152 49. Rand T, Basile A, Cejna M, Fleischmann D, Funovics M, Gschwendtner M, et al. PTA versus carboﬁlm-coated stents in infrapopliteal arteries: pilot study. Car- diovasc Intervent Radiol (2006) 29:29–38. doi:10.1007/s00270-005-0276-9 Received:12September2014;accepted:16December2014;publishedonline:07January 2015. 50. Randon C, Jacobs B, De Ryck F, Vermassen F. Angioplasty or primary stenting for infrapopliteal lesions: results of a prospective randomized trial. Cardiovasc Intervent Radiol (2010) 33:260–9. doi:10.1007/s00270-009-9765-6 Citation: Baumann F, Ozdoba C, Gröchenig E and Diehm N (2015) The impor- tance of patency in patients with critical limb ischemia undergoing endovascular revascularization for infrapopliteal arterial disease. Front. Cardiovasc. Med. 1:17. doi: 10.3389/fcvm.2014.00017 51. January 2015 \| Volume 1 \| Article 17 \| 8 Frontiers in Cardiovascular Medicine \| Vascular Medicine REFERENCES Scheinert D,Katsanos K,Zeller T,Koppensteiner R,Commeau P,Bosiers M,et al. A prospective randomized multicenter comparison of balloon angioplasty and infrapopliteal stenting with the sirolimus-eluting stent in patients with ischemic peripheral arterial disease: 1-year results from the ACHILLES trial. J Am Coll Cardiol (2012) 60:2290–5. doi:10.1016/j.jacc.2012.08.989 This article was submitted to Vascular Medicine, a section of the journal Frontiers in Cardiovascular Medicine. 52. Bosiers M, Scheinert D, Peeters P, Torsello G, Zeller T, Deloose K, et al. Random- ized comparison of everolimus-eluting versus bare-metal stents in patients with critical limb ischemia and infrapopliteal arterial occlusive disease. J Vasc Surg (2012) 55:390–8. doi:10.1016/j.jvs.2011.07.099 Copyright © 2015 Baumann, Ozdoba, Gröchenig and Diehm. 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) or licensor 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. 53. Falkowski A, Poncyljusz W,Wilk G, Szczerbo-Trojanowska M. The evaluation of primary stenting of sirolimus-eluting versus bare-metal stents in the treatment January 2015 \| Volume 1 \| Article 17 \| 8 Frontiers in Cardiovascular Medicine \| Vascular Medicine
https://openalex.org/W3198712270	https://escholarship.org/content/qt6164z345/qt6164z345.pdf?t=qzpz8c	English	null	Myocarditis occurrence with cancer immunotherapy across indications in clinical trial and post-marketing data	Scientific reports	2,021	cc-by	10,913	Title Title Myocarditis occurrence with cancer immunotherapy across indications in clinical trial and post-marketing data P li k UC San Diego UC San Diego Previously Published Works Title Myocarditis occurrence with cancer immunotherapy across indications in clinical trial and post-marketing data Permalink https://escholarship.org/uc/item/6164z345 Journal Scientific Reports, 11(1) ISSN 2045-2322 Authors Makunts, Tigran Saunders, Ila M Cohen, Isaac V et al. Publication Date 2021 DOI 10.1038/s41598-021-96467-5 Peer reviewed UC San Diego UC San Diego Previously Published Works Title Myocarditis occurrence with cancer immunotherapy across indications in clinical trial and post-marketing data Permalink https://escholarship.org/uc/item/6164z345 Journal Scientific Reports, 11(1) ISSN 2045-2322 Authors Makunts, Tigran Saunders, Ila M Cohen, Isaac V et al. Publication Date 2021 DOI 10.1038/s41598-021-96467-5 Peer reviewed UC San Diego UC San Diego Pr Myocarditis occurrence with cancer immunotherapy across indications in clinical trial and post‑marketing data OPEN Tigran Makunts1,2, Ila M. Saunders2, Isaac V. Cohen3, Mengxing Li1, Talar Moumedjian2, Masara A. Issa2, Keith Burkhart4, Peter Lee4, Sandip Pravin Patel5 & Ruben Abagyan2* Antibodies targeting the PD-1, PD-L1, and CTLA-4 immune checkpoint axis have been used in a variety of tumor types. They achieve anti-tumor activity through activating the patient’s own immune system to target immune response evading cancer cells. However, this unique mechanism of action may cause immune-related adverse events, irAEs. One of these irAEs is myocarditis which is associated with an alarming mortality rate. In this study we presented clinical cases of myocarditis from safety trial datasets submitted to the U.S. Food and Drug Administration, FDA. Additionally, we analyzed over fourteen million FDA Adverse Event Reporting System, FAERS, submissions. The statistical analysis of the FAERS data provided evidence of significantly increased reporting of myocarditis in patients administered immune checkpoint inhibitors alone, in combination with another immune checkpoint inhibitor, the kinase inhibitor axitinib, or chemotherapy, for all cancer types, when compared to patients administered chemotherapy. All combination therapies led to further increased reporting odds ratios of myocarditis. We further analyzed the occurrence of myocarditis by stratifying the reports into sub-cohorts based on specific cancer types and treatment/ control groups in major cancer immunotherapy efficacy trials and confirmed the observed trend for each cohort. The field of cancer immunotherapy has continuously gained appreciation with the success of various targeted immune checkpoint inhibitors (ICIs). Malignant cancer cells have the capacity to evade the immune system by suppressing the activation of T-cells1. This concept led to the discovery of new strategies to block the immune checkpoint breaks and re-activate the immune response1. The first immunotherapy antibody approval in 2011, ipilimumab2, targeted the cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4)2. Antibodies targeting the programmed cell death protein 1 (PD-1) receptors, pembrolizumab and nivolumab were approved in 2014, and cemiplimab in 2018. Combination therapies, such as pembrolizumab plus axitinib and nivolumab plus ipilimumab, also received approvals for various indications. Monoclonal antibodies targeting the PD-1 ligand (PD-L1) atezolizumab (2016), durvalumab (2017), and avelumab (2017)1 were also recently approved.h ( ) ( ), ( ), ( ) y pp The use of the checkpoint inhibitors has been linked to serious immune-related adverse events (irA including rare but potentially fatal cardiac toxicity such as myocarditis4. Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ myocarditis as one of the adverse reactions occurring in less than 5% of patients7. Similarly, in the prescribing information of PD-L1 blockers atezolizumab, durvalumab and avelumab, myocarditis is listed as a clinically significant irAE occurring in < 1% of the patients8–10. In a phase-III study evaluating avelumab in combination with axitinib for the treatment of advanced renal cell carcinoma (JAVELIN Renal 101), death associated with myocarditis, and necrotizing pancreatitis occurred in 3 patients in the avelumab-plus-axitinib cohort (0.7%)11. Although only a few cases of myocarditis incidence were described in clinical trials (Table 1), the severity and possible fatality of the disease remained under-characterized, prompting further investigation.hl p y p p g g The term “myocarditis” describes a wide range of pathologies that clinically manifest as an inflammatory condition of the heart muscle occurring alone or as part of a multiorgan immune-mediated disorder or reaction to exogenous or endogenous substances29. The altered immune-mediated reactions are the cause of structural and functional abnormalities in the myocardium which are responsible for a variety of injuries to the heart such as contractile impairment, chamber stiffening and conduction system irregularities. Most commonly, myocarditis is categorized according to the major histopathologic pattern which is descriptive of the different etiologies of the disease. The heterogeneity and wide spectrum of clinical manifestations present challenges to the proper diagnosis of the disease and treatment decisions30. ICI use has been reported as an etiologic factor contributing to rare, but severe cases of myocarditis, according to World Health Organization (WHO) database31. Moslehi et al. describe 101 cases of severe myocarditis following immune checkpoint inhibitor treatment across various cancer types, with a higher frequency reported for the PD-1 and PDL-1/CTLA-4 combination with respect to monotherapy31. In a large systematic review and meta-analysis of the ICI-associated irAEs, myocarditis was found to have the highest fatality rate compared to other irAEs (52 of 131 reported cases)32. Another retrospec- tive study investigating a total of 36,848 toxicities of immunotherapies reported through FAERS in 2017–2018 described a 6.3% rate of cardiovascular toxicities including myocarditis. The fatality rate of the myocarditis cases was determined to be 50%33. Overall, ipilimumab, nivolumab, pembrolizumab and combinations of PD-1/PDL-1 and CTLA-4 inhibitors are associated with myocarditis at higher rate34. In an analysis of post-marketing surveillance data, avelumab had a higher association with myocarditis out of six monotherapy ICI post-marketing reports35. In a 2016 study, Johnson et al. www.nature.com/scientificreports/ describe two case reports of lethal myocarditis accompanied with myositis in patients administered ipilimumab–nivolumab36. In another case report from 2019, Saibil et al., described an example of fatal fulminant myocarditis combined with myositis following administration of a single dose of ipilimumab–nivolumab in a patient with stage IV melanoma. The patient presented at day 16 with a history of increasing fatigue, weakness, and dyspnea ultimately progressing to respiratory failure. The histologic patterns observed were myocyte calci- fication and myocyte lysis with associated inflammatory response37.hf il The manifestations of ICI-associated myocarditis seem to differ compared to general myocarditis suggesting the presence of distinct risk factors38. Analysis of the data from a multicenter registry of 8 sites reported half of the patients with myocarditis to have experienced major adverse cardiac events which include cardiogenic shock, cardiac arrest, complete heart block and cardiovascular death. Troponin elevation and abnormal ECG were com- mon findings in most of the clinical cases. In addition, diabetes, sleep apnea and a higher body mass index were among the patient characteristics associated with a higher incidence of myocarditis compared to controls39. Also, in their analysis of adverse event reports, Zamami et al., found a significantly higher risk of myocarditis in female patients and patients 75 years and older in the context of ICI compared to general myocarditis independent of ICI treatment38. A case series identified 5 out of 8 patients to have pre-existing cardiac pathologies suggesting that myocarditis might manifest as a possible worsening of general cardiac conditions40. Co-occurrence of other immune-related adverse events such as myositis, myasthenia gravis, thyroiditis, uveitis, colitis, and hepatitis were also described in the literature32,40. As far as treatment, Mahmood et al., observed that the course of the disease was overall responsive to higher doses of steroids which were administered in 89% of cases. However, myocarditis fatalities still occurred despite steroid therapy39. Although rare, myocarditis poses a high risk to the patients due to high risk of mortality and warrants further investigation into this irAE. h d d d f h h l f h d l l l d In this study we used two data sources for a thorough evaluation for the myocarditis cases in clinical trial and postmarketing safety reports submitted to the FDA, including disease progression, preceding irAEs, demographic parameters, CTCAE41 toxicity grading, and concomitant oncology medications. www.nature.com/scientificreports/ The first source, Integrated Sum- maries of Safety42 (ISS), includes the safety information from clinical trials, submitted to the United States Food and Drug Administration (FDA) with New Molecular Entity (NME) and non-NME submissions. As the second source, we analyzed the FDA FAERS/AERS database for myocarditis reports in patients taking ICIs as monotherapy, ICI-ICI combinations, and ICI in combination with axitinib compared to chemotherapy reports irrespective of indication. We further compared and contrasted myocarditis reported frequencies repli- cating the study cohorts in the efficacy clinical trials and matching adverse event by indication, treatment, and control groups. Myocarditis occurrence with cancer immunotherapy across indications in clinical trial and post‑marketing data OPEN g p y y y Myocarditis was observed in < 1% of patients receiving ICI therapy, with cardiac rhythm disturbances as the initial presentation3. A randomized, double-blind, placebo-controlled trial evaluating the safety of ipilimumab (CA184-029), a CTLA-4 inhibitor, in adjuvant treatment of melanoma found a severe to fatal myocarditis inci- dence of 0.2%5. Myocarditis as a fatal adverse reaction was also reported for ipilimumab in combination with PD-1 blocker nivolumab as a first-line treatment for non-small-cell lung cancer (CHECKMATE-227)5. A study evaluating PD-1 blocker pembrolizumab for the treatment of classical Hodgkin Lymphoma (KEYNOTE-087) reported a myocarditis incidence of 0.5%6. The prescribing information of cemiplimab listed autoimmune 1Oak Ridge Institute of Science and Education Fellowship at Office of Clinical Pharmacology, United States Food and Drug Administration, Silver Spring, MA, USA. 2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA. 3Clinical Pharmacology and Therapeutics (CPT) Postdoctoral Training Program, University of California San Francisco, San Francisco, CA, USA. 4Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MA, USA. 5Center for Personalized Cancer Therapy, Division of Hematology and Medical Oncology, UCSD Moores Cancer Center, La Jolla, CA, USA. *email: rabagyan@health.ucsd.edu \| https://doi.org/10.1038/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Drug Initial US approval Labeled indications Efficacy trial Drug Control Myocarditis incidence Ipilimumab/YERVOY, CTLA-4 2011 Unresectable or Metastatic Melanoma, Adjuvant Treatment of Melanoma5 In combination with Nivolumab: Advanced Renal Cell Carcinoma (RCC), Microsatellite Instability-High (MSI- H) or Mismatch Repair Deficient (dMMR) Metastatic Colorectal Cancer, Hepatocellular Carcinoma Metastatic Non-Small Cell Lung Cancer (NSCLC)5 Melanoma: MDX010-2012 Metastatic NSCLC: CHECKMATE-22713 Ipilimumab mono- therapy, Ipilimumab in combination with a melanoma peptide vaccine Nivolumab, or Nivolumab + Ipili- mumab, or Nivolumab + Platinum- doublet Chemotherapy Melanoma Vaccine Monotherapy Platinum Doublet Chemotherapy From prescribing Information in Adjuvant treatment of Melanoma: severe to fatal, 0.2% (CA184-029)a In first-line Treatment of Metastatic NSCLC: In Combination with Nivolumab (CHECK- MATE-227)b Pembrolizumab/ KEYTRUDA, PD-1 2014 Melanoma, Non-Small Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Cancer, Classical Hodgkin Lymphoma (cHL), Primary Medi- astinal Large B-Cell Lymphoma, Urothelial Carcinoma, Microsatel- lite Instability-High Cancer, Gastric Cancer, Cervical Cancer, Hepa- tocellular Carcinoma, Merkel Cell Carcinoma6. Methods C d Case studies from ISS data sets. Center for Drug Evaluation and Research electronic NME and non- NME submissions, including ISS and Clinical Safety Summaries (CSS) are maintained in the Electronic Doc- ument Room43. The ISS component of the Biologic License Applications (BLAs) of interest were mined for Analysis Datasets of Adverse Events (ADAE) for the submissions of ipilimumab, pembrolizumab, nivolumab, cemiplimab, avelumab, atezolizumab, and durvalumab. A total of 24,567 reports, ICI subjects (N = 20,062) and controls (4505), were collected from BLA submissions. The ADAE sets were scanned for myocarditis, immune mediated myocarditis, and autoimmune myocarditis events, and the human subject data was used to extract demographic parameters, co-occurring adverse events (AEs), progression to myocarditis and other variables to characterize the irAE. https://doi.org/10.1038/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 \| www.nature.com/scientificreports/ g The following clinically significant irAEs occurred at an incidence of < 1% in 2616 patients who received TECENTRIQ as a single-agent and in 2421 patients who received TECENTRIQ in combination with platinum-based chemotherapy or were reported in other products in this class8. h The following clinically significant irAEs occurred at an incidence of less than 1% each in 1889 patients who received IMFINZI: aseptic meningitis, hemolytic anemia, immune thrombocytopenic purpura, myocarditis, myositis, and ocular inflammatory toxicity, including uveitis and keratitis9. i Death due to toxicity of trial treatment that occurred in 3 patients in the avelumab-plus-axitinib group (0.7%) was attributed to sudden death, myocarditis, and necrotizing pancreatitis11. j The following irAEs occurred at an incidence of less than 1% of patients who received BAVENCIO as a single agent or in 489 patients who received BAVENCIO in combination with axitinib: immune-mediated myocarditis including fatal cases, pancreatitis including fatal cases, immune-mediated myositis, psoriasis, arthritis, exfoliative dermatitis, erythema multiforme, pemphigoid, hypopituitarism, uveitis, Guillain-Barré syndrome, and systemic inflammatory response10. FDA adverse event reporting system (FAERS/AERS) and MedWatch. FAERS/AERS is a database for post marketing safety surveillance reports. It is supported by the United States Food and Drug Administra- tion (FDA). The reporting of AEs and outcomes to FAERS/AERS is done through the MedWatch44 platform, pre- dominantly on a voluntary basis. In cases when the reports are submitted to the manufacturer, the manufacturer is mandated to forward the report to FAERS/AERS.i p At the time of the study FAERS/AERS contained over fourteen million reports from the first quarter of 2004, which includes reports from prior years, to the second quarter of 2020. The reports were used to run a retrospec- tive analysis of the biologics of interest. FAERS/AERS data sets are available online at: http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryIn formation/Surveillance/AdverseDrugEffects/ucm082193.htm. Data preparation. Due to ongoing updates and changes of the format, the quarterly data sets were not all uniform. It was necessary to modify and standardize the sets, creating a consistent uniform table structure with missing columns filled with blanks. Since FAERS/AERS included reports from all over the world many of the variables including drug names were entered according to the country specific generic and brand names and spellings. Online databases were consulted to translate all drug names into single generic terms. Around 0.4% of all the FAERS/AERS reports were duplicates45–47. These are either repeated AEs in the same patient or multiple entrances for the same AE occurrence. www.nature.com/scientificreports/ Drug Initial US approval Labeled indications Efficacy trial Drug Control Myocarditis incidence Avelumab/BAVENCIO, PD-L1 2017 Metastatic Merkel Cell Carcinoma, Locally Advanced or Metastatic Urothelial Carcinoma10In combina- tion with Axitinib: first-line for advanced RCC10 Metastatic Merkel Cell Carcinoma: JAVELIN Merkel 20027 Urothelial Carcinoma: JAVELIN Solid Tumor28 Advanced RCC in com- bination with Axitinib: JAVELIN Renal 10111 Avelumab Avelumab Avelumab + axitinib Sunitinib In Advanced RCC in combination with axi- tinib (JAVELIN Renal 101): 0.2%i From prescribing informationj Drug Initial US approval Labeled indications Efficacy trial Drug Control Myocarditis incidence Avelumab/BAVENCIO, PD-L1 2017 Metastatic Merkel Cell Carcinoma, Locally Advanced or Metastatic Urothelial Carcinoma10In combina- tion with Axitinib: first-line for advanced RCC10 Metastatic Merkel Cell Carcinoma: JAVELIN Merkel 20027 Urothelial Carcinoma: JAVELIN Solid Tumor28 Advanced RCC in com- bination with Axitinib: JAVELIN Renal 10111 Avelumab Avelumab Avelumab + axitinib Sunitinib In Advanced RCC in combination with axi- tinib (JAVELIN Renal 101): 0.2%i From prescribing informationj Table 1. Summary of Myocarditis occurrence in clinical trials for immune checkpoint inhibitors. RCC renal cell carcinoma, mRCC metastatic renal cell carcinoma, NSCLC non-small cell lung cancer, SCLC small cell lung cancer, CSCC cutaneous squamous cell carcinoma. a In CA184-029, the following clinically significant irAEs were seen in less than 1% of YERVOY-treated patients unless specified: cytopenias, eosinophilia (2.1%), pancreatitis (1.3%), meningitis, pneumonitis, sarcoidosis, pericarditis, uveitis, and fatal myocarditis [see Adverse Reactions (6.1)]5. b Fatal adverse reactions occurred in 1.7% of patients; these included events of pneumonitis (4 patients), myocarditis, acute kidney injury, shock, hyperglycemia, multi- system organ failure, and renal failure5. c Other clinically important adverse reactions that occurred in less than 10% of patients on KEYNOTE-087 included infusion reactions (9%), hyperthyroidism (3%), pneumonitis (3%), uveitis and myositis (1% each), and myelitis and myocarditis (0.5% each)6. d Of the 11 patients (2.6%) in the pembrolizumab–axitinib group who died from adverse events, 4 (0.9%) died from treatment-related adverse events (from myasthenia gravis, myocarditis, necrotizing fasciitis, and pneumonitis, in 1 patient each)16. e Fatal adverse reactions occurred in 1.7% of patients; these included events of pneumonitis (4 patients), myocarditis, acute kidney injury, shock, hyperglycemia, multi-system organ failure, and renal failure5. f LIBTAYO was permanently discontinued due to adverse reactions in 5% of patients; adverse reactions resulting in permanent discontinuation were pneumonitis, autoimmune myocarditis, hepatitis, aseptic meningitis, complex regional pain syndrome, cough, and muscular weakness7. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 1. Summary of Myocarditis occurrence in clinical trials for immune checkpoint inhibitors. RCC renal cell carcinoma, mRCC metastatic renal cell carcinoma, NSCLC non-small cell lung cancer, SCLC small cell lung cancer, CSCC cutaneous squamous cell carcinoma. a In CA184-029, the following clinically significant irAEs were seen in less than 1% of YERVOY-treated patients unless specified: cytopenias, eosinophilia (2.1%), pancreatitis (1.3%), meningitis, pneumonitis, sarcoidosis, pericarditis, uveitis, and fatal myocarditis [see Adverse Reactions (6.1)]5. b Fatal adverse reactions occurred in 1.7% of patients; these included events of pneumonitis (4 patients), myocarditis, acute kidney injury, shock, hyperglycemia, multi- system organ failure, and renal failure5. c Other clinically important adverse reactions that occurred in less than 10% of patients on KEYNOTE-087 included infusion reactions (9%), hyperthyroidism (3%), pneumonitis (3%), uveitis and myositis (1% each), and myelitis and myocarditis (0.5% each)6. d Of the 11 patients (2.6%) in the pembrolizumab–axitinib group who died from adverse events, 4 (0.9%) died from treatment-related adverse events (from myasthenia gravis, myocarditis, necrotizing fasciitis, and pneumonitis, in 1 patient each)16. e Fatal adverse reactions occurred in 1.7% of patients; these included events of pneumonitis (4 patients), myocarditis, acute kidney injury, shock, hyperglycemia, multi-system organ failure, and renal failure5. f LIBTAYO was permanently discontinued due to adverse reactions in 5% of patients; adverse reactions resulting in permanent discontinuation were pneumonitis, autoimmune myocarditis, hepatitis, aseptic meningitis, complex regional pain syndrome, cough, and muscular weakness7. g The following clinically significant irAEs occurred at an incidence of < 1% in 2616 patients who received TECENTRIQ as a single-agent and in 2421 patients who received TECENTRIQ in combination with platinum-based chemotherapy or were reported in other products in this class8. h The following clinically significant irAEs occurred at an incidence of less than 1% each in 1889 patients who received IMFINZI: aseptic meningitis, hemolytic anemia, immune thrombocytopenic purpura, myocarditis, myositis, and ocular inflammatory toxicity, including uveitis and keratitis9. i Death due to toxicity of trial treatment that occurred in 3 patients in the avelumab-plus-axitinib group (0.7%) was attributed to sudden death, myocarditis, and necrotizing pancreatitis11. j The following irAEs occurred at an incidence of less than 1% of patients who received BAVENCIO as a single agent or in 489 patients who received BAVENCIO in combination with axitinib: immune-mediated myocarditis including fatal cases, pancreatitis including fatal cases, immune-mediated myositis, psoriasis, arthritis, exfoliative dermatitis, erythema multiforme, pemphigoid, hypopituitarism, uveitis, Guillain-Barré syndrome, and systemic inflammatory response10. www.nature.com/scientificreports/ In combination with Axitinib: first-line treat- ment against advanced/ metastatic Renal Cell Carcinoma (mRCC) (https://www.fda.gov/ drugs/drug-approvals- and-databases/fda-appro ves-pembrolizumab- plus-axitinib-advanced- renal-cell-carcinoma) Melanoma: KEYNOTE-00614 Classical Hodgkin Lymphoma: Phase II KEYNOTE-08715 mRCC: KEY- NOTE-42616 NSCLC: KEYNOTE 18917 NSCLC: KEY- NOTE—40718 Pembrolizumab Pembrolizumab Pembrolizumab + axi- tinib Pembrolizumab + peme- trexed + platinum-based chemotherapy Pembrolizumab + car- boplatin + paclitaxel or nab-paclitaxel Ipilimumab Single arm, Non-rand- omized Sunitinib Placebo + peme- trexed + platinum-based chemotherapy Placebo + carbopl- atin + paclitaxel or nab-paclitaxel In Classical Hodgkin Lymphoma: 0.5% (KEYNOTE-087)c In mRCC: Of the 11 patients who died from adverse events in the combination group, 1 died from myocarditis Nivolumab/OPDIVO, PD-1 2014 Unresectable or Metastatic Melanoma, Adjuvant Treatment of Melanoma, Metastatic NSCLC, Small Cell Lung Cancer, Advanced RCC, cHL, Squamous Cell Carcinoma of the Head and Neck, Urothelial Carcinoma, Microsatel- lite Instability-High or Mismatch Repair Deficient Metastatic Colorectal Cancer, Hepatocellular Carci- noma, Esophageal Squa- mous Cell Carcinoma (ESCC)19 Advanced Melanoma: CHECKMATE-03720 Metastatic NSCLC in combination with Ipilimumab: CHECK- MATE-22713 Nivolumab Nivolumab, or Nivolumab + Ipili- mumab, or Nivolumab + Platinum- doublet Chemotherapy Either Dacarbazine or Carboplatin and Paclitaxel Platinum Doublet Chemotherapy In metastatic NSCLC: (CHECKMATE-227)e Cemiplimab/LIBTAYO, PD-1 2018 Metastatic Cutaneous Squamous Cell Carci- noma (CSCC) or locally advanced CSCC7 Study 1423 and 154021 Cemiplimab, Cemi- plimab + anti-cancer therapy (radiotherapy, cyclophosphamide, docetaxel, carboplatin, GM-CSF, paclitaxel, pemetrexed) From prescribing informationf Atezolizumab/TECEN- TRIQ, PD-L1 2016 Urothelial Carcinoma, NSCLC, Locally Advanced or Metastatic Triple-Negative Breast Cancer, Small Cell Lung Cancer (SCLC), Hepato- cellular Carcinoma8 Urothelial Carcinoma: IMvigor21022 Non-squamous NSCLC: Impower15023 Atezolizumab Atezolizumab in Combi- nation with Carbopl- atin + Paclitaxel with or without Bevacizumab Carboplatin + pacli- taxel + bevacizumab From prescribing informationg Durvalumab/IMFINZI, PD-L1 2017 Urothelial Carcinoma, NSCLC, SCLC9 Urothelial Carcinoma: Study 110824 NSCLC: PACIFIC25 SCLC: CASPIAN26 Durvalumab Durvalumab Durvalumab ± tremeli- mumab with platinum- based chemotherapy (carboplatin or cispl- atin + etoposide) Placebo Platinum-based chemo- therapy From prescribing informationh Continued https://doi.org/10.1038/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ g The following clinically significant irAEs occurred at an incidence of < 1% in 2616 patients who received TECENTRIQ as a single-agent and in 2421 patients who received TECENTRIQ in combination with platinum-based chemotherapy or were reported in other products in this class8. h The following clinically significant irAEs occurred at an incidence of less than 1% each in 1889 patients who received IMFINZI: aseptic meningitis, hemolytic anemia, immune thrombocytopenic purpura, myocarditis, myositis, and ocular inflammatory toxicity, including uveitis and keratitis9. i Death due to toxicity of trial treatment that occurred in 3 patients in the avelumab-plus-axitinib group (0.7%) was attributed to sudden death, myocarditis, and necrotizing pancreatitis11. j The following irAEs occurred at an incidence of less than 1% of patients who received BAVENCIO as a single agent or in 489 patients who received BAVENCIO in combination with axitinib: immune-mediated myocarditis including fatal cases, pancreatitis including fatal cases, immune-mediated myositis, psoriasis, arthritis, exfoliative dermatitis, erythema multiforme, pemphigoid, hypopituitarism, uveitis, Guillain-Barré syndrome, and systemic inflammatory response10. Table 1. Summary of Myocarditis occurrence in clinical trials for immune checkpoint inhibitors. RCC renal cell carcinoma, mRCC metastatic renal cell carcinoma, NSCLC non-small cell lung cancer, SCLC small cell lung cancer, CSCC cutaneous squamous cell carcinoma. a In CA184-029, the following clinically significant irAEs were seen in less than 1% of YERVOY-treated patients unless specified: cytopenias, eosinophilia (2.1%), pancreatitis (1.3%), meningitis, pneumonitis, sarcoidosis, pericarditis, uveitis, and fatal myocarditis [see Adverse Reactions (6.1)]5. b Fatal adverse reactions occurred in 1.7% of patients; these included events of pneumonitis (4 patients), myocarditis, acute kidney injury, shock, hyperglycemia, multi- system organ failure, and renal failure5. c Other clinically important adverse reactions that occurred in less than 10% of patients on KEYNOTE-087 included infusion reactions (9%), hyperthyroidism (3%), pneumonitis (3%), uveitis and myositis (1% each), and myelitis and myocarditis (0.5% each)6. d Of the 11 patients (2.6%) in the pembrolizumab–axitinib group who died from adverse events, 4 (0.9%) died from treatment-related adverse events (from myasthenia gravis, myocarditis, necrotizing fasciitis, and pneumonitis, in 1 patient each)16. e Fatal adverse reactions occurred in 1.7% of patients; these included events of pneumonitis (4 patients), myocarditis, acute kidney injury, shock, hyperglycemia, multi-system organ failure, and renal failure5. f LIBTAYO was permanently discontinued due to adverse reactions in 5% of patients; adverse reactions resulting in permanent discontinuation were pneumonitis, autoimmune myocarditis, hepatitis, aseptic meningitis, complex regional pain syndrome, cough, and muscular weakness7. www.nature.com/scientificreports/ The duplicates were identified and deleted. Study outcomes. The outcome of interest was defined as an adverse event of myocarditis, immune medi- ated myocarditis, or autoimmune myocarditis. Infection related myocarditis terms such as viral, bacterial, and fungal myocarditis were excluded from the analysis. Cohort selection. At the time of the analysis the public database of FAERS/AERS contained 14,202,841 total reports. The following cohorts were compiled for ICI patients, (1) monotherapy: ipilimumab (n = 8267), nivolumab (n = 27,149), pembrolizumab (n = 13,476), cemiplimab (n = 161), atezolizumab (n = 2397), avelumab (n = 305), and durvalumab (n = 1710); (2) anti-PD-1/CTLA-4 combinations: ipilimumab + nivolumab (n = 7970), ipilimumab + pembrolizumab (n = 225); (3) ICI + axitinib: pembrolizumab + axitinib (n = 207), avelumab + axi- tinib (n = 94). https://doi.org/10.1038/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 \| www.nature.com/scientificreports/ Figure 1. Selection of initial cohorts for ICI monotherapy, anti-PD-1 + CTLA-4, ICI + KI, positive control, and control cohorts. Figure 1. Selection of initial cohorts for ICI monotherapy, anti-PD-1 + CTLA-4, ICI + KI, positive control, an control cohorts. Figure 1. Selection of initial cohorts for ICI monotherapy, anti-PD-1 + CTLA-4, ICI + KI, positive control, and control cohorts. The following cohorts were compiled as positive controls, and controls used for reporting odds ratio calcula- tion (ROR-controls), (1) positive control: anthracyclines with or without chemotherapy (n = 134,001), (2) ROR- control: chemotherapy regimens, excluding ICIs and anthracyclines (n = 1,065,158) (Fig. 1 and Supplementary Table S1). Anthracyclines were analyzed separate from chemotherapy as a positive control, since they have been historically associated with myocarditis adverse events48–50. Additionally, myocarditis occurrence in FAERS/ AERS was calculated for clozapine monotherapy reports as a non-oncology reference point due to its known association with the myocarditis51–53. Reported frequencies for the myocarditis reports in the listed cohorts were calculated for an odds ratio analysis to estimate statistical significance of increased reporting. Anthracyclines with or without chemotherapy, rather than clozapine, were chosen as the positive control to preserve oncology indication uniform in the cohorts.i To investigate indication specific myocarditis reports in FAERS/AERS, a separate set of cohorts was cre- ated based on indications and treatments in efficacy trials all ICIs (Fig. www.nature.com/scientificreports/ 2 and Supplementary Table S2): (1) Melanoma—ipilimumab (n = 4659), ipilimumab + melanoma vaccine (n = 5), melanoma vaccine (n = 602), pembrolizumab (n = 2686), nivolumab (n = 3239), nivolumab + ipilimumab (n = 3493), dacarbazine (n = 83), carboplatin + paclitaxel (n = 35), (2) NSCLC—nivolumab (n = 9432), nivolumab + ipilimumab (n = 511), nivolumab + platinum doublet (n = 155), control-platinum doublet (n = 1564), atezolizumab (n = 1098), ate- zolizumab + carboplatin + paclitaxel (n = 52), atezolizumab + carboplatin + paclitaxel + bevacizumab (n = 207), carboplatin + paclitaxel + bevacizumab (n = 724), durvalumab (n = 1278), durvalumab + tremelimumab + (car- boplatin or cisplatin) + etoposide (n = 17), (carboplatin or cisplatin) + etoposide (n = 344), pembrolizumab + pem- etrexed + (carboplatin or cisplatin) (n = 892), pemetrexed + (carboplatin or cisplatin) (n = 1292), pembroli- zumab + carboplatin + paclitaxel (n = 616), carboplatin + paclitaxel (n = 1299), (3) SCLC—durvalumab (n = 6), durvalumab + tremelimumab + (carboplatin or cisplatin) + etoposide (n = 220), (4) RCC—pembrolizumab + axi- tinib (n = 163), avelumab + axitinib (n = 52), sunitinib (13,624), (5) Lymphoma—pembrolizumab (n = 98), (6) CSCC—cemiplimab (n = 122), cemiplimab + radiology (n = 1), (7) Urothelial carcinoma—avelumab (n = 3), (8) Merkel call carcinoma—avelumab (n = 127). Results Myocarditis event cases in ISS. There were 16 cases of myocarditis out of 20,062 ICI subjects and 1 case out of 4505 in a control/chemotherapy group in ISS. The myocarditis odds ratio (OR) calculation shows an eleva- tion for the ICI group (OR 3.6), though this observation was not statistically significant (95% CI [0.5–27.1]). https://doi.org/10.1038/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 \| www.nature.com/scientificreports/ Figure 2. FAERS/AERS cohorts based on indications and treatments in efficacy trials for all ICIs. The two groups on each sub-cohort indicate the treatment vs control used in the efficacy trials. NSCLC non-small cell lung cancer, SCLC small cell lung cancer, RCC renal cell carcinoma, MCC Merkel cell carcinoma, CSCC cutaneous squamous cell carcinoma. Figure 2. FAERS/AERS cohorts based on indications and treatments in efficacy trials for all ICIs. The two groups on each sub-cohort indicate the treatment vs control used in the efficacy trials. NSCLC non-small cell lung cancer, SCLC small cell lung cancer, RCC renal cell carcinoma, MCC Merkel cell carcinoma, CSCC cutaneous squamous cell carcinoma. Interestingly, in 14 out of 16 patients who developed myocarditis with ICI, the complication occurred after discontinuation of therapy, independent of overall treatment duration which ranged from 1 to 578 days (Table 2 and Fig. 3). Interestingly, in 14 out of 16 patients who developed myocarditis with ICI, the complication occurred after discontinuation of therapy, independent of overall treatment duration which ranged from 1 to 578 days (Table 2 and Fig. 3).h py p g y and Fig. 3). The most common AEs that occurred prior to and during the onset of myocarditis were cardiac, hepatic, pulmonary, and endocrine irAEs (Table 2, Fig. 4). g The most common AEs that occurred prior to and during the onset of myocarditis were cardiac, hepatic ulmonary, and endocrine irAEs (Table 2, Fig. 4). Myocarditis events in FAERS/AERS ICI treatment reports (all cancers). All ICIs were signifi- cantly associated with increased reporting myocarditis events: ipilimumab reporting odds ratio (ROR) 6.5, 95% CI [4.2, 10.0], nivolumab (20.1 [17.1, 23.7]), pembrolizumab (24.1 [19.7, 29.4]), cemiplimab (64.0 [23.6, 173.4]), atezolizumab (24.3 [16.0, 37.1]), avelumab (16.6 [4.1, 66.8]), durvalumab (10.3 [4.9, 21.8]). ICI–ICI and ICI–axitinib cohorts had a significant increase in myocarditis reporting compared to ipilimumab, pembroli- zumab, nivolumab, or avelumab monotherapy: ipilimumab + nivolumab (46.2 [38.2, 55.9]), ipilimumab + pem- brolizumab (45.5 [16.8, 122.7]), pembrolizumab + axitinib (36.9 [11.8, 115.9]), avelumab + axitinib (55.6 [13.4, 222.3]). Results To compare avelumab and pembrolizumab monotherapy to axitinib monotherapy, the FAERS/AERS database was searched for axitinib monotherapy terms. Interestingly only one report of myocarditis in 5492 axitinib monotherapy reports was found. Anthracyclines ± chemotherapy cohort, selected as a positive control, had a significant association with myocarditis reporting (3.7 [3.1, 4.4]) (Fig. 5). It should be emphasized that the reported frequencies of myocarditis reports (Fig. 5a) and the reporting odds ratios (Fig. 5b) do not represent actual population frequencies, but a statistically significant increased reporting of this irAE to the FAERS/AERS and should be interpreted as such. Myocarditis events in FAERS/AERS ICI treatment reports (separated by cancer type). When FAERS/AERS reports were organized into cohorts based on the indication, treatment and control groups in the ICI efficacy trials (Table 1 and Fig. 2), myocarditis reports were present in nearly all indication cohorts. There were no reports of myocarditis in any of the control chemotherapy cohorts, thus the statistical difference between cohorts was not evaluated (Fig. 6). The observed trend was similar to the analysis done with all can- cer types. ICI–ICI combination, ICI–chemotherapy combination, and ICI–kinase inhibitor combinations had a higher number of myocarditis reporting compared to ICI monotherapy.hf g y p g p py The separate dataset with differentiated cohorts by individual ICIs and types of cancer was further analyzed for co-occurring AEs and for death by any cause outcomes. Co-occurring AE analysis was performed for each individual myocarditis case in FAERS/AERS (Supplementary Table S3). 39.9% of all myocarditis cases were associated with death by any cause (Supplementary Table S4). Additionally, there was a significant overlap with the co-occurring AEs observed in the case series (Table 2, Fig. 4, Supplementary S4). Noteworthy was the Scientific Reports \| (2021) 11:17324 \| https://doi.org/10.1038/s41598-021-96467-5 www.nature.com/scientificreports/ Subject number Treatment (dose)54 Indication Treatment duration Time to myocarditis onset after first drug exposure Adverse events by grade Grade 1 AE Grade 2 AE Grade 3 AE Grade 4 AE Grade 5 AE (resulting in death) 1 Carboplatin AUC of 6 mg. Results mL/min q3w + + pem- etrexed 500 mg/ m2 q3w NCSLC 81 days 59 days Conjunctivitis Cough Dysgeusia Dysgeusia Dyspnea Herpes simplex Mucosal inflam- mation Nausea Pyrexia Rash Tachycardia Dyspepsia Fatigue Lethargy Myocarditis None reported None reported 2 Atezolizumab 1200 mg q3w + cisplatin 75 mg/m2 q3w + pem- etrexed 500 mg/ m2 q3w NCSLC 22 days 42 days C-reactive pro- tein increased Oxygen satura- tion decreased None reported Decreased appetite General physical health deterio- ration Myocarditis Transient ischemic attack Lung infection None reported 3 Cemiplimab (3 mg/kg Q2W) CSCC 57 days 58 days Alanine ami- notransferase increased Aspartate ami- notransferase increased Back pain Blood alkaline phosphatase increased Blood creatine phosphokinase increased Blood creatine phosphokinase MB increased Oral contusion Sensitivity to weather change Conjunctivitis Eye contusion Eye swelling Visual impair- ment Myocarditis None reported None reported 4 Durvalumab (10 mg/kg Q2W) Listed as SCLC and solid tumors 1 day 4 days Abdominal pain [TR] Ascites [TR] (DW) Back pain Nausea [TR] Oedema periph- eral [TR] Troponin increased [TR] Vomiting [TR] Ascites Dyspnea [TR] Fatigue [TR] Hyperglycemia Myocarditis Myocarditis Pancreatic carcinoma Troponin increased [TR] None reported Pancreatic carcinoma 5 Ipilimumab (10 mg/kg q3w) Melanoma 22 days 28 days Diarrhea [+++] Fatigue Musculoskeletal pain Pain in extrem- ity Cholecystitis Groin pain Hemoglobin decreased Injection-site reaction Periarthritis Pyrexia Allergic rhinitis [+ +] Hepatitis [+++] (DW) Myocarditis [+++] (DW) Pneumonitis [+++] (DW) None reported None reported 6 Ipilimumab (1 mg/kg) Melanoma 578 days 27 days Sinus bradycar- dia [+] (DR) Ventricular extrasystoles [+] (DR) Blood creati- nine increase (DR) Confusional state (DR) Supraventricu- lar arrhythmia Aspartate ami- notransferase increased Blood bilirubin increased Colitis [++] Diarrhea [+] (DW) Hypophos- phatemia Hypotension (DR) Leukopenia Lymphopenia Myocarditis None reported None reported Continued https://doi.org/10.1038/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 \| www.nature.com/scientificreports/ Subject number Treatment (dose)54 Indication Treatment duration Time to myocarditis onset after first drug exposure Adverse events by grade Grade 1 AE Grade 2 AE Grade 3 AE Grade 4 AE Grade 5 AE (resulting in death) 7 Ipilimumab (1 mg/ kg) + nivolumab (3 mg/kg) Bladder cancer 64 days 89 days Acute kidney injury Alanine ami- notransferase increased [TR] Anemia [TR] Aspartate ami- notransferase increased [TR] Blood albumin decreased Blood alkaline phosphatase increased Blood calcium decreased Blood creatine increased Blood phospho- rus decreased Blood urea increased Depression Dry mouth [TR] Dry skin [TR] Hyperthyroid- ism [TR] Hypomagne- saemia Hypomagne- saemia Edema periph- eral Oral candidiasis [TR] Pelvic pain Troponin I increased [TR] Tumor hemor- rhage Weight decreased Angina pectoris [TR] Blood bicarbo- nate increased [TR] Blood creatine phosphokinase increased [TR] Blood creatine phosphokinase MB increased [TR] Blood gases abnormal [TR] Blood lactic acid decreased Blood lactic acid decreased Blood potas- sium increased Carbon dioxide increased [TR] Dysgeusia [TR] Escherichia infection Lymphocyte count decreased Malaise [TR] Nausea Neutrophil count increased [TR] Pelvic pain Urinary tract infection Weight decreased Angina pectoris [TR] Blood creatine phosphokinase increased [TR] Blood creatine phosphokinase MB increased [TR] Constipation [TR] Dry mouth [TR] Dyspnea Myocarditis [TR] Nausea Oral candidiasis [TR] Pelvic pain Stridor [TR] Troponin I increased [TR] Vomiting [TR] (DD) Myocarditis [TR] Malignant neoplasm progression 8 Pembrolizumab (2 mg/kg Q3W) MCC 1 day 26 days Anemia [TR] Asthenia [TR] Bundle branch block left [TR] Burning sensa- tion [TR] Delirium [TR] Disorientation [TR] Dizziness [TR] Eyelid ptosis [TR] Fall [TR] Leukocytosis [TR] Ophthalmople- gia [TR] Oral candidiasis [TR] Proteinuria [TR] Acute kidney injury [TR] Atrial fibrilla- tion [TR] Fatigue [TR] Hypertension [TR] Malnutrition [TR] Acute myocar- dial infarction [TR] Alanine ami- notransferase increased [TR] Aspartate ami- notransferase increased [TR] Blood creatine phosphokinase increased [TR] Cardiac failure acute [TR] Encephalopathy [TR] Hyponatremia [TR] Ventricular arrhythmia [TR] Ventricular tachycardia [TR] Hyperglycemia [TR] Myocarditis [TR] Small intestinal hemorrhage [TR] None reported Continued https://doi.org/10.1038/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 \| www.nature.com/scientificreports/ Subject number Treatment (dose)54 Indication Treatment duration Time to myocarditis onset after first drug exposure Adverse events by grade Grade 1 AE Grade 2 AE Grade 3 AE Grade 4 AE Grade 5 AE (resulting in death) 9 Pembrolizumab (200 mg Q3W) NSCLC 540 days 557 days Abdominal pain upper[TR] Alanine ami- notransferase increased [TR] Aspartate ami- notransferase increased [TR] Blood alkaline phosphatase increased [TR] Cardiac failure [TR] Cough Decreased appetite Dyspnea Eczema [TR] Pruritus [TR] Cough Diarrhea Papule Acidosis Myocarditis [TR] None reported None reported 10 Pembrolizumab (200 mg Q3W) Bladder cancer 129 days 141 days Atrioventricu- lar block first degree Blood alkaline phosphatase increased Blood bilirubin increased Bone pain Decreased appetite Fatigue Lymphadenopa- thy Pleural effusion Pruritus None reported Hepatic enzyme increased [TR] Myocarditis [TR] Scrotal oedema None reported None reported 11 Pembrolizumab (200 mg Q3W) Bladder cancer 23 days 34 days Blood thyroid stimulating hor- mone increased [TR] None reported Back pain [TR] Eyelid ptosis [TR] Fatigue [TR] Hepatitis [TR] Pneumonia [TR] Thyroiditis [TR] Myocarditis [TR] Myositis [TR] 12 Pembrolizumab (200 mg Q3W) Melanoma 127 days 138 days Weight decreased [TR] Iodine defi- ciency Myocarditis [TR] Myocarditis [TR] None reported None reported 13 Pembrolizumab (200 mg Q3W) HL 1 day 15 days Diarrhea [TR] Headache [TR] Tachycardia [TR] Thrombocyto- penia Transaminases increased [TR] Bacteremia Dyspnea [TR] Myositis [TR] Weight decreased [TR] Myocarditis [TR] None reported 14 Pembroli- zumab (200 mg Q3W) + Axitinib (5 m BID) RCC 17 days 17 days Dysphonia [TR] Chest pain Fatigue [TR] Musculoskeletal chest pain None reported None reported Myocarditis [TR] 15 Pembroli- zumab (200 mg Q3W) + Axitinib (5 m BID) RCC 43 days 46 days Clostridium difficile colitis Erythema Insomnia Pneumonia Diarrhea [TR] Decreased appetite [TR] Electrolyte imbalance [TR] Hepatic func- tion abnormal Myocarditis [TR] None reported 16 Avelumab (20 mg/kg Q2W) Thymoma 15 days 18 days Dizziness Pyrexia Weight increased None reported Autoimmune disorder [TR] (DW) Blood creatine phosphokinase increased [TR] (DW) None reported None reported 17 Avelumab (10 mg/kg Q2W) Head and neck cancer 197 days 207 days Fatigue Myocarditis Pleural effusion Hypothyroid- ism Myocarditis Pleural effusion None reported None reported None reported Table 2. Results Case series. [+++] = Certain AERELL; [++] = Probable AERELL; [+] = Possible AERELL; [TR] = Treatment related, plausibility unspecified. DR dose reduced, DW drug withdrawn, DD dose delayed. Cases are part of the approval packages for the listed ICIs (see PharmaPendium). Table 2. Case series. [+++] = Certain AERELL; [++] = Probable AERELL; [+] = Possible AERELL; [TR] = Treatment related, plausibility unspecified. DR dose reduced, DW drug withdrawn, DD dose delayed. Cases are part of the approval packages for the listed ICIs (see PharmaPendium). https://doi.org/10.1038/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 \| www.nature.com/scientificreports/ co-occurrence of myocarditis with myositis (17.6%) and myasthenia gravis (8.2%), in addition to cardiac failure (10.4%), pneumonitis (4, 7), and increased troponin (4.3%) (Supplementary Table S5). Discussion In this study, we evaluated the myocarditis cases in the clinical trials for ICIs, using the ISS42 data submitted to the FDA and the FDA FAERS/AERS database for reports in patients receiving ICIs as monotherapy in ICI ICI Figure 3. Visualization of the time to end of treatment due to an AE and time to myocarditis X-axis represents the cases numbered in Table 2. Figure 4. Progression of myocarditis cases with preceding AEs and AEs co-occurring at time of myocarditis. Figure 3. Visualization of the time to end of treatment due to an AE and time to myocarditis X-axis represents the cases numbered in Table 2. Figure 3. Visualization of the time to end of treatment due to an AE and time to myocarditis X-axis represe the cases numbered in Table 2. Figure 4. Progression of myocarditis cases with preceding AEs and AEs co-occurring at time of myocarditis. Figure 4. Progression of myocarditis cases with preceding AEs and AEs co-occurring at time of myocarditis. Figure 4. Progression of myocarditis cases with preceding AEs and AEs co-occurring at time of myocarditis co-occurrence of myocarditis with myositis (17.6%) and myasthenia gravis (8.2%), in addition to cardiac failure (10.4%), pneumonitis (4, 7), and increased troponin (4.3%) (Supplementary Table S5). co-occurrence of myocarditis with myositis (17.6%) and myasthenia gravis (8.2%), in addition to cardiac failure (10.4%), pneumonitis (4, 7), and increased troponin (4.3%) (Supplementary Table S5). www.nature.com/scientificreports/ (b) Reporting odds ratios were calculated comparing reported frequencies of myocarditis reports in ICI monotherapy, ICI combination and ICI with axitinib cohorts to myocarditis frequencies in chemotherapy cohorts. Anthracyclines ± chemotherapy cohort used as a positive control. given that the elimination half-lives of ICIs range from 6.1 days (avelumab) to 27.3 days (pembrolizumab), continued vigilance and monitoring of irAEs is critical for patient safety, well after the ICI is discontinued56. The delay in discontinuation of the ICI after myocarditis occurrence may be attributed to the challenges related to diagnosing this rare irAE. Although it takes time to diagnose some of the more complex adverse events, in ISS/ ADAE these events are marked/recorded with the date when they were first noticed or suspected, independent of definitive diagnosis date. Since the true therapeutic target of ICI is a T-cell, and irAEs likely represent endogenous immunologic phenomena, it is possible that the administration of ICI leading to immune myocarditis may not have been the most proximal administration to the time of symptomatic deterioration. Of note, the myocarditis incidence of 0.08% seen in the ISS data, is consistent with prior studies among patients receiving ICIs which have demonstrated an incidence of myocarditis ranging from 0.04 to 1.14%31,36,39,55,57,58. Deducing a statistically significant conclusion regarding associations between disease histology or various thera- pies and the development of myocarditis is limited by the small number of the ISS myocarditis cases.i p p y y y By analyzing the FDA FAERS/AERS database, we confirmed the association between myocarditis and ICIs with the frequency of myocarditis ranging from 0.25 to 2.48% in patients receiving ICIs as monotherapy, in combination with ICI, in combination with axitinib, and in combination with chemotherapy regimens. We demonstrated the association of myocarditis was stronger with combined ICIs as compared to ICI monotherapy which in agreement with prior studies35,59; patients receiving ipilimumab with nivolumab demonstrated the highest reported risk of myocarditis. The all-cause reported mortality rate was 39.9% in all the ICI patients who experienced myocarditis. Melanoma patients with myocarditis due to ipilimumab with nivolumab use had a higher rate of all-cause mortality (53.4%) compared to ICI monotherapy (Supplementary Table S4)35,59. g y p py pp y In contrast to the disproportionality and Bayesian analyses completed by Fan et al.35, we determined that avelumab monotherapy did not have as strong of an association with myocarditis, while avelumab combined with axitinib did demonstrate a stronger association. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. (a) Reported frequencies of myocarditis events for patients administered monotherapy: ipilimumab (n = 8267), nivolumab (n = 27,149), pembrolizumab (n = 13,476), cemiplimab (n = 161), atezolizumab (n = 2397), avelumab (n = 305), and durvalumab (n = 1710), ipilimumab + nivolumab (n = 7970), ipilimumab + pembrolizumab (n = 225), pembrolizumab + axitinib (n = 207), avelumab + axitinib (n = 94), anthracyclines with or without chemotherapy (n = 134,001), chemotherapy and chemotherapy combinations, excluding ICIs and anthracyclines (n = 1,065,158), clozapine (n = 50,558. (b) Reporting odds ratios were calculated comparing reported frequencies of myocarditis reports in ICI monotherapy, ICI combination and ICI with axitinib cohorts to myocarditis frequencies in chemotherapy cohorts. Anthracyclines ± chemotherapy cohort used as a positive control. Figure 5. (a) Reported frequencies of myocarditis events for patients administered monotherapy: i ili b ( 8267) i l b ( 27 149) b li b ( 13 476) i li b ( 161) Figure 5. (a) Reported frequencies of myocarditis events for patients administered monotherapy: ipilimumab (n = 8267), nivolumab (n = 27,149), pembrolizumab (n = 13,476), cemiplimab (n = 161), atezolizumab (n = 2397), avelumab (n = 305), and durvalumab (n = 1710), ipilimumab + nivolumab (n = 7970), ipilimumab + pembrolizumab (n = 225), pembrolizumab + axitinib (n = 207), avelumab + axitinib (n = 94), anthracyclines with or without chemotherapy (n = 134,001), chemotherapy and chemotherapy combinations, excluding ICIs and anthracyclines (n = 1,065,158), clozapine (n = 50,558. (b) Reporting odds ratios were calculated comparing reported frequencies of myocarditis reports in ICI monotherapy, ICI combination and ICI with axitinib cohorts to myocarditis frequencies in chemotherapy cohorts. Anthracyclines ± chemotherapy cohort used as a positive control. Figure 5. (a) Reported frequencies of myocarditis events for patients administered monotherapy: ipilimumab (n = 8267), nivolumab (n = 27,149), pembrolizumab (n = 13,476), cemiplimab (n = 161), atezolizumab (n = 2397), avelumab (n = 305), and durvalumab (n = 1710), ipilimumab + nivolumab (n = 7970), ipilimumab + pembrolizumab (n = 225), pembrolizumab + axitinib (n = 207), avelumab + axitinib (n = 94), anthracyclines with or without chemotherapy (n = 134,001), chemotherapy and chemotherapy combinations, excluding ICIs and anthracyclines (n = 1,065,158), clozapine (n = 50,558. Discussion In this study, we evaluated the myocarditis cases in the clinical trials for ICIs, using the ISS42 data submitted to the FDA, and the FDA FAERS/AERS database for reports in patients receiving ICIs as monotherapy, in ICI–ICI combinations, ICI in combinations with chemotherapy regimens, and in combinations with axitinib. This is the first comprehensive analysis of the ISS42 reports of ICIs. We found that the development of myocarditis occurred earliest on day 4 and the latest on day 557, with a median of 38 days, and this result is consistent with findings in the literature that report that the majority of cases of myocarditis present approximately one to two months after ICI initiation55. Most notably, myocarditis occurred 11 days (range 1–25 days) after the ICI was discontinued; https://doi.org/10.1038/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 \| Scientific Reports \| (2021) 11:17324 \| www.nature.com/scientificreports/ The disparity in the results may be due to the fact that our analysis included ICI monotherapy reports, while Fan and colleagues used ICI reports where concomitant drugs were used and ICI was the primary suspect which allows for significant bias and error, especially when search- ing for a rare or unexpected event. Furthermore, we organized the reports into cohorts by specific indication, and quantified the myocarditis association by using positive and negative controls with the same type of cancer, which is rarely done in disproportionality analysis studies. Additionally, we analyzed the co-occurring adverse events and observed myositis to be the most common AE reported with myocarditis, suggesting a stronger etiological connection between the two irAEs that may not be explained by general immune activation alone. Scientific Reports \| (2021) 11:17324 \| https://doi.org/10.1038/s41598-021-96467-5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. Reported frequencies of myocarditis events in FAERS/AERS database in cohorts based on efficacy trial indications, treatments and control groups. NSCLC non-small cell lung cancer, SCLC small cell lung cancer, RCC renal cell carcinoma, RCC renal cell carcinoma, CSCC cutaneous squamous cell carcinoma. Figure 6. Reported frequencies of myocarditis events in FAERS/AERS database in cohorts based on efficacy trial indications, treatments and control groups. NSCLC non-small cell lung cancer, SCLC small cell lung cancer, RCC renal cell carcinoma, RCC renal cell carcinoma, CSCC cutaneous squamous cell carcinoma. Figure 6. Reported frequencies of myocarditis events in FAERS/AERS database in cohorts based on efficacy trial indications, treatments and control groups. NSCLC non-small cell lung cancer, SCLC small cell lung cancer, RCC renal cell carcinoma, RCC renal cell carcinoma, CSCC cutaneous squamous cell carcinoma. The association between myocarditis and myositis has been previously noted in previous immunotherapy60,61 utoimmune disease62,63, and infection case studies64,65. Taken together, the results from the ISS and the FDA FAERS/AERS databases highlight the need for prompt recognition, diagnosis, and management of myocarditis in patients receiving ICIs, with additional vigilance with ICIs combination therapies, from treatment initiation through several weeks after ICI discontinuation.h p g ft The monitoring and management of cardiac irAEs has been well described by Palaskas and Spallarosa; a diagnostic workup including the use of laboratory values (Troponin I, N-terminal pro B-type natriuretic (BNP) peptide, BNP), imaging (12-lead electrocardiogram, echocardiogram, cardiac magnetic resonance, telemetry monitoring), and procedures (endomyocardial biopsy and coronary angiography) is recommended in patients with suspected myocarditis55,66. https://doi.org/10.1038/s41598-021-96467-5 www.nature.com/scientificreports/ While the diagnostic workup is performed, ICI therapy should be discontinued, and prompt initiation of corticosteroids (1000 mg intravenous (IV) methylprednisolone for three days followed by 1 mg/kg IV/oral prednisone) is recommended. If the diagnostic workup demonstrates definite, probable, or possible myocarditis, corticosteroids should be continued and tapered off over four to six weeks. Of particular importance is attention to the electrocardiographic changes that occur in myocarditis, such as arrythmias67–69 is the predominant mechanism of morbidity and mortality and close consultation with cardiology colleagues and in particular electrophysiology subspecialists is key in the multidisciplinary care of these patients.ii p p y gy p y p y p In summary, we confirmed statistically significant association of ICI use with myocarditis using FARS/AERS data and stratified this association by specific cancer types and by ICI combination therapies. We found and an increased reporting of myocarditis cases for patients treated with ICI–ICI, ICI–axitinib, and ICI–chemotherapy combinations. Study limitations. Adverse event reporting to FAERS/AERS is voluntary and reports are not always clini- cally adjudicated for causality. The calculated frequencies do not represent the actual population but rather the reported frequency of myocarditis AEs out of all reported ICI AEs in the FAERS/AERS database. This reported frequency definition needs to be kept in mind while evaluating those frequencies, as the numbers may be exag- gerated and do not represent the actual number of cases in the total ICI-administered population. Studies have shown that there may be significant underreporting and overreporting of adverse events70,71. Absence of lab values and complete medical records, including comprehensive information, concurrent medications, presence of a pacemaker, and comorbidities may introduce uncertainties to our analysis. However, using postmarketing surveillance data remains an important tool in identifying a statistically significant signal, especially for very rare https://doi.org/10.1038/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 \| www.nature.com/scientificreports/ adverse events such as myocarditis which was virtually non-existent in the clinical trial data. Additionally, both in ISS and FAERS/AERS data, a noteworthy limitation is the variability in the way the adverse events were coded. There is a need for harmonization of nomenclature, a consensus, and having the ability for algorithms to cluster terms to better understanding temporal kinetics. adverse events such as myocarditis which was virtually non-existent in the clinical trial data. Additionally, both in ISS and FAERS/AERS data, a noteworthy limitation is the variability in the way the adverse events were coded. www.nature.com/scientificreports/ There is a need for harmonization of nomenclature, a consensus, and having the ability for algorithms to cluster terms to better understanding temporal kinetics. Data availabilityh There was no direct human participation in this study. The data sets utilized were de-identified. Institutional Review Board requirements do not apply under 45 CFR 46.102. Cases used in the case-series section were included in the approval package: https://www.pharmapendium.com. FAERS/AERS datasets are available to the public online: https://www.fda.gov/drugs/questions-and-answers-fdas-adverse-event-reporting-system-faers/ fda-adverse-event-reporting-system-faers-latest-quarterly-data-files. Received: 4 May 2021; Accepted: 9 August 2021 References 1. Assal, A., Kaner, J., Pendurti, G. & Zang, X. 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Author contributions T.M., M.L., I.V.C., T.Mo. & M.A.I. performed the research, R.A., T.M., I.M.S. & S.P.P. designed the study and, R.A., T.M., I.M.S., K.B., S.P.P., P.L. drafted the manuscript and reviewed the final version. R.A. and M.L. pro- cessed the data sets. T.M., M.L., I.V.C., T.Mo. & M.A.I. performed the research, R.A., T.M., I.M.S. & S.P.P. designed the study and, R.A., T.M., I.M.S., K.B., S.P.P., P.L. drafted the manuscript and reviewed the final version. R.A. and M.L. pro- cessed the data sets. Acknowledgements g We thank Dr. Da Shi and members of Abagyan lab for contributions to processing the FAERS and AERS data files and supporting the computer environment. www.nature.com/scientificreports/ 11, 972 https://doi.org/10.3389/fphar.2020.00972 (2020).i 67. Bonaca, M. P. et al. Myocarditis in the setting of cancer therapeutics: Proposed case definitions for emerging clinical syndromes in cardio-oncology. Circulation 140(2), 80–91. https://doi.org/10.1161/CIRCULATIONAHA.118.034497 (2019).f 68. Moslehi, J. J. Cardiovascular toxic effects of targeted cancer therapies. N. Engl. J. Med. 375(15), 1457–1467. https://doi.org/10.1056/ NEJMra1100265 (2016).h J ( ) 9. Thompson, J. A. et al. NCCN guidelines insights: Management of immunotherapy-related toxicities, Version 1.2020. J. Natl. Compr Cancer Netw. 18(3), 230–241. https://doi.org/10.6004/jnccn.2020.0012 (2020). https://doi.org/10.1038/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 \| Competing interests i p g Dr. Patel receives scientific advisory income from: Amgen, AstraZeneca, Bristol-Myers Squibb, Eli Lilly, Genen- tech, Illumina, Merck, Rakuten, Paradigm, Tempus. Dr. Patel’s university receives research funding from: Bris- tol-Myers Squibb, Eli Lilly, Incyte, AstraZeneca/MedImmune, Merck, Pfizer, Roche/Genentech, Xcovery. Fate Therapeutics, Genocea, Iovance. Dr. Saunders is currently affiliated with Horizon CME, and has been on advisory boards of Genentech, Partner Therapeutics, and Takeda Inc. All other authors declare no conflict of financial or non-financial interest. © The Author(s) 2021 Additional informationh Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-96467-5. Correspondence and requests for materials should be addressed to R.A. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the 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/s41598-021-96467-5 Scientific Reports \| (2021) 11:17324 \|
https://openalex.org/W4224881413	https://www.researchsquare.com/article/rs-1573274/latest.pdf	English	null	6-benzylaminopurine and kinetin modulations during in vitro propagation of Quercus robur (L.): an assessment of anatomical, biochemical, and physiological profiling of shoots	Research Square (Research Square)	2,022	cc-by	12,025	6-benzylaminopurine and kinetin modulations during in vitro propagation of Quercus robur (L.): an assessment of anatomical, biochemical, and physiological profiling of shoots Page 1/29 6-benzylaminopurine and kinetin modulations during in vitro propagation of Quercus robur (L.): an assessment of anatomical, biochemical, and physiological profiling of shoots João Paulo Rodrigues Martins (  jprmartinss@yahoo.com.br ) Polish Academy of Sciences Institute of Dendrology Kornik: Polska Akademia Nauk Instytut Dendrolog w Korniku https://orcid.org/0000-0003-0554-6793 Mikołaj Krzysztof Wawrzyniak Institute of Dendrology Polish Academy of Sciences: Instytut Dendrologii Polskiej Akademii Nauk Juan Manuel Ley-López Institute of Dendrology Polish Academy of Sciences: Instytut Dendrologii Polskiej Akademii Nauk Ewa Marzena Kalemba Institute of Dendrology Polish Academy of Sciences: Instytut Dendrologii Polskiej Akademii Nauk Marcel Merlo Mendes Federal University of Espirito Santo: Universidade Federal do Espirito Santo Paweł Chmielarz Institute of Dendrology Polish Academy of Sciences: Instytut Dendrologii Polskiej Akademii Nauk Research Article Keywords: cytokinin, morphophysiological disturbances, oaks, plant anatomy, plant physiology, woody plant species Posted Date: April 26th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1573274/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 Plant Cell, Tissue and Organ Culture (PCTOC) on June 2nd, 2022. See the published version at https://doi.org/10.1007/s11240-022-02339-9 Mikołaj Krzysztof Wawrzyniak Institute of Dendrology Polish Academy of Sciences: Instytut Dendrologii Polskiej Akademii Nauk Juan Manuel Ley-López Institute of Dendrology Polish Academy of Sciences: Instytut Dendrologii Polskiej Akademii Nauk Ewa Marzena Kalemba Institute of Dendrology Polish Academy of Sciences: Instytut Dendrologii Polskiej Akademii Nauk Marcel Merlo Mendes Federal University of Espirito Santo: Universidade Federal do Espirito Santo Paweł Chmielarz Institute of Dendrology Polish Academy of Sciences: Instytut Dendrologii Polskiej Akademii Nauk Research Article Posted Date: April 26th, 2022 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 Plant Cell, Tissue and Organ Culture (PCTOC) on June 2nd, 2022. See the published version at https://doi.org/10.1007/s11240-022-02339-9. Page 1/29 Page 1/29 Abstract For many woody species, such as Quercus robur, cytokinins in the culture medium are required to maintain in vitro plant material. Among synthetic cytokinins, 6-benzylaminopurine (BAP) and kinetin (KIN) are the most frequently used. In addition to inducing shoots, cytokinins can cause morphophysiological disorders. Therefore, we aimed to investigate the anatomical, biochemical, and physiological alterations and profiles of Q. robur shoots exposed to two cytokinins, applied alone and in combination. Shoots previously established in vitro were transferred to WPM culture media supplemented with BAP at concentrations of 0, 1.25, and 3.50 µM combined with KIN at concentrations of 0, 0.62, and 1.25 µM totaling 9 treatments. Anatomical, physiological, and biochemical analyses were performed after 40 d of culture. BAP induced the formation of new buds with anatomically underdeveloped leaves; induced shoot-tip necrosis, which is considered a response to the inefficient transport of water and nutrients; reduced the thickness of the cell walls of phloem fibers; and decreased the content of phenolic compounds and photosynthetic pigments. These responses were less pronounced with co-exposure to KIN. In contrast, KIN alone stimulated a larger area of secondary xylem and more lignified cell walls. BAP can induce shoots with underdeveloped anatomical and biochemical characteristics. Shoots that grew with KIN alone had stem and leaf anatomical characteristics, indicating greater commitment to cellular differentiation than proliferation. When both cytokinins are combined, KIN can partially mitigate the deleterious effects of BAP on in vitro growth. Introduction Quercus robur (L.) (Fagaceae) is an economically important hardwood tree species that is extensively used as construction material. However, the propagation of Q. robur by seeds may be challenging because of their short period of viability (Ntuli et al. 2011). An alternative for the propagation of Q. robur is via vegetative methods. This allows the maintenance of high-value genotypes and is useful for gene conservation (Savill and Kanowski, 1993). Among the vegetative methods, plant tissue culture techniques offer a valuable option for the large-scale propagation of economically interesting woody species, including Quercus (Pandey and Tamta 2012; Baccarin et al. 2015; Wojtania et al. 2015; Gentile et al. 2017; Fadladeen and Toma 2020). During in vitro propagation, synthetic cytokinins are one of the main groups of plant growth regulators used to supplement the medium for induction of de novo shoot regeneration in Quercus shoots (Chalupa 1988; Puddephat et al. 1997; Martínez et al. 2017; Pandey et al. 2018). Although 6-benzylaminopurine (BAP) is the most common cytokinin used for the in vitro propagation of Quercus, there have also been reports of kinetin (KIN) usage (Chalupa 1988; Pandey and Tamta 2012; Fadladeen and Toma 2020). Pandey and Tamta (2012) observed that the previous use of KIN later influenced the rooting and subsequent survival of in vitro-propagated plants. However, studies on the effects of cytokinins on de novo shoot regeneration effectiveness, an approach to compare the two types, is frequently documented in woody species (Chalupa 1988; Sant'Ana et al. 2018; Fadladeen and Toma 2020). Nevertheless, the Page 2/29 combined use of two types of cytokinins sometimes yields better results than a single cytokinin (Sharma et al. 2017; Bhat et al. 2022). Although cytokinins might be crucial for driving or improving in vitro responses, the shoots may show physiological and anatomical disorders induced by them (Dobránszki and Mendler-Drienyovszki 2014; Rosa et al., 2018). Under exogenous cytokinin exposure, plants may present with alterations in shoot length (visibly shorter shoots), decreased chlorophyll content, chlorotic leaves, shoot-tip necrosis, and low performance of the photosynthetic apparatus (Gentile et al. 2017; Lizárraga et al. 2017; Rosa et al. 2018; Silva et al. 2020; Murvanidze et al. 2022). Morphological disorders induced in vitro by synthetic cytokinins, such as shoot-tip necrosis, have been previously reported in woody plant species, including Quercus (Vieitez et al. 1985, 1997, 2009; Martínez et al. 2017; Surakshitha et al. 2019). However, according to Bairu et al. Introduction (2009), there are differing opinions on the effects of cytokinins on shoot-tip necrosis. This phenomenon is manifested by visible symptoms, in which the apical shoot becomes brown and later dies (McCown and Seller 1987). Morphophysiological disorders might be a stress response and can be characterized by studies that connect the anatomy, physiology, and biochemistry of in vitro explants. Analyses involving the quantitative stem and leaf anatomy of shoots are valuable tools for defining how in vitro conditions can influence the success of all micropropagation steps (Martins et al. 2015; Manokari et al. 2021b, 2022). In addition to anatomical studies, a deeper approach to physiological and biochemical changes may be useful for stress characterization. Alterations in the antioxidant activity and content of photosynthetic pigments and phenolic compounds are considered good indicators of stress in plants under ex vitro and in vitro conditions (Wojtania et al. 2015; Chalker-Scott and Fuchigami 2018; Martins et al. 2018b, 2020). Therefore, in addition to developing a micropropagation protocol, investigating the anatomical and physiological status of in vitro shoots may be crucial for further research. Many studies on the in vitro culture of woody plant species have focused on multiplication rates during micropropagation (Chalupa 1988; Sant'Ana et al. 2018; Bhat et al. 2022). In this way, many researchers have not considered the anatomical and physiological status of in vitro shoots at a deeper level. The morphophysiological status of in vitro-propagated shoots can interfere significantly during the acclimatization phase (Martins et al., 2018a; Manokari et al. 2021b). In this study, we investigated the morphophysiological disorders that may occur during in vitro regeneration of Q. robur explants. Therefore, we proposed a comprehensive approach to assess the anatomical, biochemical, and physiological profiles of the in vitro-propagated shoots of Q. robur. We aimed to investigate the morphophysiological changes in Q. robur in vitro growing shoots exposed to two cytokinins (BAP and KIN), applied alone and in combination. The collected data were analyzed through additional data processing using multivariate analyses of Pearson correlation and principal component analysis (PCA). Material And Methods In vitro culture with BAP and KIN Shoots (approximately 2 cm) obtained under the aforementioned conditions were used as explants. Shoots were transferred to 320 mL glass containers containing 50 mL WPM with 30 g L−1 sucrose and solidified with 7 g L−1 agar. The treatments consisted of medium supplemented with three levels of BAP (0, 1.25, and 3.5 μM) combined with three levels of KIN (0, 0.62, and 1.25 μM) (Sigma-Aldrich, St. Louis, USA), totaling 9 treatments. Shoots cultured in cytokinin-free medium (0 μM BAP + 0 μM KIN) were used as the controls. The experiment was conducted using four shoots per container. The media's pH was adjusted to 5.7 before autoclaving at 120 °C for 20 min. After shoot inoculation on the medium in a laminar flow cabinet, the plant material was kept in a growth chamber for 40 d under the conditions mentioned above. Plant material and in vitro establishment and culture Branches measuring 1.5 m long were collected from selected Q. robur trees. The collected branches were cut into 30–40 cm fragments that were 2–3 cm in diameter and disinfected with sodium hypochlorite (commercial solution with 10% activated chlorine) for 20 min. The fragments of branches were cultured in plastic pots with water, under growth room conditions at 23 °C, in 90% humidity, and photoperiod of 16 h dark/8 h light. After 30 d, green epicormic shoots regenerated from the branches were used as explant sources to initiate the in vitro culture. The epicormic shoots were divided into approximately 2 cm fragments (with 1–2 buds each, without leaves) and disinfected with mercury chloride (0.1% for 3 min). After disinfection, the explants were washed four times with sterile distilled water for 1 min, placed on Woody Plant Medium (WPM) (Lloyd and McCown 1981) with 3.5 µM BAP (Sigma-Aldrich, St. Louis, USA) (Puddephat et al. 1997) and solidified with 7 g L−1 agar. The medium's pH was adjusted to 5.7 before autoclaving at 120 °C for 20 min. After transferring the explants to the in vitro culture medium, the plant material was kept under controlled conditions in a phytotron (growth chamber), with a light intensity of 80 µmol m−2s−1 of photosynthetically active radiation (PAR), 16 h light/8 h dark photoperiod, at 23 °C. The explants were transferred to fresh culture medium every 40 d to obtain the necessary number of shoots with similar morphology. Quantification of photosynthetic pigment contents Pigment extraction was performed using 0.030–0.039 g of six independent plant samples (n = 6). The material was placed in 2 mL Eppendorf containing 1 mL 80% (v/v) acetone and maintained for 48 h in the dark at 4 °C. Chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid (Car) contents were quantified using a microplate reader (Infinite M200 pro, Tecan, Switzerland). The absorbance was measured at λ = 665, 645, and 480 nm for Chl a, Chl b, and Car, respectively. The Chl content was estimated as proposed by Arnon (1949) and expressed as micrograms of pigment per gram of fresh weight (μg g−1 FW). Stem and leaf anatomy of in vitro Q. robur shoots After 40 d of culture, five shoots from each treatment were sampled randomly and stored in 50% ethanol. The anatomical characterization of stems was performed by examining cross-sections of stems between 0.5 and 0.8 cm from the shoot base. The leaf anatomy was determined by cross sections of the first fully expanded pair of leaves from the apex. Cross-sections were prepared with the aid of a double razor and safranin–astra blue staining solution. The leaf venation pattern (vein distribution) was characterized by diaphanization in 5% sodium hydroxide solution (NaOH) and staining with 1% safranin solution (Kraus and Arduin 1997; Martins et al. 2020). The slides were photographed using a digital camera (Zeiss AxioCam MRc 5, Germany) coupled to a Zeiss Axioskop microscope (Carl Zeiss, Germany). Anatomical measurements (n = 5) were carried out using Imagetool® software calibrated with a microscopic ruler. In the stem cross sections, the total transverse stem (μm2) and xylem area (μm2) were measured. The anatomical traits analyzed in the leaves were the thickness of the adaxial (μm) and abaxial epidermis (μm), palisade (μm), spongy parenchyma (μm), cell walls of the phloem fibers (μm), and the number of vessel elements. The leaf and stem cross sections were stained with berberine hemisulfate and aniline blue solutions. This was done to visualize lignin distribution in the tissues (Brundrett et al. 1988). Staining procedures and slide assembly were performed as described by Martins et al. (2020). Sections were observed under a fluorescence microscope (Zeiss Axioskop, Germany). Images were captured with a Zeiss AxioCam MRc 5 camera using UV light with an excitation/emission spectrum of 385 nm. Analysis of the multiplication rate The multiplication rate was estimated by verifying the budding percentage response and number of shoots per explant. The analysis was performed after 40 d of growth using 40 explants from each treatment, which were randomly collected and divided into five parcels (n = 5). In addition, the probability of shoot-tip necrosis was analyzed. Each jar in which at least one shoot-tip exhibited necrosis was classified as under the presence of necrosis. Probability was calculated using 10 different jars (n = 10). Page 4/29 Page 4/29 Chlorophyll a fluorescence analysis Photosynthetic apparatus performance was measured using an FMS 2+ pulse-modulated chlorophyll fluorimeter (Hansatech, King's Lynn, Norfolk, UK) according to the experimental protocol described by Kramer et al. (2004). Measurements were performed on six plants from each treatment at 40 d of growth. Dark adaptation and reading procedures were performed as described by Martins et al. (2020). The maximum quantum yield of primary photochemistry [Fv/Fm = 1 – (Fo – Fm)], quantum yield for energy dissipation [Fo/Fm = 1 – (Fv – Fm)], and non-photochemical quenching coefficient [qNP = (Fm – Fm)/(Fm – Fo)] were calculated automatically by the device. Antioxidant activity of phenolic compounds Determination of the antioxidant activity in the total phenolic and non-tannin phenolic extracts was carried out using a CUPric reducing antioxidant capacity (CUPRAC) assay, in which the main reagent, copper (II)-neocuproine (2,9-dimethyl1,10-phenanthroline), oxidizes antioxidants to generate a colored product (Apak et al. 2004). The reaction was performed in 1 M ammonium acetate buffer (pH 7) containing 10 mM copper chloride and 7.5 mM neocuproine. Absorbance was measured at 450 nm and calculated based on the calibration curve obtained with Trolox in the 0–25 µM range (R2 = 0.9952) and expressed as Trolox equivalent antioxidant activity (TEAC). Extraction of phenolic compounds Leaf samples containing 0.1 g material were homogenized in methanol:water:hydrogen chloride (79:20:1) solvent at 4 °C. Hydrogen chloride was added to prevent oxidation. Clear extracts were obtained after 15 min of centrifugation at 16,000 × g and were further diluted for quantification. Total phenolic content (TPC) was determined spectrophotometrically (A1) using the Folin-Ciocalteu (F-C) reagent (Singleton et al. 1999). A mixture containing 20 µL diluted sample extract, 40 µL 10% FC reagent, and 160 µL 700 mM sodium carbonate was incubated for 1 h in the dark and measured at 765 nm using an Infinite M200 PRO Page 5/29 (Tecan) plate reader and Magellan software. One milliliter of diluted extract was incubated with 0.05 g hide powder (60 min) to adsorb tannins and centrifuged for 5 min at 16,000 × g (Ainsworth and Gillespie 2007). The non-tannin phenolic content was determined spectrophotometrically (A2) using the procedure described above. Serial dilutions of 0–6 mM gallic acid in methanol was used to create a standard curve (R2 = 0.9998) expressing the phenolic compounds as gallic acid equivalents (GAE). Statistical analysis The experimental design was completely randomized using a 3 × 3 factorial scheme (BAP concentrations of 0, 1.25, and 3.5 μM × KIN concentrations of 0, 0.62, and 1.25 μM). The data were subjected to two-way analysis of variance (ANOVA), and the means were compared using Tukey’s test at 5% significance. For additional analyses, a correlation matrix and PCA were performed. Correlation matrices were created using Pearson's method in the corrplot package. The function PCA was implemented with "FactoMineR" version 2.3, factoextra version 1.0.7, corrplot version 0.84 R packages (Le et al. 2008; Wei and Simko 2017; Kassambara and Mundt 2020). Growth traits Page 6/29 Cytokinins considerably influenced the in vitro regeneration process (Fig. 1). More precisely, explants grown in cytokinin-free medium (0 µM BAP + 0 µM KIN) exhibited leaves with necrotic lesions (Fig. 1A). Shoots cultured under BAP-free conditions did not form any shoot buds at the base of the explants (de novo buds), regardless of KIN concentration. In contrast, BAP supplementation had a positive effect on the budding response and number of new shoots. Among the treatments with 3.5 µM BAP, explants grown with 1.25 µM KIN displayed a 29% reduction in budding response (Figs. 1J-K). Shoot-tip necrosis was visible in explants exposed to BAP. This phenomenon was enhanced by increasing the BAP concentration, irrespective of the KIN concentration applied (Fig. 1L). Influence of cytokinins on stem and leaf anatomy In contrast, a reduction in the thickness of this parenchyma was observed in shoots cultured with 3.5 µM BAP + 1.25 µM KIN (Figs 4, 5F). Both BAP and KIN concentrations influenced the palisade/spongy ratio, but acted independently. The palisade/spongy ratio decreased as the BAP concentration increased (R2 = 0.86), whereas KIN exposure increased the palisade/spongy ratio (Figs. 4, 5B). μM BAP had thicker spongy parenchyma when co-exposed to 1.25 μM KIN. In contrast, a reduction in the thickness of this parenchyma was observed in shoots cultured with 3.5 µM BAP + 1.25 µM KIN (Figs 4, 5F). Both BAP and KIN concentrations influenced the palisade/spongy ratio, but acted independently. The palisade/spongy ratio decreased as the BAP concentration increased (R2 = 0.86), whereas KIN exposure increased the palisade/spongy ratio (Figs. 4, 5B). However, concerning the anatomical traits of leaves, the number of vessel elements and the thickness of the cell walls of phloem fibers were assumed to be influenced by BAP and KIN. The highest number of vessel elements was observed in leaves grown in a medium containing 1.25 µM KIN and without BAP (Figs 4 and 5G). Furthermore, leaves that grew under BAP exposure displayed considerably decreased cell wall thickness of phloem fibers. By comparing 0 or 1.25 µM BAP treatments, leaves grown under 0.62 µM KIN showed greater thickness for the cell walls of phloem fibers (Figs 4, 5C). The deposition of lignin in the cross-sections of stems and leaves was visualized using berberine hemisulfate and aniline blue solutions, which emitted fluorescence from the cell walls of xylem and phloem fibers. The area of fluorescence signal was reduced in plants treated with BAP. Interestingly, the fluorescence signal in the leaves was lower (Fig. 6). Photosynthetic pigment contents BAP and KIN affected the content of photosynthetic pigments in Q. robur shoots cultured in vitro. More precisely, BAP and KIN concentrations affected all parameters related to Chl a and Chl b. Among the treatments without KIN, shoots cultured with 3.5 µM BAP showed a significant reduction in Chl a, Chl b, Chl total content, and Chl a/b ratio. A decrease in Chl content and the Chl a/b ratio was also detected in shoots grown in media with 0.62 or 1.25 µM KIN BAP (1.25 and 3.5 µM) (Figs. 5D, E, I, J). Likewise, the Chl a and Chl b contents of carotenoids were also modulated by the BAP and KIN concentrations; however, they acted independently. The Car content increased (R2 = 0.85) concomitantly with the KIN concentrations, but decreased (R2 = 0.97) with increasing BAP concentration (Fig. 5H). Influence of cytokinins on stem and leaf anatomy Although transversal stem cross-sections of Q. robur shoots were performed in the same region of the organ, apparent differences in stem morphology were observed among the treatments. Stem width (total transverse stem area) was affected only by BAP, and it increased linearly (R2 = 0.98) as a function of BAP concentration (Figs. 2A-J). Importantly, KIN concentration was the primary modulator of the xylem area. Shoots grown under 1.25 µM KIN had the largest xylem area in the cross-sections of stems (Figs. 2A-I, L). Both BAP and KIN affected the xylem area/total transverse stem area ratios. However, the effects of both cytokinins were independent of each other. A linear reduction in the xylem area/total transverse stem area ratio was strongly related to increasing BAP concentrations (R2 = 0.95). Among the KIN concentrations, shoots cultivated with 1.25 µM KIN achieved the highest xylem area/total transverse stem area ratio (Figs. 2A-I, K). The leaves showed morphological differences. Although we did not measure leaf size, the leaves grown with KIN alone were clearly larger. In contrast, shoots cultured without exogenous cytokinin supplementation or BAP treatment had smaller leaves. Venation patterns also changed. Shoots grown in BAP-free medium showed leaves with thicker veins (Fig. 3). Under the tested conditions, Q. robur leaves exhibited stomata with a hipostomatic distribution wherein the epidermis had one cell layer on both the adaxial and abaxial sides. The mesophyll revealed dorsiventral organization, with one layer of palisade parenchyma on the adaxial leaf side and three layers of spongy parenchyma on the abaxial side. The vascular bundles were collateral and surrounded by an almost uninterrupted ring of phloem fibers (Fig. 4). The treatments had an impact on the leaf anatomy. The thickness of both the adaxial and abaxial sides of the epidermis did not change and reached 11.3 µm and 10.4 µm on average, respectively (Fig. 4). The thickness of the palisade parenchyma was affected by BAP and KIN concentrations; however, they acted separately. Shoots exposed to KIN had thicker palisade parenchyma, whereas leaves with thinner palisade parenchyma were observed after BAP supplementation (Figs. 4, 5A). The thickness of the spongy parenchyma was modified using both the BAP and KIN. Leaves grown without BAP showed similar spongy parenchyma thickness among treatments with KIN. The leaves of shoots grown with 1.25 Page 7/29 Page 7/29 Page 7/29 μM BAP had thicker spongy parenchyma when co-exposed to 1.25 μM KIN. Correlation and principal component analysis (PCA) Correlation analysis between all measured parameters was performed. Growth traits related to de novo shoot regeneration induction (budding and number of new shoots) showed a negative relationship with anatomical traits (palisade parenchyma, palisade/spongy ratio, and thickness of cell walls of phloem fibers) and phenolic content (total phenolics and non-tannin phenolics). In addition, the non- tannin TEAC showed a negative relationship with Chl a/b. The leaf anatomical traits were positively correlated with the phenolic content (total phenolics and non-tannin phenolics) (Fig. 9A). PCA was performed using the parameters that most contributed to the explanation of the data; thus, spongy parenchyma, adaxial and abaxial epidermis, stem width, and qNP were excluded from the analysis. The two principal components explained 55% of the total variance in the dataset, with axes 1 and 2 explaining 40.4% and 14.6% of the variance, respectively. The traits related to growth (budding, number of new shoots, and shoot-tip necrosis (STN)), anatomy (anatomical traits of stems and leaves), antioxidant capacity (TEAC and non-tannin phenolics TEAC), and content of photosynthetic pigments (Chl a, Chl b, Ch a/b, Chl total, and Car) were correlated with axis 1 of the analysis (average > 0.7). The photochemical parameters (Fv/Fm and Fo/Fm) obtained through Chl a fluorescence showed a high correlation with axis 2 (average > 0.6). Through PCA analysis, three distinct groups were revealed, and they showed correlations of the analyzed traits in this study with the treatments. The first cluster showed the treatments without BAP supplementation (0 μM BAP + 0 μM KIN, 0 μM BAP + 0.62 μM KIN, and 0 μM BAP + 1.25 μM KIN), which were plotted as a function of axis 1, on the right side. This demonstrates a correlation with anatomical traits, phenolic content, and photosynthetic pigment content. The second cluster grouped the treatments with 1.25 μM BAP supplementation (1.25 μM BAP + 0 μM KIN, 1.25 μM BAP + 0.62 μM KIN, and 1.25 μM BAP + 1.25 μM KIN) and this was plotted as a function of axis 2. The second cluster demonstrated its correlation with Chl a fluorescence parameters. The treatments with 3.5 μM BAP supplementation were grouped in the third cluster (3.5 μM BAP + 0 μM KIN, 3.5 μM BAP + 0.62 μM KIN, and 3.5 μM BAP + 1.25 μM KIN), and were also plotted in axis 1, but on the left side. Phenolic content and Trolox equivalent antioxidant capacity Phenolic content and antioxidant capacity were investigated in leaves modified with BAP and KIN. Application of 0 and 0.62 µM KIN reduced the content of total phenolics and non-tannin phenolics when shoots were co-exposed to BAP. Among the treatments with 1.25 µM KIN, the contents of the total phenolics and non-tannins were similar but lower than those in the control plants grown without cytokinin supplementation. The highest total antioxidant capacity, expressed as the Trolox equivalent antioxidant capacity (TEAC), was observed in the leaves of shoots cultured with 3.5 µM BAP and without KIN. After the elimination of condensed tannins, BAP alone was found to stimulate the antioxidant response, together with an increase in its concentration (Figs 7A-D). fl Page 8/29 Page 8/29 Chlorophyll a fluorescence Cytokinin treatments have been documented to modulate chlorophyll a fluorescence. Fv/Fm and Fo/Fm were affected only by BAP concentrations. Shoots grown in the medium supplemented with BAP had higher Fv/Fm values. Nevertheless, a tendency of decrease at BAP concentrations higher than 1.25 μM was revealed (Fig. 8A). The Fo/Fm value was higher when BAP was not added to the medium (Fig. 8B). qNP was impacted by both BAP and KIN. The lowest qNP was observed in plants cultured in the control KIN-free and BAP-free media. In contrast, the highest qNP was detected in the leaves of shoots grown on the medium supplemented with only 1.25 μM BAP (Fig. 8C). Discussion This study reports the impact of BAP and KIN alone and their synergistic effect on the in vitro regeneration process as well as on the morphophysiology of Q. robur shoots. Shoots cultured with BAP showed anatomical and biochemical characteristics that denote a less advanced level of secondary growth (transition status between primary and secondary growth). In contrast, shoots cultured in medium without BAP (control and KIN supplementation) had tissues with highly differentiated cells and clear secondary growth morphology. These characteristics were correlated with de novo shoot regeneration efficiency. BAP was more efficient in promoting shoot bud formation at the base of the explants. The effective response of BAP to in vitro multiplication of Quercus species has been reported previously (Puddephat et al. 1997; Tamta et al. 2008; Pandey and Tamta 2012). BAP shows a more prolonged stimulation of cell division because it is not easily broken down by plants and is more stable and resistant to oxidation (Agustina et al. 2020). Such characteristics may contribute to the effectiveness of this synthetic cytokinin in stimulating bud formation, as observed in our study. The efficiency of BAP in our study correlated with a lower level of cell determination in the tissues. During in vitro culture, the composition of the medium can modulate cells to stages with variable differentiation levels, including multipotent, pluripotent, and totipotent levels (Baccarin et al., 2015). This response can regulate plant tissues with lower cellular determination and higher competence acquisition. In addition, shoots of Q. robur cultured with BAP showed stems with a larger investment in parenchymatic cells, that is induction of adventitious buds. Parenchyma cells in shoot bases may act as a pluripotent cell niche for direct organogenesis (induction of adventitious buds) (Graner et al. 2019). The explants of Q. robur were unable to induce shoot buds in the control medium (cytokinin-free) or under KIN supplementation as the only exogenous cytokinin source. This species is not only exogenous cytokinin-dependent for shoot organogenesis induction, but also synthetic cytokinin type-dependent for the effectiveness of the regeneration process. Similar results have been previously reported for Q. robur and Q. aegilops (Chalupa 1988; Fadladeen and Toma 2020). In the present study, the low efficiency of de novo shoot regeneration in Q. robur was predominantly linked to anatomical traits. Correlation and principal component analysis (PCA) PCA analysis showed a high correlation among de novo shoot regeneration capacity, shoot-tip necrosis, and antioxidant capacity (Fig. 9B). Page 9/29 Discussion Shoots cultured in a medium without BAP were characterized by tissues of the stems with cells more committed to differentiation (high cellular determination and low competence acquisition) than proliferation, which leads to a less efficient morphogenetic status for the induction of shoots at the base of the stem. Cell proliferation is required for de novo shoot regeneration (Fehér, 2019). In this context, BAP favored cell proliferation in Q. robur explants. In addition, it has been suggested that the low efficiency of KIN in shoot induction could be correlated to the activity of the enzyme system that catalyzes the degradation of cytokinins, such as cytokinin oxidase/dehydrogenase (Schmülling et al. 2003; Nisler et al. 2016; Rosa et al. 2018). With respect to the anatomical traits of stems, shoots grown with 1.25 µM KIN, mainly in the absence of BAP, presented a larger area of secondary xylem and lignified cells. In contrast, BAP induced shoots with Page 10/29 Page 10/29 a reduced xylem area/total transverse stem area ratio in Q. robur explants. More developed xylem can transport water and mineral nutrients better to the whole aerial part, in addition to providing mechanical support (Melnyk 2017; Cabello and Chan 2019; Qi et al. 2020). However, the low mobility of nutrients may lead to physiological disorders in vitro, such as shoot-tip necrosis (Silva et al. 2020). These authors also verified that the incidence of shoot-tip necrosis was higher in shoots with non-lignified tissues (softer tissues) than in those with more lignified tissues. These findings are in agreement with those of the present study. The probability of shoot-tip necrosis showed a negative relationship with the xylem area/total transverse stem area ratio in Q. robur explants. The xylem area fraction is related to the hydraulic efficiency of stems (Cabello and Chan 2019; Zhang et al. 2021). This is because xylem hydraulic conductivity is associated with the number and diameter of vessel elements (based on the Hagen–Poiseuille equation), and even a slight reduction in these xylem features can affect the transport of water and nutrients (Quintana-Pulido et al. 2018; Martins et al. 2019; 2021). Thus, we suggest that shoot-tip necrosis is a morphophysiological response due to inefficient transport of water and nutrients through the shoot from the base to the tip. Likewise, the anatomical traits of stems and leaves also exhibited characteristics that indicated a lower level of tissue development. Manokari et al. Discussion (2021a, b) demonstrated that leaves grown under BAP showed underdeveloped structural traits, such as reduced/underdeveloped xylem and phloem elements and tissues of support (collenchyma and sclerenchyma). In the present study, the epidermal cells were similar in thickness; however, the cells of the chlorophyll parenchyma were affected. Cytokinin type affected the elongation of palisade parenchyma cells. Leaves grown with BAP had palisade parenchyma cells that were more compact. In contrast, KIN induced longer cells. This decline in the palisade parenchyma thickness may be correlated to a lower efficiency of water transport promoted by the xylem vessels, which can influence the cell elongation process. Cell elongation is promoted by cell expansion and directly interferes with water uptake (Schmalstig and Geiger 1985; Silva-Cunha et al. 2021). However, the spongy parenchyma thickness did not show a clear response to the function of the treatments. Nevertheless, when the palisade/spongy bone ratio was analyzed, a clear pattern of anatomical response was observed. Addition of BAP to the medium induced a lower palisade/spongy ratio, mainly in the absence of KIN. This decreased palisade/spongy ratio can indicate a reduced development level of leaf anatomy, which means a larger area with larger interstitial spaces among parenchyma cells (Luan et al. 2021; Manokari et al. 2022). The effects of cytokinin treatments on the formation of leaf conduction tissues were also documented. The number of vessel elements in the midrib differed among the treatments with and without KIN, which means that KIN can stimulate the development of leaves with higher hydraulic conductivity, as discussed before. However, a closer observation of the leaf venation pattern, including the vein density and caliber of the vessels, raised the hypothesis that BAP modulates the formation of conduction tissues. More precisely, BAP limited the development of leaf veins, mainly in leaves grown under 3.5 µM. The declining capacity of hydraulic conductivity in plants reflects leaf xylem formation (Falchi et al. 2020). The Page 11/29 Page 11/29 quantification of the number of vessel elements and/or vein density can indicate the efficiency of water and nutrient transport (Martins et al. 2021; Sakurai and Miklavcic 2021). Our study revealed that BAP negatively influenced the thickness of the cell walls of the phloem fibers (sclerenchyma). This response was linked to the total phenolic content, as confirmed by PCA and correlation analysis. Discussion Phenolic content was significantly positively correlated with lignin production, one of the cell wall components of all vascular plants (Zhao et al. 2016; Chalker-Scott and Fuchigami 2018; Tikhomirova et al. 2018). In leaves, the proper development of sclerenchyma cell walls is essential for maintaining shape and functioning (Martins et al. 2015). As part of vascular bundles, sclerenchyma cell walls can support the tissues responsible for transporting water and nutrients throughout the plant (Bao et al. 2012). Thus, leaves with lower cell wall thickness, in response to low lignin deposition, can impair conduction tissues. Therefore, the anatomical traits (stem and leaf) of shoots grown with BAP, especially at 3.5 µM, can indicate low-efficiency hydraulic conductivity in the whole plant body, which could correlate with shoot-tip necrosis. The total phenolic and non-tannin phenolic contents were negatively correlated with budding percentage and number of new shoots. Wojtania et al. (2015) suggested that phenolic content may inhibit shoot formation in vitro. These authors also revealed that the addition of BAP lowered phenolic production compared with the BAP-absence medium. This corroborates our findings based on PCA analysis. However, we also showed that budding induction is related to anatomical traits. Our work also found changes in Chl and Car contents. Decreases in the content of photosynthetic pigments in leaves cultured in vitro supplemented with excess cytokinin are frequently reported (Gentile et al. 2017; Martins et al. 2018b; Murvanidze et al. 2022). This decline in the content of photosynthetic pigments may be related to physiological stress, which results in alterations in reactive oxygen species production and increased antioxidant activity (Dakah et al. 2014; Gentile et al. 2017). Q. robur shoots cultivated with excess cytokinin showed clear signs of physiological disorders observed, such as s reduced Chl a/b ratio and increased antioxidant activity (TEAC), in addition to lesser content of photosynthetic pigments. A reduction in the Chl total content, followed by a reduction in the Chl a/b ratio, indicates high Chl a degradation and possible damage to the density of reaction centers of photosystem II (PSII) (Martins et al. 2021). We hypothesize that excess cytokinin can induce oxidative stress, leading to changes in Chl content. Our hypothesis is supported by the negative relationship between the content of photosynthetic pigments and the Chl a/b ratio and measured antioxidant capacity. Discussion Shoots that grew with 3.5 µM BAP combined with 0.62 or 1.25 µM KIN did not present an antioxidant activity as high as the shoots cultured with 3.5 µM BAP alone (KIN free). This could be interpreted as an indirect response to the Car content. Carotenoids can act as antioxidants by scavenging reactive oxygen species (Kim et al. 2021). Martins et al. (2021) also verified that a reduced Car content could negatively affect the Chl content as well as the Chl a/b ratio. Therefore, even though 3.5 µM BAP induced shoots with a lower Car content, this response was less pronounced with KIN co-exposure. This could have Page 12/29 Page 12/29 contributed to the smaller alterations in the Chl proportion, which was negatively correlated with antioxidant activity. Cytokinin treatment also influenced the photosynthetic apparatus performance. The addition of BAP promoted slight changes (approximately 5%) in the parameters related to potential quantum yield (Fv/Fm and Fo/Fm). Leaves under typical conditions usually present range values of Fv/Fm between 0.7–0.8 and Fo/Fm between 0.2–0.3. However, an Fv/Fm below 0.6 indicates photoinhibition as a stress response (Guidi et al. 2019). Thus, shoots under in vitro conditions did not show strong signs of photoinhibition based on the quantum yield of PSII. Nevertheless, the results suggest that Q. robur shoots must grow under ideal exogenous cytokinin supplementation because either too low or too high concentrations reduces the potential photochemical efficiency of PSII, and more energy is lost through dissipation. Further analyses showed the importance of exogenous cytokinin supplementation in the photosynthetic apparatus performance of Q. robur shoots in vitro. Shoots cultured with BAP and/or KIN had qNP values ≥ 0.58. These values indicate that the plants have a high photoprotective capacity. The qNP parameter can vary between 0 and 1. It is related to the capacity of a plant to protect itself from photodamage due to excess energy (photoinhibition) (Lurie et al. 1994; Kumar and Parsad 2015; Maoka 2020). In the present study, the drastic reduction in qNP in the shoots of the control treatment (cytokinin-free) can denote the lowest efficiency of plants to minimize any photoenergy-induced damage to the reaction centers. This low photoprotective capacity could be correlated with the leaf senescence process, since shoots of the control treatment showed leaves with necrotic lesions. Discussion Although plant hormones interact with each other to regulate leaf senescence, an increase in endogenous cytokinin levels can delay the processes involved in leaf senescence (Schippers et al., 2007; Xiao et al. 2017). Through correlation and PCA analyses, it was possible to prove that the ability to induce de novo shoot regeneration is negatively correlated with the development of plant tissues at the cellular and biochemical levels. The use of BAP in the culture medium generated shoots with organs that clearly showed underdeveloped anatomical and biochemical characteristics. The preservation of cells at a lower level of determination is essential to allow greater cell proliferation and the formation of new shoots. Therefore, the results suggest that the key to competence acquisition and induction of new shoots might involve preserving cells to stages with multipotent and/or pluripotent characteristics. However, the formation of tissues with less specialized cells also impairs shoot quality due to the malformation of conduction tissues, which can lead to shoot-tip necrosis. In contrast, in the in vitro culture without BAP, the formation of tissues showed a higher cellular determination stage (highly differentiated and specialized cells), which led to a lower de novo shoot regeneration capacity. Conclusion BAP can induce shoots with underdeveloped anatomical and biochemical characteristics. A lower level of cellular determination in tissues was linked to de novo shoot regeneration efficiency. However, the BAP Page 13/29 Page 13/29 shoots also presented clear signs of physiological stress, such as low photosynthetic pigment content and higher antioxidant activity. An increase in BAP concentration can enhance the shoot-tip necrosis phenomenon. In contrast, shoots grown with KIN alone had stem and leaf anatomical characteristics that indicated a greater commitment to cellular differentiation than proliferation and showed a clear secondary growth morphology. When both cytokinins are combined (KIN and BAP), KIN can partially mitigate the deleterious effects of BAP on in vitro growth. Acknowledgments: The authors acknowledge the Ulam Programme scholarship (PPN/ULM/2019/1/00037) granted by Polish National Agency for Academic exchange. The authors also acknowledge Andreia Barcelos Passos Lima Gontijo, Lorenzo Toscano Conde, and Ludmila Cristina Oliveira for providing chemical reagents related to plant anatomy. 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Ciên Agrotec 42: 372– 380. https://doi.org/10.1590/1413-70542018424011018 54. Sant’Ana CRDO, Paiva R, Reis MVD, Silva DPCD, Silva LC (2018) In vitro propagation of Campomanesia rufa: An endangered fruit species. Ciên Agrotec 42: 372– 380. https://doi.org/10.1590/1413-70542018424011018 55. Savill PS, Kanowski PJ (1993) Tree improvement programs for European oaks: goals and strategies. Ann Sci For 50: 368s–383s. https://doi.org/10.1051/forest:19930741 55. Savill PS, Kanowski PJ (1993) Tree improvement programs for European oaks: goals and strategies. Ann Sci For 50: 368s–383s. https://doi.org/10.1051/forest:19930741 Page 18/29 56. Schippers JHM, Jing HC, Hille J, Dijkwel PP (2007) Developmental and hormonal control of leaf senescence, senescence processes in plants. In: Gan S (eds.) Senescence processes in plants. Oxford, Blackwell, pp 145–170. 57. Schmalstig JG, Geiger DR (1985) Phloem unloading in developing leaves of sugar beet: I. Evidence for pathway through the symplast. Plant Physiol 79: 237–241. https://doi.org/10.1104/pp.79.1.237 57. Schmalstig JG, Geiger DR (1985) Phloem unloading in developing leaves of sugar beet: I. Evidence for pathway through the symplast. Plant Physiol 79: 237–241. https://doi.org/10.1104/pp.79.1.237 58. References Schmülling T, Werner T, Riefler M, Krupková E, Bartrina y Manns I (2003) Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. J Plant Res 116(3): 241–252. https://doi.org/10.1007/s10265-003-0096-4 58. Schmülling T, Werner T, Riefler M, Krupková E, Bartrina y Manns I (2003) Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. J Plant Res 116(3): 241–252. https://doi.org/10.1007/s10265-003-0096-4 59. Sharma U, Kataria V, Shekhawat NS (2017) In vitro propagation, ex vitro rooting and leaf micromorphology of Bauhinia racemosa Lam.: a leguminous tree with medicinal values. Physiol Mol Biol Plant, 23(4): 969–977. https://doi.org/10.1007/s12298-017-0459-2 59. Sharma U, Kataria V, Shekhawat NS (2017) In vitro propagation, ex vitro rooting and leaf micromorphology of Bauhinia racemosa Lam.: a leguminous tree with medicinal values. Physiol Mol Biol Plant, 23(4): 969–977. https://doi.org/10.1007/s12298-017-0459-2 60. Silva JAT, Nezami-Alanagh E, Barreal ME, Kher MM, Wicaksono A, Gulyás A, Hidvégi N, Magyar- Tábori K, Mendler-Drienyovszki N, Márton L, Landín M, Gallego PP, Driver JA, Dobránszki, J. (2020). Shoot tip necrosis of in vitro plant cultures: a reappraisal of possible causes and solutions. Planta 252: 47. https://doi.org/10.1007/s00425-020-03449-4 61. Silva-Cunha LF, Oliveira VP, Nascimento AWS, Silva BRS, Batista BL, Alsahli AA, Lobato AKDS (2021) Leaf application of 24‐epibrassinolide mitigates cadmium toxicity in young Eucalyptus urophylla plants by modulating leaf anatomy and gas exchange. Physiol Plant 173(1): 67– 87. https://doi.org/10.1111/ppl.13182 62. Singleton VL, Orthofer R, Lamuela-Raventos RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol. 299: 152– 178. https://doi.org/10.1016/S0076-6879(99)99017-1 63. Surakshitha NC, Soorianathasundaram K, Ganga M, Raveendran M (2019) Alleviating shoot tip necrosis during in vitro propagation of grape cv. Red Globe. Sci Hort 248: 118– 125. https://doi.org/10.1016/j.scienta.2019.01.013 63. Surakshitha NC, Soorianathasundaram K, Ganga M, Raveendran M (2019) Alleviating shoot tip necrosis during in vitro propagation of grape cv. Red Globe. Sci Hort 248: 118– 125. https://doi.org/10.1016/j.scienta.2019.01.013 64. Tamta S, Palni LMS, Purohit VK, Nandi SK (2008) In vitro propagation of brown oak (Quercus semecarpifolia Sm.) from seedling explants. In Vitro Cell Dev Biol Plant 44(2): 136– 141. https://doi.org/10.1007/s11627-008-9138-x 64. Tamta S, Palni LMS, Purohit VK, Nandi SK (2008) In vitro propagation of brown oak (Quercus semecarpifolia Sm.) from seedling explants. In Vitro Cell Dev Biol Plant 44(2): 136– 141. https://doi.org/10.1007/s11627-008-9138-x 65. Tikhomirova LI, Bazarnova NG, Sinitsyna AA (2018) Histochemical study of xylem cells in in vitro culture of Iris sibirica L. References Russ J Bioorg. Chem 44(7): 860– 869. https://doi.org/10.1134/S1068162018070129 65. Tikhomirova LI, Bazarnova NG, Sinitsyna AA (2018) Histochemical study of xylem cells in in vitro culture of Iris sibirica L. Russ J Bioorg. Chem 44(7): 860– 869. https://doi.org/10.1134/S1068162018070129 66. Vieitez AM, Carmen San-Jose M, Vieitez E (1985) In vitro plantlet regeneration from juvenile and mature Quercus robur, L. J Hort Sci 60(1): 99– 106. https://doi.org/10.1080/14620316.1985.11515607 66. Vieitez AM, Carmen San-Jose M, Vieitez E (1985) In vitro plantlet regeneration from juvenile and mature Quercus robur, L. J Hort Sci 60(1): 99– 106. https://doi.org/10.1080/14620316.1985.11515607 67. Vieitez AM, Corredoira E, Ballester A, Muñoz F, Durán J, Ibarra M (2009) In vitro regeneration of the important North American oak species Quercus alba, Quercus bicolor and Quercus rubra. Plant Cell Tiss Organ Cult 98(2), 135-145. https://doi.org/10.1007/s11240-009-9546-6 67. Vieitez AM, Corredoira E, Ballester A, Muñoz F, Durán J, Ibarra M (2009) In vitro regeneration of the important North American oak species Quercus alba, Quercus bicolor and Quercus rubra. Plant Cell Tiss Organ Cult 98(2), 135-145. https://doi.org/10.1007/s11240-009-9546-6 Page 19/29 68. Vieitez AM, Pintos F, San-Jose MC, Ballester A (1993) In vitro shoot proliferation determined by explant orientation of juvenile and mature Quercus rubra L. Tree Physiol 12: 107– 117. https://doi.org/10.1093/treephys/12.2.107 69. Wei T, Simko VR (2017) package "corrplot": Visualization of a Correlation Matrix (Version 084). Available from https://githubcom/taiyun/corrplot 70. Wojtania A, Skrzypek E, Gabryszewska E (2015) Effect of cytokinin, sucrose and nitrogen salts concentrations on the growth and development and phenolics content in Magnolia × soulangiana 'Coates' shoots in vitro. Acta Sci Pol Hortorum Cultus 14(3): 51–62. 71. Xiao XO, Zeng YM, Cao BH, Lei JJ, Chen QH, Meng CM, Cheng YJ (2017) PSAG12-IPT overexpression in eggplant delays leaf senescence and induces abiotic stress tolerance. J Hort Sci Biotechnol 92(4): 349–357. https://doi.org/10.1080/14620316.2017.1287529 72. Zhang Y, Jyske T, Pumpanen J, Hölttä T, Gao Q, Berninger F, Duan B (2021) Adaptation of Abies fargesii var. faxoniana (Rehder et E.H. Wilson) Tang S Liu seedlings to high altitude in a subalpine forest in southwestern China with special reference to phloem and xylem traits. Ann For Sci 78: 85. https://doi.org/10.1007/s13595-021-01095-8 73. Zhao S, Wen J, Wang H, Zhang Z, Li X (2016) Changes in lignin content and activity of related enzymes in the endocarp during the walnut shell development period. Hort Plant J 2(3): 141– 146. https://doi.org/10.1016/j.hpj.2016.08.003 73. References Zhao S, Wen J, Wang H, Zhang Z, Li X (2016) Changes in lignin content and activity of related enzymes in the endocarp during the walnut shell development period. Hort Plant J 2(3): 141– 146. https://doi.org/10.1016/j.hpj.2016.08.003 Figures Page 20/29 Page 20/29 Figure 1 The visual aspect of Q. robur in vitro shoot growth after 40 d as a function of cytokinins (BAP, KIN) treatments. (A-I). The arrows detailed morphology of shoot-tip necrosis formed in vitro. Budding (%) (J), number of new shoots (K), and probability of shoot tip necrosis (L) of Q. robur during in vitro growth. Figure 1 The visual aspect of Q. robur in vitro shoot growth after 40 d as a function of cytokinins (BAP, KIN) treatments. (A-I). The arrows detailed morphology of shoot-tip necrosis formed in vitro. Budding (%) (J), number of new shoots (K), and probability of shoot tip necrosis (L) of Q. robur during in vitro growth. For each growth trait, the means ± SE followed by the same letter (uppercase letters comparing BAP concentrations and lowercase letters comparing KIN concentrations at each BAP concentration) do not differ significantly according to Tukey’s test (p<0.05). Bar = 2 cm Page 21/29 Figure 2 Cross-sections of stems of Q. robur after 40 d in medium containing BAP (0, 1.25, and 3.5 μM) and KIN (0, 0.65, and 1.25 μM) during in vitro culture. Stem cross-sections stained by safranin and astra-blue solutions. For each anatomical trait, the means ± SE (n = 5) followed by the same letter do not differ significantly according to Tukey’s test (p<0.05). cp – cortical parenchyma, fb – fibers, pi – pith, ph – phloem, xy – xylem. Bars = 500 μm Figure 2 Figure 2 Cross-sections of stems of Q. robur after 40 d in medium containing BAP (0, 1.25, and 3.5 μM) and KIN (0, 0.65, and 1.25 μM) during in vitro culture. Stem cross-sections stained by safranin and astra-blue solutions. For each anatomical trait, the means ± SE (n = 5) followed by the same letter do not differ significantly according to Tukey’s test (p<0.05). cp – cortical parenchyma, fb – fibers, pi – pith, ph – phloem, xy – xylem. Bars = 500 μm Cross-sections of stems of Q. robur after 40 d in medium containing BAP (0, 1.25, and 3.5 μM) and KIN (0, 0.65, and 1.25 μM) during in vitro culture. Stem cross-sections stained by safranin and astra-blue solutions. For each anatomical trait, the means ± SE (n = 5) followed by the same letter do not differ significantly according to Tukey’s test (p<0.05). cp – cortical parenchyma, fb – fibers, pi – pith, ph – phloem, xy – xylem. Bars = 500 μm Page 22/29 Page 22/29 Page 22/29 Figure 3 Venation pattern of leaves of Q. robur shoots grown in medium containing different combinations of BAP (0, 1.25, and 3.5 μM) and KIN (0, 0.65, and 1.25 μM) during in vitro culture. Bars = 1 cm (A-C, G-I, and M-O) and 500 μm (D-F, J-L, and P-S). Figure 3 Venation pattern of leaves of Q. robur shoots grown in medium containing different combinations of BAP (0, 1.25, and 3.5 μM) and KIN (0, 0.65, and 1.25 μM) during in vitro culture. Bars = 1 cm (A-C, G-I, and M-O) and 500 μm (D-F, J-L, and P-S). Page 23/29 Figure 4 Cross-sections of Q. robur leaves at 40 d in medium containing different combinations of BAP (0, 1.25, and 3.5 μM) and KIN (0, 0.65, and 1.25 μM) during in vitro culture. Stem cross-sections stained by safranin and astra-blue solutions. ab – abaxial epidermis, ad – adaxial epidermis, pp – palisade parenchyma, ph – phloem, pf – phloem fibers, sp – spongy parenchyma, ve – vessel element. Bars = 100 μm (A-C, G-I, and M-O) and 50 μm (D-F, J-L, and P-S). Figure 4 Cross-sections of Q. robur leaves at 40 d in medium containing different combinations of BAP (0, 1.25, and 3.5 μM) and KIN (0, 0.65, and 1.25 μM) during in vitro culture. Stem cross-sections stained by safranin and astra-blue solutions. Figure 2 ab – abaxial epidermis, ad – adaxial epidermis, pp – palisade parenchyma, ph – phloem, pf – phloem fibers, sp – spongy parenchyma, ve – vessel element. Bars = 100 μm (A-C, G-I, and M-O) and 50 μm (D-F, J-L, and P-S). Page 24/29 Page 24/29 Figure 5 Anatomical traits and photosynthetic pigment contents of Q. ro (BAP, KIN) during in vitro culture. For each growth trait, the mean content, means (± SE), n = 6, followed by the same letter (upperc concentrations at each KIN concentration and lowercase letters Figure 5 Anatomical traits and photosynthetic pigment contents of Q. robur leaves as a function of cytokinins (BAP, KIN) during in vitro culture. For each growth trait, the means (± SE), n = 5, or for each pigment content, means (± SE), n = 6, followed by the same letter (uppercase letters comparing BAP concentrations at each KIN concentration and lowercase letters comparing KIN concentrations at each BAP concentration), do not differ significantly according to Tukey’s test (p<0.05). Figure 5 Figure 5 Anatomical traits and photosynthetic pigment contents of Q. robur leaves as a function of cytokinins (BAP, KIN) during in vitro culture. For each growth trait, the means (± SE), n = 5, or for each pigment content, means (± SE), n = 6, followed by the same letter (uppercase letters comparing BAP concentrations at each KIN concentration and lowercase letters comparing KIN concentrations at each BAP concentration), do not differ significantly according to Tukey’s test (p<0.05). Page 25/29 Page 25/29 Figure 6 Stem and leaf cross-sections of Q. robur shoots grown in a medium with different concentrations of BAP (0, 1.25, and 3.5 µM) and KIN (0, 0.62, and 1.25 µM). Cross-sections stained by berberine hemi-sulfate and aniline blue solutions. pf – phloem fibers; xy – xylem. Bars = 100 μm (A-C, G-I, and M-O) and 200 μm (D-F, J-L, and P-S). Figure 6 Stem and leaf cross-sections of Q. robur shoots grown in a medium with different concentrations of BAP (0, 1.25, and 3.5 µM) and KIN (0, 0.62, and 1.25 µM). Cross-sections stained by berberine hemi-sulfate and aniline blue solutions. pf – phloem fibers; xy – xylem. Bars = 100 μm (A-C, G-I, and M-O) and 200 μm (D-F, J-L, and P-S). Page 26/29 Page 26/29 Figure 7 Phenolic content and antioxidant capacity of Q. robur leaves as a function of cytokinins (BAP, KIN) during in vitro culture. For each anatomical trait, means (± SE), n = 5, followed by the same letter (uppercase letters comparing BAP concentrations at each KIN concentration and lowercase letters comparing KIN concentrations at each BAP concentration), do not differ significantly according to Tukey’s test (p<0.05). Figure 7 Phenolic content and antioxidant capacity of Q. robur leaves as a function of cytokinins (BAP, KIN) during in vitro culture. For each anatomical trait, means (± SE), n = 5, followed by the same letter (uppercase letters comparing BAP concentrations at each KIN concentration and lowercase letters comparing KIN concentrations at each BAP concentration), do not differ significantly according to Tukey’s test (p<0.05). Page 27/29 Page 27/29 Page 27/29 Figure 8 Parameters of modulated chlorophyll a fluorescenc KIN) during in vitro culture. For each parameter, me (uppercase letters comparing BAP concentrations a each BAP concentration), do not differ significantly Figure 8 Parameters of modulated chlorophyll a fluorescenc KIN) during in vitro culture. For each parameter, mea (uppercase letters comparing BAP concentrations a each BAP concentration), do not differ significantly Page 28/29 Figure 8 Parameters of modulated chlorophyll a fluorescence of Q. robur leaves as a function of cytokinins (BAP, KIN) during in vitro culture. For each parameter, means (± SE), n = 6, followed by the same letter (uppercase letters comparing BAP concentrations and lowercase letters comparing KIN concentrations at each BAP concentration), do not differ significantly according to Tukey’s test (p<0.05). Figure 8 Figure 8 Parameters of modulated chlorophyll a fluorescence of Q. robur leaves as a function of cytokinins (BAP, KIN) during in vitro culture. For each parameter, means (± SE), n = 6, followed by the same letter (uppercase letters comparing BAP concentrations and lowercase letters comparing KIN concentrations at each BAP concentration), do not differ significantly according to Tukey’s test (p<0.05). Page 28/29 Page 28/29 Figure 9 Pearson correlation coefficient and principal component analysis of growth, anatomical, and biochemical traits of Q. robur shoots as a function of cytokinin treatments during in vitro culture. Figure 9 Pearson correlation coefficient and principal component analysis of growth, anatomical, and biochemical traits of Q. robur shoots as a function of cytokinin treatments during in vitro culture. Pearson correlation coefficient and principal component analysis of growth, anatomical, and biochemical traits of Q. robur shoots as a function of cytokinin treatments during in vitro culture. Page 29/29 Page 29/29 Page 29/29
https://openalex.org/W2512149058	http://www.accessdata.fda.gov/drugsatfda_docs/label/2013/125084s242lbl.pdf	English	null	Immunotherapy and colon cancer	Integrative cancer science and therapeutics	2,016	cc-by	23,205	-ADVERSE REACTIONS--  Locally or regionally advanced squamous cell carcinoma of the head and neck in combination with radiation therapy. (1.1, 14.1)  Recurrent locoregional disease or metastatic squamous cell carcinoma of the head and neck in combination with platinum-based therapy with 5-FU. (1.1, 14.1) To report SUSPECTED ADVERSE REACTIONS, contact Bristol-Myers Squibb at 1-800-721-5072 or FDA at 1-800-FDA-1088 or www.fda.gov/medwatch  Recurrent or metastatic squamous cell carcinoma of the head and neck progressing after platinum-based therapy. (1.1, 14.1) --RECENT MAJOR CHANGES ---------------------------RECENT MAJOR CHANGES-------------------------- Indications and Usage Colorectal Cancer (1.2) 07/2012 Dosage and Administration Colorectal Cancer (2.2) 07/2012 Warnings and Precautions Use of Erbitux in Combination With Radiation and Cisplatin (5.5) 03/2013 K-Ras Testing in Metastatic or Advanced Colorectal Cancer Patients (5.7) 07/2012 ---------------------------INDICATIONS AND USAGE---------------------------- ® Erbitux® is an epidermal growth factor receptor (EGFR) antagonist indicated for treatment of: Head and Neck Cancer This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda Limitation of Use: Erbitux is not indicated for treatment of K-Ras mutation- positive colorectal cancer. (5.7, 14.2) HIGHLIGHTS OF PRESCRIBING INFORMATION These highlights do not include all the information needed to use ERBITUX safely and effectively. See full prescribing information for ERBITUX. --CONTRAINDICATIONS- --RECENT MAJOR CHANGES-------------------------- ---RECENT MAJOR CHANGES--------------------- WARNING: SERIOUS INFUSION REACTIONS and CARDIOPULMONARY ARREST See full prescribing information for complete boxed warning.  Serious infusion reactions, some fatal, occurred in approximately 3% of patients. (5.1)  Cardiopulmonary arrest and/or sudden death occurred in 2% of patients with squamous cell carcinoma of the head and neck treated with Erbitux and radiation therapy and in 3% of patients with squamous cell carcinoma of the head and neck treated with cetuximab in combination with platinum-based therapy with 5-fluorouracil (5-FU). Closely monitor serum electrolytes, including serum magnesium, potassium, and calcium, during and after Erbitux administration. (5.2, 5.6) --DOSAGE AND ADMINISTRATION-- ------------------------DOSAGE AND ADMINISTRATION--------------------- --DOSAGE AND ADMINISTRATION- ------------------------DOSAGE AND ADMINISTRATION  Premedicate with an H1 antagonist. (2.3) ERBITUX® (cetuximab) injection, for intravenous infusion Initial U.S. Approval: 2004 ERBITUX® (cetuximab) injection, for intravenous infusion Initial U.S. Approval: 2004  Administer 400 mg/m2 initial dose as a 120-minute intravenous infusion followed by 250 mg/m2 weekly infused over 60 minutes. (2.1, 2.2)  Initiate Erbitux one week prior to initiation of radiation therapy. Complete Erbitux administration 1 hour prior to platinum-based therapy with 5-FU (2.1) and FOLFIRI (2.2). --DOSAGE FORMS AND STRENGTHS--  200 mg/100 mL, single-use vial (3) Colorectal Cancer K-Ras mutation-negative (wild-type), EGFR-expressing, metastatic colorectal cancer as determined by FDA-approved tests  in combination with FOLFIRI for first-line treatment,  in combination with irinotecan in patients who are refractory to irinotecan-based chemotherapy, See 17 for PATIENT COUNSELING INFORMATION  as a single agent in patients who have failed oxaliplatin- and irinotecan-based chemotherapy or who are intolerant to irinotecan. (1.2, 5.7, 12.1, 14.2) Revised: 03/2013 FULL PRESCRIBING INFORMATION: CONTENTS* WARNING: SERIOUS INFUSION REACTIONS AND CARDIOPULMONARY ARREST 1 INDICATIONS AND USAGE 1.1 Squamous Cell Carcinoma of the Head and Neck (SCCHN) 1.2 K-Ras Mutation-negative, EGFR-expressing Colorectal Cancer 2 DOSAGE AND ADMINISTRATION 2.1 Squamous Cell Carcinoma of the Head and Neck 2.2 Colorectal Cancer 2.3 Recommended Premedication 2.4 Dose Modifications 2.5 Preparation for Administration 3 DOSAGE FORMS AND STRENGTHS 4 CONTRAINDICATIONS 5 WARNINGS AND PRECAUTIONS 5.1 Infusion Reactions 5.2 Cardiopulmonary Arrest 5.3 Pulmonary Toxicity 5.4 Dermatologic Toxicity 5.5 Use of Erbitux in Combination With Radiation and Cisplatin FULL PRESCRIBING INFORMATION: CONTENTS* WARNING: SERIOUS INFUSION REACTIONS AND CARDIOPULMONARY ARREST 1 INDICATIONS AND USAGE 1.1 Squamous Cell Carcinoma of the Head and Neck (SCCHN) 1.2 K-Ras Mutation-negative, EGFR-expressing Colorectal Cancer 2 DOSAGE AND ADMINISTRATION 2.1 Squamous Cell Carcinoma of the Head and Neck 2.2 Colorectal Cancer 2.3 Recommended Premedication 2.4 Dose Modifications 2.5 Preparation for Administration 3 DOSAGE FORMS AND STRENGTHS 4 CONTRAINDICATIONS 5 WARNINGS AND PRECAUTIONS 5.1 Infusion Reactions 5.2 Cardiopulmonary Arrest 5.3 Pulmonary Toxicity 5.4 Dermatologic Toxicity 5.5 Use of Erbitux in Combination With Radiation and Cisplatin 5.6 Hypomagnesemia and Electrolyte Abnormalities 5.7 K-Ras Testing in Metastatic or Advanced Colorectal Cancer Patients 5.8 Epidermal Growth Factor Receptor (EGFR) Expression and Response 6 ADVERSE REACTIONS 6.1 Clinical Trials Experience 6.2 Immunogenicity 6.3 Postmarketing Experience 7 DRUG INTERACTIONS 8 USE IN SPECIFIC POPULATIONS 8.1 Pregnancy 8.3 Nursing Mothers 8.4 Pediatric Use 8.5 Geriatric Use 10 OVERDOSAGE 11 DESCRIPTION 12 CLINICAL PHARMACOLOGY 12.1 Mechanism of Action 12.2 Pharmacodynamics 12.3 Pharmacokinetics 2 1 Reference ID: 3270603 13 NONCLINICAL TOXICOLOGY 14.2 Colorectal Cancer 14 13.1 Carcinogenesis, Mutagenesis, Impairment of Fertility 13.2 Animal Pharmacology and/or Toxicology CLINICAL STUDIES 16 17 HOW SUPPLIED/STORAGE AND HANDLING PATIENT COUNSELING INFORMATION 14.1 Squamous Cell Carcinoma of the Head and Neck * Sections or subsections omitted from the full prescribing information (SCCHN) are not listed This label may not be the latest approved by FDA. Colorectal Cancer For current labeling information, please visit https://www.fda.gov/drugsatfda 13 NONCLINICAL TOXICOLOGY 14.2 Colorectal Cancer 14 13.1 Carcinogenesis, Mutagenesis, Impairment of Fertility 13.2 Animal Pharmacology and/or Toxicology CLINICAL STUDIES 16 17 HOW SUPPLIED/STORAGE AND HANDLING PATIENT COUNSELING INFORMATION 14.1 Squamous Cell Carcinoma of the Head and Neck * Sections or subsections omitted from the full prescribing information (SCCHN) are not listed This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 13 NONCLINICAL TOXICOLOGY 14.2 Colorectal Cancer 14 13.1 Carcinogenesis, Mutagenesis, Impairment of Fertility 13.2 Animal Pharmacology and/or Toxicology CLINICAL STUDIES 16 17 HOW SUPPLIED/STORAGE AND HANDLING PATIENT COUNSELING INFORMATION 14.1 Squamous Cell Carcinoma of the Head and Neck * Sections or subsections omitted from the full prescribing information (SCCHN) are not listed This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 2 2 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA 1 FULL PRESCRIBING INFORMATION 2 WARNING: SERIOUS INFUSION REACTIONS and 3 CARDIOPULMONARY ARREST 4 Infusion Reactions: Serious infusion reactions occurred with the administration of 5 Erbitux in approximately 3% of patients in clinical trials, with fatal outcome 6 reported in less than 1 in 1000. [See Warnings and Precautions (5.1), Adverse 7 Reactions (6).] Immediately interrupt and permanently discontinue Erbitux infusion 8 for serious infusion reactions. [See Dosage and Administration (2.4), Warnings and 9 Precautions (5.1).] 10 Cardiopulmonary Arrest: Cardiopulmonary arrest and/or sudden death occurred 11 in 2% of patients with squamous cell carcinoma of the head and neck treated with 12 Erbitux and radiation therapy in Study 1 and in 3% of patients with squamous cell 13 carcinoma of the head and neck treated with European Union (EU)-approved 14 cetuximab in combination with platinum-based therapy with 5-fluorouracil (5-FU) 15 in Study 2. Closely monitor serum electrolytes, including serum magnesium, 16 potassium, and calcium, during and after Erbitux administration. [See Warnings 17 and Precautions (5.2, 5.6), Clinical Studies (14.1).] 2 WARNING: SERIOUS INFUSION REACTIONS and 3 CARDIOPULMONARY ARREST 4 Infusion Reactions: Serious infusion reactions occurred with the administration of 5 Erbitux in approximately 3% of patients in clinical trials, with fatal outcome 6 reported in less than 1 in 1000. [See Warnings and Precautions (5.1), Adverse 7 Reactions (6).] Immediately interrupt and permanently discontinue Erbitux infusion 8 for serious infusion reactions. [See Dosage and Administration (2.4), Warnings and 9 Precautions (5.1).] 10 Cardiopulmonary Arrest: Cardiopulmonary arrest and/or sudden death occurred 11 in 2% of patients with squamous cell carcinoma of the head and neck treated with 12 Erbitux and radiation therapy in Study 1 and in 3% of patients with squamous cell 13 carcinoma of the head and neck treated with European Union (EU)-approved 14 cetuximab in combination with platinum-based therapy with 5-fluorouracil (5-FU) 15 in Study 2. Closely monitor serum electrolytes, including serum magnesium, 16 potassium, and calcium, during and after Erbitux administration. [See Warnings 17 and Precautions (5.2, 5.6), Clinical Studies (14.1).] Reference ID: 3270603 WARNING: SERIOUS INFUSION REACTIONS and CARDIOPULMONARY ARREST 10 Cardiopulmonary Arrest: Cardiopulmonary arrest and/or sudden death occurred 11 in 2% of patients with squamous cell carcinoma of the head and neck treated with 12 Erbitux and radiation therapy in Study 1 and in 3% of patients with squamous cell 13 carcinoma of the head and neck treated with European Union (EU)-approved 14 cetuximab in combination with platinum-based therapy with 5-fluorouracil (5-FU) 15 in Study 2. Closely monitor serum electrolytes, including serum magnesium, 16 potassium, and calcium, during and after Erbitux administration. [See Warnings 17 and Precautions (5.2, 5.6), Clinical Studies (14.1).] 30 1.2 K-Ras Mutation-negative, EGFR-expressing Colorectal 31 Cancer 30 1.2 K-Ras Mutation-negative, EGFR-expressing Colorectal 31 Cancer 32 Erbitux is indicated for the treatment of K-Ras mutation-negative (wild-type), epidermal 33 growth factor receptor (EGFR)-expressing, metastatic colorectal cancer (mCRC) as 34 determined by FDA-approved tests for this use [see Dosage and Administration (2.2), 35 Warnings and Precautions (5.7), Clinical Studies (14.2)] 36  in combination with FOLFIRI (irinotecan, 5-fluorouracil, leucovorin) for first 37 line treatment, 38  in combination with irinotecan in patients who are refractory to irinotecan 39 based chemotherapy, 40  as a single agent in patients who have failed oxaliplatin- and irinotecan-based 41 chemotherapy or who are intolerant to irinotecan. [See Warnings and 42 Precautions (5.7), Clinical Pharmacology (12.1), Clinical Studies (14.2).] 43 Limitation of Use: Erbitux is not indicated for treatment of K-Ras mutation-positive 44 colorectal cancer [see Warnings and Precautions (5.7), Clinical Studies (14.2)]. 45 2 DOSAGE AND ADMINISTRATION 46 2.1 Squamous Cell Carcinoma of the Head and Neck 47 Erbitux in combination with radiation therapy or in combination with platinum-based 48 therapy with 5-FU: 32 Erbitux is indicated for the treatment of K-Ras mutation-negative (wild-type), epidermal 33 growth factor receptor (EGFR)-expressing, metastatic colorectal cancer (mCRC) as 34 determined by FDA-approved tests for this use [see Dosage and Administration (2.2), 35 Warnings and Precautions (5.7), Clinical Studies (14.2)] 36  in combination with FOLFIRI (irinotecan, 5-fluorouracil, leucovorin) for first 37 line treatment, 36  in combination with FOLFIRI (irinotecan, 5-fluorouracil, leucovorin) for first 37 line treatment, 38  in combination with irinotecan in patients who are refractory to irinotecan 39 based chemotherapy, 38  in combination with irinotecan in patients who are refractory to irinotecan 39 based chemotherapy, 40  as a single agent in patients who have failed oxaliplatin- and irinotecan-based 41 chemotherapy or who are intolerant to irinotecan. [See Warnings and 42 Precautions (5.7), Clinical Pharmacology (12.1), Clinical Studies (14.2).] 40  as a single agent in patients who have failed oxaliplatin- and irinotecan-based 41 chemotherapy or who are intolerant to irinotecan. [See Warnings and 42 Precautions (5.7), Clinical Pharmacology (12.1), Clinical Studies (14.2).] 40  as a single agent in patients who have failed oxaliplatin- and irinotecan-based 41 chemotherapy or who are intolerant to irinotecan. [See Warnings and 42 Precautions (5.7), Clinical Pharmacology (12.1), Clinical Studies (14.2).] 43 Limitation of Use: Erbitux is not indicated for treatment of K-Ras mutation-positive 44 colorectal cancer [see Warnings and Precautions (5.7), Clinical Studies (14.2)]. 18 1 INDICATIONS AND USAGE 19 1.1 Squamous Cell Carcinoma of the Head and Neck 20 (SCCHN) 24 Erbitux is indicated in combination with platinum-based therapy with 5-FU for the first 25 line treatment of patients with recurrent locoregional disease or metastatic squamous cell 26 carcinoma of the head and neck. [See Clinical Studies (14.1).] 24 Erbitux is indicated in combination with platinum-based therapy with 5-FU for the first 25 line treatment of patients with recurrent locoregional disease or metastatic squamous cell 26 carcinoma of the head and neck. [See Clinical Studies (14.1).] 3 Reference ID: 3270603 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 30 1.2 K-Ras Mutation-negative, EGFR-expressing Colorectal 31 Cancer 45 2 DOSAGE AND ADMINISTRATION 46 2.1 Squamous Cell Carcinoma of the Head and Neck 47 Erbitux in combination with radiation therapy or in combination with platinum-based 48 therapy with 5-FU: 49  The recommended initial dose is 400 mg/m2 administered one week prior to 50 initiation of a course of radiation therapy or on the day of initiation of platinum 51 based therapy with 5-FU as a 120-minute intravenous infusion (maximum 52 infusion rate 10 mg/min). Complete Erbitux administration 1 hour prior to 53 platinum-based therapy with 5-FU. 54  The recommended subsequent weekly dose (all other infusions) is 250 mg/m2 55 infused over 60 minutes (maximum infusion rate 10 mg/min) for the duration of 56 radiation therapy (6–7 weeks) or until disease progression or unacceptable 57 toxicity when administered in combination with platinum-based therapy with 58 5-FU. Complete Erbitux administration 1 hour prior to radiation therapy or 59 platinum-based therapy with 5-FU. 43 Limitation of Use: Erbitux is not indicated for treatment of K-Ras mutation-positive 44 colorectal cancer [see Warnings and Precautions (5.7), Clinical Studies (14.2)]. 43 Limitation of Use: Erbitux is not indicated for treatment of K-Ras mutation-positive 44 colorectal cancer [see Warnings and Precautions (5.7), Clinical Studies (14.2)]. 43 Limitation of Use: Erbitux is not indicated for treatment of K-Ras mutation-positive 44 colorectal cancer [see Warnings and Precautions (5.7), Clinical Studies (14.2)]. Reference ID: 3270603 6 2.1 Squamous Cell Carcinoma of the Head and Nec 47 Erbitux in combination with radiation therapy or in combination with platinum-based 48 therapy with 5-FU: 47 Erbitux in combination with radiation therapy or in combination with platinum-based 48 therapy with 5-FU: 4 4 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 60 Erbitux monotherapy: 61  The recommended initial dose is 400 mg/m2 administered as a 120-minute 62 intravenous infusion (maximum infusion rate 10 mg/min). 63  The recommended subsequent weekly dose (all other infusions) is 250 mg/m2 64 infused over 60 minutes (maximum infusion rate 10 mg/min) until disease 65 progression or unacceptable toxicity. 66 2.2 Colorectal Cancer 67  Determine K-Ras mutation and EGFR-expression status using FDA-approved 68 tests prior to initiating treatment. Only patients whose tumors are K-Ras mutation 69 negative (wild-type) should receive Erbitux. 70  The recommended initial dose, either as monotherapy or in combination with 71 irinotecan or FOLFIRI (irinotecan, 5-fluorouracil, leucovorin), is 400 mg/m2 72 administered as a 120-minute intravenous infusion (maximum infusion rate 73 10 mg/min). Complete Erbitux administration 1 hour prior to FOLFIRI. 74  The recommended subsequent weekly dose, either as monotherapy or in 75 combination with irinotecan or FOLFIRI, is 250 mg/m2 infused over 60 minutes 76 (maximum infusion rate 10 mg/min) until disease progression or unacceptable 77 toxicity. Complete Erbitux administration 1 hour prior to FOLFIRI. 78 2.3 Recommended Premedication 79 Premedicate with an H1 antagonist (eg, 50 mg of diphenhydramine) intravenously 80 30–60 minutes prior to the first dose; premedication should be administered for 81 subsequent Erbitux doses based upon clinical judgment and presence/severity of prior 82 infusion reactions. 83 2.4 Dose Modifications 84 Infusion Reactions 85 Reduce the infusion rate by 50% for NCI CTC Grade 1 or 2 and non serious NCI CTC 60 Erbitux monotherapy: For current labeling information, please visit https://www.fda.gov/drugsatfda nt labeling information, please visit https://www.fda.gov/drugsatfda 87 Immediately and permanently discontinue Erbitux for serious infusion reactions, 88 requiring medical intervention and/or hospitalization. [See Warnings and Precautions 89 (5.1).] 90 Dermatologic Toxicity 91 Recommended dose modifications for severe (NCI CTC Grade 3 or 4) acneiform rash are 92 specified in Table 1. [See Warnings and Precautions (5.4).] Table 1: Erbitux Dose Modification Guidelines for Rash Severe Acneiform Erbitux Dose Rash Erbitux Outcome Modification 1st occurrence Delay infusion 1 to 2 weeks Improvement Continue at 250 mg/m2 No Improvement Discontinue Erbitux 2nd occurrence Delay infusion 1 to 2 weeks Improvement Reduce dose to 200 mg/m2 No Improvement Discontinue Erbitux 3rd occurrence Delay infusion 1 to 2 weeks Improvement Reduce dose to 150 mg/m2 No Improvement Discontinue Erbitux 4th occurrence Discontinue Erbitux 93 2.5 Preparation for Administration 91 Recommended dose modifications for severe (NCI CTC Grade 3 or 4) acneiform rash are 92 specified in Table 1. [See Warnings and Precautions (5.4).] Table 1: Erbitux Dose Modification Guidelines for Rash Table 1: Erbitux Dose Modification Guidelines for Rash Severe Acneiform Erbitux Dose Rash Erbitux Outcome Modification 1st occurrence Delay infusion 1 to 2 weeks Improvement Continue at 250 mg/m2 No Improvement Discontinue Erbitux 2nd occurrence Delay infusion 1 to 2 weeks Improvement Reduce dose to 200 mg/m2 No Improvement Discontinue Erbitux 3rd occurrence Delay infusion 1 to 2 weeks Improvement Reduce dose to 150 mg/m2 No Improvement Discontinue Erbitux 4th occurrence Discontinue Erbitux 60 Erbitux monotherapy: 60 Erbitux monotherapy: 61  The recommended initial dose is 400 mg/m2 administered as a 120-minute 62 intravenous infusion (maximum infusion rate 10 mg/min). 63  The recommended subsequent weekly dose (all other infusions) is 250 mg/m2 64 infused over 60 minutes (maximum infusion rate 10 mg/min) until disease 65 progression or unacceptable toxicity. 79 Premedicate with an H1 antagonist (eg, 50 mg of diphenhydramine) intravenously 80 30–60 minutes prior to the first dose; premedication should be administered for 81 subsequent Erbitux doses based upon clinical judgment and presence/severity of prior 82 infusion reactions. 85 Reduce the infusion rate by 50% for NCI CTC Grade 1 or 2 and non-serious NCI CTC 86 Grade 3 infusion reaction. 5 Reference ID: 3270603 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 87 Immediately and permanently discontinue Erbitux for serious infusion reactions, 88 requiring medical intervention and/or hospitalization. [See Warnings and Precautions 89 (5.1).] 90 Dermatologic Toxicity 91 Recommended dose modifications for severe (NCI CTC Grade 3 or 4) acneiform rash are 92 specified in Table 1. [See Warnings and Precautions (5.4).] Table 1: Erbitux Dose Modification Guidelines for Rash Severe Acneiform Erbitux Dose Rash Erbitux Outcome Modification 1st occurrence Delay infusion 1 to 2 weeks Improvement Continue at 250 mg/m2 No Improvement Discontinue Erbitux 2nd occurrence Delay infusion 1 to 2 weeks Improvement Reduce dose to 200 mg/m2 No Improvement Discontinue Erbitux 3rd occurrence Delay infusion 1 to 2 weeks Improvement Reduce dose to 150 mg/m2 No Improvement Discontinue Erbitux 4th occurrence Discontinue Erbitux 93 2.5 Preparation for Administration 94 Do not administer Erbitux as an intravenous push or bolus. 95 Administer via infusion pump or syringe pump. Do not exceed an infusion rate of 96 10 mg/min. 97 Administer through a low protein binding 0.22-micrometer in-line filter. 98 Parenteral drug products should be inspected visually for particulate matter and 99 discoloration prior to administration, whenever solution and container permit. 100 The solution should be clear and colorless and may contain a small amount of easily 101 visible, white, amorphous, cetuximab particulates. Do not shake or dilute. This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. 107 5 WARNINGS AND PRECAUTIONS 109 Serious infusion reactions, requiring medical intervention and immediate, permanent 110 discontinuation of Erbitux included rapid onset of airway obstruction (bronchospasm, 111 stridor, hoarseness), hypotension, shock, loss of consciousness, myocardial infarction, 112 and/or cardiac arrest. Severe (NCI CTC Grades 3 and 4) infusion reactions occurred in 113 2–5% of 1373 patients in Studies 1, 3, 5, and 6 receiving Erbitux, with fatal outcome in 114 1 patient. [See Clinical Studies (14.1, 14.2).] 109 Serious infusion reactions, requiring medical intervention and immediate, permanent 110 discontinuation of Erbitux included rapid onset of airway obstruction (bronchospasm, 111 stridor, hoarseness), hypotension, shock, loss of consciousness, myocardial infarction, 112 and/or cardiac arrest. Severe (NCI CTC Grades 3 and 4) infusion reactions occurred in 113 2–5% of 1373 patients in Studies 1, 3, 5, and 6 receiving Erbitux, with fatal outcome in 114 1 patient. [See Clinical Studies (14.1, 14.2).] 115 Approximately 90% of severe infusion reactions occurred with the first infusion despite 116 premedication with antihistamines. 115 Approximately 90% of severe infusion reactions occurred with the first infusion despite 116 premedication with antihistamines. 115 Approximately 90% of severe infusion reactions occurred with the first infusion despite 116 premedication with antihistamines. 117 Monitor patients for 1 hour following Erbitux infusions in a setting with resuscitation 118 equipment and other agents necessary to treat anaphylaxis (eg, epinephrine, 119 corticosteroids, intravenous antihistamines, bronchodilators, and oxygen). Monitor longer 120 to confirm resolution of the event in patients requiring treatment for infusion reactions. 121 Immediately and permanently discontinue Erbitux in patients with serious infusion 122 reactions. [See Boxed Warning, Dosage and Administration (2.4).] 121 Immediately and permanently discontinue Erbitux in patients with serious infusion 122 reactions. [See Boxed Warning, Dosage and Administration (2.4).] 122 reactions. [See Boxed Warning, Dosage and Administration (2.4).] 7 Administer through a low protein binding 0.22-micrometer in-line filter. 98 Parenteral drug products should be inspected visually for particulate matter and 99 discoloration prior to administration, whenever solution and container permit. 98 Parenteral drug products should be inspected visually for particulate matter and 99 discoloration prior to administration, whenever solution and container permit. 100 The solution should be clear and colorless and may contain a small amount of easily 101 visible, white, amorphous, cetuximab particulates. Do not shake or dilute. 100 The solution should be clear and colorless and may contain a small amount of easily 101 visible, white, amorphous, cetuximab particulates. Do not shake or dilute. 6 6 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 102 3 DOSAGE FORMS AND STRENGTHS 103 100 mg/50 mL, single-use vial 104 200 mg/100 mL, single-use vial 105 4 CONTRAINDICATIONS 106 None. 107 5 WARNINGS AND PRECAUTIONS 108 5.1 Infusion Reactions 109 Serious infusion reactions, requiring medical intervention and immediate, permanent 110 discontinuation of Erbitux included rapid onset of airway obstruction (bronchospasm, 111 stridor, hoarseness), hypotension, shock, loss of consciousness, myocardial infarction, 112 and/or cardiac arrest. Severe (NCI CTC Grades 3 and 4) infusion reactions occurred in 113 2–5% of 1373 patients in Studies 1, 3, 5, and 6 receiving Erbitux, with fatal outcome in 114 1 patient. [See Clinical Studies (14.1, 14.2).] 115 Approximately 90% of severe infusion reactions occurred with the first infusion despite 116 premedication with antihistamines. 117 Monitor patients for 1 hour following Erbitux infusions in a setting with resuscitation 118 equipment and other agents necessary to treat anaphylaxis (eg, epinephrine, 119 corticosteroids, intravenous antihistamines, bronchodilators, and oxygen). Monitor longer 120 to confirm resolution of the event in patients requiring treatment for infusion reactions. 121 Immediately and permanently discontinue Erbitux in patients with serious infusion 122 reactions. [See Boxed Warning, Dosage and Administration (2.4).] 123 5.2 Cardiopulmonary Arrest 124 Cardiopulmonary arrest and/or sudden death occurred in 4 (2%) of 208 patients treated 125 with radiation therapy and Erbitux as compared to none of 212 patients treated with 126 radiation therapy alone in Study 1. Three patients with prior history of coronary artery 102 3 DOSAGE FORMS AND STRENGTHS 103 100 mg/50 mL, single-use vial 141 5.3 142 Interstitial lung disease (ILD), including 1 fatality, occurred in 4 of 1570 (<0.5%) patients 143 receiving Erbitux in Studies 1, 3, and 6, as well as other studies, in colorectal cancer and 144 head and neck cancer. Interrupt Erbitux for acute onset or worsening of pulmonary 145 symptoms. Permanently discontinue Erbitux for confirmed ILD. 146 5.4 Dermatologic Toxicity 147 Dermatologic toxicities, including acneiform rash, skin drying and fissuring, paronychial 148 inflammation, infectious sequelae (for example, S. aureus sepsis, abscess formation, 149 cellulitis, blepharitis, conjunctivitis, keratitis/ulcerative keratitis with decreased visual 150 acuity, cheilitis), and hypertrichosis occurred in patients receiving Erbitux therapy. 151 Acneiform rash occurred in 76–88% of 1373 patients receiving Erbitux in Studies 1, 3, 5, 152 and 6. Severe acneiform rash occurred in 1–17% of patients. 153 Acneiform rash usually developed within the first two weeks of therapy and resolved in a 154 majority of the patients after cessation of treatment, although in nearly half, the event 155 continued beyond 28 days. Monitor patients receiving Erbitux for dermatologic toxicities 156 and infectious sequelae. Instruct patients to limit sun exposure during Erbitux therapy. 157 [See Dosage and Administration (2.4).] 123 5.2 Closely 139 monitor serum electrolytes, including serum magnesium, potassium, and calcium, during 140 and after Erbitux. [See Boxed Warning, Warnings and Precautions (5.6).] 141 5.3 Pulmonary Toxicity 142 Interstitial lung disease (ILD), including 1 fatality, occurred in 4 of 1570 (<0.5%) patients 143 receiving Erbitux in Studies 1, 3, and 6, as well as other studies, in colorectal cancer and 144 head and neck cancer. Interrupt Erbitux for acute onset or worsening of pulmonary 145 symptoms. Permanently discontinue Erbitux for confirmed ILD. 146 5.4 Dermatologic Toxicity 147 Dermatologic toxicities, including acneiform rash, skin drying and fissuring, paronychial 148 inflammation, infectious sequelae (for example, S. aureus sepsis, abscess formation, 149 cellulitis, blepharitis, conjunctivitis, keratitis/ulcerative keratitis with decreased visual 150 acuity, cheilitis), and hypertrichosis occurred in patients receiving Erbitux therapy. 151 Acneiform rash occurred in 76–88% of 1373 patients receiving Erbitux in Studies 1, 3, 5, 152 and 6. Severe acneiform rash occurred in 1–17% of patients. 153 Acneiform rash usually developed within the first two weeks of therapy and resolved in a 154 majority of the patients after cessation of treatment, although in nearly half, the event 155 continued beyond 28 days. Monitor patients receiving Erbitux for dermatologic toxicities 156 and infectious sequelae. Instruct patients to limit sun exposure during Erbitux therapy. 157 [See Dosage and Administration (2.4).] 123 5.2 124 Cardiopulmonary arrest and/or sudden death occurred in 4 (2%) of 208 p 124 Cardiopulmonary arrest and/or sudden death occurred in 4 (2%) of 208 patients treated 125 with radiation therapy and Erbitux as compared to none of 212 patients treated with 126 radiation therapy alone in Study 1. Three patients with prior history of coronary artery 127 disease died at home, with myocardial infarction as the presumed cause of death. One of 128 these patients had arrhythmia and one had congestive heart failure. Death occurred 27, 129 32, and 43 days after the last dose of Erbitux. One patient with no prior history of 7 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. y pp y For current labeling information, please visit https://www.fda.gov/drugsatfda 130 coronary artery disease died one day after the last dose of Erbitux. In Study 2, fatal 131 cardiac disorders and/or sudden death occurred in 7 (3%) of 219 patients treated with 132 EU-approved cetuximab and platinum-based therapy with 5-FU as compared to 4 (2%) of 133 215 patients treated with chemotherapy alone. Five of these 7 patients in the 134 chemotherapy plus cetuximab arm received concomitant cisplatin and 2 patients received 135 concomitant carboplatin. All 4 patients in the chemotherapy-alone arm received cisplatin. 136 Carefully consider use of Erbitux in combination with radiation therapy or platinum 137 based therapy with 5-FU in head and neck cancer patients with a history of coronary 138 artery disease, congestive heart failure, or arrhythmias in light of these risks. Closely 139 monitor serum electrolytes, including serum magnesium, potassium, and calcium, during 140 and after Erbitux. [See Boxed Warning, Warnings and Precautions (5.6).] 132 EU-approved cetuximab and platinum-based therapy with 5-FU as compared to 4 (2%) of 133 215 patients treated with chemotherapy alone. Five of these 7 patients in the 134 chemotherapy plus cetuximab arm received concomitant cisplatin and 2 patients received 135 concomitant carboplatin. All 4 patients in the chemotherapy-alone arm received cisplatin. 136 Carefully consider use of Erbitux in combination with radiation therapy or platinum 137 based therapy with 5-FU in head and neck cancer patients with a history of coronary 138 artery disease, congestive heart failure, or arrhythmias in light of these risks. 6 5.4 Dermatologic Toxicity 147 Dermatologic toxicities, including acneiform rash, skin drying and fissuring, paronychial 148 inflammation, infectious sequelae (for example, S. aureus sepsis, abscess formation, 149 cellulitis, blepharitis, conjunctivitis, keratitis/ulcerative keratitis with decreased visual 150 acuity, cheilitis), and hypertrichosis occurred in patients receiving Erbitux therapy. 151 Acneiform rash occurred in 76–88% of 1373 patients receiving Erbitux in Studies 1, 3, 5, 152 and 6. Severe acneiform rash occurred in 1–17% of patients. 147 Dermatologic toxicities, including acneiform rash, skin drying and fissuring, paronychial 148 inflammation, infectious sequelae (for example, S. aureus sepsis, abscess formation, 149 cellulitis, blepharitis, conjunctivitis, keratitis/ulcerative keratitis with decreased visual 150 acuity, cheilitis), and hypertrichosis occurred in patients receiving Erbitux therapy. 151 Acneiform rash occurred in 76–88% of 1373 patients receiving Erbitux in Studies 1, 3, 5, 152 and 6. Severe acneiform rash occurred in 1–17% of patients. 153 Acneiform rash usually developed within the first two weeks of therapy and resolved in a 154 majority of the patients after cessation of treatment, although in nearly half, the event 155 continued beyond 28 days. Monitor patients receiving Erbitux for dermatologic toxicities 156 and infectious sequelae. Instruct patients to limit sun exposure during Erbitux therapy. 157 [See Dosage and Administration (2.4).] 153 Acneiform rash usually developed within the first two weeks of therapy and resolved in a 154 majority of the patients after cessation of treatment, although in nearly half, the event 155 continued beyond 28 days. Monitor patients receiving Erbitux for dermatologic toxicities 156 and infectious sequelae. Instruct patients to limit sun exposure during Erbitux therapy. 157 [See Dosage and Administration (2.4).] 8 8 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 158 5.5 Use of Erbitux in Combination With Radiation and 159 Cisplatin 160 In a controlled study, 940 patients with locally advanced SCCHN were randomized 1:1 to 161 receive either Erbitux in combination with radiation therapy and cisplatin or radiation 162 therapy and cisplatin alone. The addition of Erbitux resulted in an increase in the 163 incidence of Grade 3–4 mucositis, radiation recall syndrome, acneiform rash, cardiac 164 events, and electrolyte disturbances compared to radiation and cisplatin alone. Adverse 165 reactions with fatal outcome were reported in 20 patients (4.4%) in the Erbitux 166 combination arm and 14 patients (3.0%) in the control arm. Nine patients in the Erbitux 167 arm (2.0%) experienced myocardial ischemia compared to 4 patients (0.9%) in the 168 control arm. The main efficacy outcome of the study was progression-free survival (PFS). 169 The addition of Erbitux to radiation and cisplatin did not improve PFS. 170 5.6 170 5.6 Hypomagnesemia and Electrolyte Abnormalities 171 In patients evaluated during clinical trials, hypomagnesemia occurred in 55% of 172 365 patients receiving Erbitux in Study 5 and two other clinical trials in colorectal 173 cancer and head and neck cancer, respectively, and was severe (NCI CTC Grades 3 and 174 4) in 6–17%. 175 In Study 2, where EU-approved cetuximab was administered in combination with 176 platinum-based therapy, the addition of cetuximab to cisplatin and 5-FU resulted in an 177 increased incidence of hypomagnesemia (14% vs. 6%) and of Grade 3–4 178 hypomagnesemia (7% vs. 2%) compared to cisplatin and 5-FU alone. In contrast, the 179 incidences of hypomagnesemia were similar for those who received cetuximab, 180 carboplatin, and 5-FU compared to carboplatin and 5-FU (4% vs. 4%). No patient 181 experienced Grade 3–4 hypomagnesemia in either arm in the carboplatin subgroup. 182 The onset of hypomagnesemia and accompanying electrolyte abnormalities occurred 183 days to months after initiation of Erbitux. Periodically monitor patients for 184 hypomagnesemia, hypocalcemia, and hypokalemia, during and for at least 8 weeks 185 following the completion of Erbitux. Replete electrolytes as necessary. 171 In patients evaluated during clinical trials, hypomagnesemia occurred in 55% of 172 365 patients receiving Erbitux in Study 5 and two other clinical trials in colorectal 173 cancer and head and neck cancer, respectively, and was severe (NCI CTC Grades 3 and 174 4) in 6–17%. 175 In Study 2, where EU-approved cetuximab was administered in combination with 176 platinum-based therapy, the addition of cetuximab to cisplatin and 5-FU resulted in an 177 increased incidence of hypomagnesemia (14% vs. 6%) and of Grade 3–4 178 hypomagnesemia (7% vs. 2%) compared to cisplatin and 5-FU alone. In contrast, the 179 incidences of hypomagnesemia were similar for those who received cetuximab, 180 carboplatin, and 5-FU compared to carboplatin and 5-FU (4% vs. 4%). No patient 181 experienced Grade 3–4 hypomagnesemia in either arm in the carboplatin subgroup. 175 In Study 2, where EU-approved cetuximab was administered in combination with 176 platinum-based therapy, the addition of cetuximab to cisplatin and 5-FU resulted in an 177 increased incidence of hypomagnesemia (14% vs. 6%) and of Grade 3–4 178 hypomagnesemia (7% vs. 2%) compared to cisplatin and 5-FU alone. In contrast, the 179 incidences of hypomagnesemia were similar for those who received cetuximab, 180 carboplatin, and 5-FU compared to carboplatin and 5-FU (4% vs. 4%). 170 5.6 No patient 181 experienced Grade 3–4 hypomagnesemia in either arm in the carboplatin subgroup. 182 The onset of hypomagnesemia and accompanying electrolyte abnormalities occurred 183 days to months after initiation of Erbitux. Periodically monitor patients for 184 hypomagnesemia, hypocalcemia, and hypokalemia, during and for at least 8 weeks 185 following the completion of Erbitux. Replete electrolytes as necessary. 9 9 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 186 5.7 K-Ras Testing in Metastatic or Advanced Colorectal 187 Cancer Patients 188 Determination of K-Ras mutational status in colorectal tumors using an FDA-approved 189 test indicated for this use is necessary for selection of patients for treatment with Erbitux. 190 Erbitux is indicated only for patients with EGFR-expressing K-Ras mutation-negative 191 (wild-type) mCRC. Erbitux is not an effective treatment for patients with colorectal 192 cancer that harbor somatic mutations in codons 12 and 13 (exon 2). Studies 4 and 5, 193 conducted in patients with colorectal cancer, demonstrated a benefit with Erbitux 194 treatment only in the subset of patients whose tumors were K-Ras mutation-negative 195 (wild-type). Erbitux is not effective for the treatment of K-Ras mutation-positive 196 colorectal cancer as determined by an FDA-approved test for this use. [See Indications 197 and Usage (1.2), Clinical Pharmacology (12.1), Clinical Studies (14.2).] 198 Perform the assessment for K-Ras mutation status in colorectal cancer in laboratories 199 with demonstrated proficiency in the specific technology being utilized. Improper assay 200 performance can lead to unreliable test results. 198 Perform the assessment for K-Ras mutation status in colorectal cancer in laboratories 199 with demonstrated proficiency in the specific technology being utilized. Improper assay 200 performance can lead to unreliable test results. 198 Perform the assessment for K-Ras mutation status in colorectal cancer in laboratories 199 with demonstrated proficiency in the specific technology being utilized. Improper assay 200 performance can lead to unreliable test results. 203 5.8 Epidermal Growth Factor Receptor (EGFR) Expression 204 and Response 231 6.1 Clinical Trials Experience 232 Because clinical trials are conducted under widely varying conditions, adverse reaction 233 rates observed in the clinical trials of a drug cannot be directly compared to rates in the 234 clinical trials of another drug and may not reflect the rates observed in practice. 235 The data below reflect exposure to Erbitux in 1373 patients with SCCHN or colorectal 236 cancer in randomized Phase 3 (Studies 1 and 5) or Phase 2 (Studies 3 and 6) trials treated 237 at the recommended dose and schedule for medians of 7 to 14 weeks. [See Clinical 238 Studies (14).] 239 Infusion reactions: Infusion reactions, which included pyrexia, chills, rigors, 215 The following adverse reactions are discussed in greater detail in other sections of the 216 label: 217  Infusion reactions [See Boxed Warning, Warnings and Precautions (5.1).] 218  Cardiopulmonary arrest [See Boxed Warning, Warnings and Precautions (5.2).] 219  Pulmonary toxicity [See Warnings and Precautions (5.3).] 220  Dermatologic toxicity [See Warnings and Precautions (5.4).] 221  Hypomagnesemia and Electrolyte Abnormalities [See Warnings and Precautions 222 (5.6).] 223 The most common adverse reactions in Erbitux clinical trials (incidence 25%) include 224 cutaneous adverse reactions (including rash, pruritus, and nail changes), headache, 225 diarrhea, and infection. 226 The most serious adverse reactions with Erbitux are infusion reactions, cardiopulmonary 223 The most common adverse reactions in Erbitux clinical trials (incidence 25%) include 224 cutaneous adverse reactions (including rash, pruritus, and nail changes), headache, 225 diarrhea, and infection. 226 The most serious adverse reactions with Erbitux are infusion reactions, cardiopulmonary 227 arrest, dermatologic toxicity and radiation dermatitis, sepsis, renal failure, interstitial lung 228 disease, and pulmonary embolus. 226 The most serious adverse reactions with Erbitux are infusion reactions, cardiopulmonary 227 arrest, dermatologic toxicity and radiation dermatitis, sepsis, renal failure, interstitial lung 228 disease, and pulmonary embolus. 229 Across Studies 1, 3, 5, and 6, Erbitux was discontinued in 3–10% of patients because of 230 adverse reactions. 229 Across Studies 1, 3, 5, and 6, Erbitux was discontinued in 3–10% of patients because of 230 adverse reactions. 203 5.8 Epidermal Growth Factor Receptor (EGFR) Expression 204 and Response 205 Because expression of EGFR has been detected in nearly all SCCHN tumor specimens, 206 patients enrolled in the head and neck cancer clinical studies were not required to have 207 immunohistochemical evidence of EGFR tumor expression prior to study entry. 208 Patients enrolled in the colorectal cancer clinical studies were required to have 209 immunohistochemical evidence of EGFR tumor expression. Primary tumor or tumor 210 from a metastatic site was tested with the DakoCytomation EGFR pharmDx™ test kit. 211 Specimens were scored based on the percentage of cells expressing EGFR and intensity 212 (barely/faint, weak-to-moderate, and strong). Response rate did not correlate with either 213 the percentage of positive cells or the intensity of EGFR expression. 208 Patients enrolled in the colorectal cancer clinical studies were required to have 209 immunohistochemical evidence of EGFR tumor expression. Primary tumor or tumor 210 from a metastatic site was tested with the DakoCytomation EGFR pharmDx™ test kit. 211 Specimens were scored based on the percentage of cells expressing EGFR and intensity 212 (barely/faint, weak-to-moderate, and strong). Response rate did not correlate with either 213 the percentage of positive cells or the intensity of EGFR expression. 10 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda For current labeling information, please visit https://www.fda.gov/drugsatfda 214 6 ADVERSE REACTIONS 215 The following adverse reactions are discussed in greater detail in other sections of the 216 label: 217  Infusion reactions [See Boxed Warning, Warnings and Precautions (5.1).] 218  Cardiopulmonary arrest [See Boxed Warning, Warnings and Precautions (5.2).] 219  Pulmonary toxicity [See Warnings and Precautions (5.3).] 220  Dermatologic toxicity [See Warnings and Precautions (5.4).] 221  Hypomagnesemia and Electrolyte Abnormalities [See Warnings and Precautions 222 (5.6).] 223 The most common adverse reactions in Erbitux clinical trials (incidence 25%) include 224 cutaneous adverse reactions (including rash, pruritus, and nail changes), headache, 225 diarrhea, and infection. 226 The most serious adverse reactions with Erbitux are infusion reactions, cardiopulmonary 227 arrest, dermatologic toxicity and radiation dermatitis, sepsis, renal failure, interstitial lung 228 disease, and pulmonary embolus. 229 Across Studies 1, 3, 5, and 6, Erbitux was discontinued in 3–10% of patients because of 230 adverse reactions. 231 6.1 Clinical Trials Experience 231 6.1 Clinical Trials Experience 232 Because clinical trials are conducted under widely varying conditions, adverse reaction 233 rates observed in the clinical trials of a drug cannot be directly compared to rates in the 234 clinical trials of another drug and may not reflect the rates observed in practice. 235 The data below reflect exposure to Erbitux in 1373 patients with SCCHN or colorectal 236 cancer in randomized Phase 3 (Studies 1 and 5) or Phase 2 (Studies 3 and 6) trials treated 237 at the recommended dose and schedule for medians of 7 to 14 weeks. [See Clinical 238 Studies (14).] 239 Infusion reactions: Infusion reactions, which included pyrexia, chills, rigors, 240 dyspnea, bronchospasm, angioedema, urticaria, hypertension, and hypotension occurred 241 in 15–21% of patients across studies. Grades 3 and 4 infusion reactions occurred in 2–5% 242 of patients; infusion reactions were fatal in 1 patient. 232 Because clinical trials are conducted under widely varying conditions, adverse reaction 233 rates observed in the clinical trials of a drug cannot be directly compared to rates in the 234 clinical trials of another drug and may not reflect the rates observed in practice. 232 Because clinical trials are conducted under widely varying conditions, adverse reaction 233 rates observed in the clinical trials of a drug cannot be directly compared to rates in the 234 clinical trials of another drug and may not reflect the rates observed in practice. 235 The data below reflect exposure to Erbitux in 1373 patients with SCCHN or colorectal 236 cancer in randomized Phase 3 (Studies 1 and 5) or Phase 2 (Studies 3 and 6) trials treated 237 at the recommended dose and schedule for medians of 7 to 14 weeks. [See Clinical 238 Studies (14).] 235 The data below reflect exposure to Erbitux in 1373 patients with SCCHN or colorectal 236 cancer in randomized Phase 3 (Studies 1 and 5) or Phase 2 (Studies 3 and 6) trials treated 237 at the recommended dose and schedule for medians of 7 to 14 weeks. [See Clinical 238 Studies (14).] 235 The data below reflect exposure to Erbitux in 1373 patients with SCCHN or colorectal 236 cancer in randomized Phase 3 (Studies 1 and 5) or Phase 2 (Studies 3 and 6) trials treated 237 at the recommended dose and schedule for medians of 7 to 14 weeks. 246 Squamous Cell Carcinoma of the Head and Neck 246 Squamous Cell Carcinoma of the Head and Neck 247 Erbitux in Combination with Radiation Therapy 248 Table 2 contains selected adverse reactions in 420 patients receiving radiation therapy 249 either alone or with Erbitux for locally or regionally advanced SCCHN in Study 1. 250 Erbitux was administered at the recommended dose and schedule (400 mg/m2 initial 251 dose, followed by 250 mg/m2 weekly). Patients received a median of 8 infusions (range 252 1–11). Table 2: Incidence of Selected Adverse Reactions (10%) in Patients with Locoregionally Advanced SCCHN Erbitux plus Radiation Radiation Therapy Alone (n=208) (n=212) Body System Grades Grades Grades Grades Preferred Term 1–4 3 and 4 1–4 3 and 4 % of Patients Body as a Whole Asthenia 56 4 49 5 Fevera 29 1 13 1 Headache 19 <1 8 <1 Infusion Reactionb 15 3 2 0 Infection 13 1 9 1 Chillsa 16 0 5 0 Digestive Nausea 49 2 37 2 Emesis 29 2 23 4 Diarrhea 19 2 13 1 Dyspepsia 14 0 9 1 Metabolic/Nutritional Weight Loss 84 11 72 7 Dehydration 25 6 19 8 Alanine Transaminase, highc 43 2 21 1 Aspartate Transaminase, highc 38 1 24 1 231 6.1 Clinical Trials Experience [See Clinical 238 Studies (14).] 235 The data below reflect exposure to Erbitux in 1373 patients with SCCHN or colorectal 236 cancer in randomized Phase 3 (Studies 1 and 5) or Phase 2 (Studies 3 and 6) trials treated 237 at the recommended dose and schedule for medians of 7 to 14 weeks. [See Clinical 238 Studies (14).] 239 Infusion reactions: Infusion reactions, which included pyrexia, chills, rigors, 240 dyspnea, bronchospasm, angioedema, urticaria, hypertension, and hypotension occurred 241 in 15–21% of patients across studies. Grades 3 and 4 infusion reactions occurred in 2–5% 242 of patients; infusion reactions were fatal in 1 patient. 239 Infusion reactions: Infusion reactions, which included pyrexia, chills, rigors, 240 dyspnea, bronchospasm, angioedema, urticaria, hypertension, and hypotension occurred 241 in 15–21% of patients across studies. Grades 3 and 4 infusion reactions occurred in 2–5% 242 of patients; infusion reactions were fatal in 1 patient. 11 Reference ID: 3270603 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 243 Infections: The incidence of infection was variable across studies, ranging from 244 13–35%. Sepsis occurred in 1–4% of patients. 243 Infections: The incidence of infection was variable across studies, ranging from 244 13–35%. Sepsis occurred in 1–4% of patients. 245 Renal: Renal failure occurred in 1% of patients with colorectal cancer. 245 Renal: Renal failure occurred in 1% of patients with colorectal cancer. 247 Erbitux in Combination with Radiation Therapy b Infusion reaction is defined as any event described at any time during the clinical study as “allergic reaction” or “anaphylactoid reaction”, or any event occurring on the first day of dosing described as “allergic reaction”, “anaphylactoid reaction”, “fever”, “chills”, “chills and fever”, or “dyspnea”. c Based on laboratory measurements, not on reported adverse reactions, the number of subjects with tested samples varied from 205–206 for Erbitux plus Radiation arm; 209–210 for Radiation alone. d c Based on laboratory measurements, not on reported adverse reactions, the number of subjects with tested samples varied from 205–206 for Erbitux plus Radiation arm; 209–210 for Radiation alone. d d Acneiform rash is defined as any event described as “acne”, “rash”, “maculopapular rash”, “pustular rash”, “dry skin”, or “exfoliative dermatitis”. 253 The incidence and severity of mucositis, stomatitis, and xerostomia were similar in both 253 The incidence and severity of mucositis, stomatitis, and xerostomia were similar in both 254 arms of the study 254 arms of the study. 247 Erbitux in Combination with Radiation Therapy 248 Table 2 contains selected adverse reactions in 420 patients receiving radiation therapy 249 either alone or with Erbitux for locally or regionally advanced SCCHN in Study 1. 250 Erbitux was administered at the recommended dose and schedule (400 mg/m2 initial 251 dose, followed by 250 mg/m2 weekly). Patients received a median of 8 infusions (range 252 1–11). Table 2: Incidence of Selected Adverse Reactions (10%) in Patients with Locoregionally Advanced SCCHN Erbitux plus Radiation Radiation Therapy Alone (n=208) (n=212) Body System Grades Grades Grades Grades Preferred Term 1–4 3 and 4 1–4 3 and 4 % of Patients Body as a Whole Asthenia 56 4 49 5 Fevera 29 1 13 1 Headache 19 <1 8 <1 Infusion Reactionb 15 3 2 0 Infection 13 1 9 1 Chillsa 16 0 5 0 Digestive Nausea 49 2 37 2 Emesis 29 2 23 4 Diarrhea 19 2 13 1 Dyspepsia 14 0 9 1 Metabolic/Nutritional Weight Loss 84 11 72 7 Dehydration 25 6 19 8 Alanine Transaminase, highc 43 2 21 1 Aspartate Transaminase, highc 38 1 24 1 Incidence of Selected Adverse Reactions (10%) in Patients with Locoregionally Advanced SCCHN 12 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda y pp y For current labeling information, please visit https://www.fda.gov/drugsatfd Table 2: Incidence of Selected Adverse Reactions (10%) in Patients with Locoregionally Advanced SCCHN Erbitux plus Radiation Radiation Therapy Alone (n=208) (n=212) Body System Grades Grades Grades Grades Preferred Term 1–4 3 and 4 1–4 3 and 4 % of Patients Alkaline Phosphatase, highc 33 <1 24 0 Respiratory Pharyngitis 26 3 19 4 Skin/Appendages d Acneiform Rash 87 17 10 1 Radiation Dermatitis 86 23 90 18 Application Site Reaction 18 0 12 1 Pruritus 16 0 4 0 a Includes cases also reported as infusion reaction. Incidence of Selected Adverse Reactions (10%) in Patients with Locoregionally Advanced SCCHN b Infusion reaction is defined as any event described at any time during the clinical study as “allergic reaction” or “anaphylactoid reaction”, or any event occurring on the first day of dosing described as “allergic reaction”, “anaphylactoid reaction”, “fever”, “chills”, “chills and fever”, or “dyspnea”. 255 Late Radiation Toxicity 256 The overall incidence of late radiation toxicities (any grade) was higher in Erbitux in 257 combination with radiation therapy compared with radiation therapy alone. The following 258 sites were affected: salivary glands (65% versus 56%), larynx (52% versus 36%), 259 subcutaneous tissue (49% versus 45%), mucous membrane (48% versus 39%), esophagus 260 (44% versus 35%), skin (42% versus 33%). The incidence of Grade 3 or 4 late radiation 261 toxicities was similar between the radiation therapy alone and the Erbitux plus radiation 262 treatment groups. 13 13 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. y pp y For current labeling information, please visit https://www.fda.gov/drugsatfda 263 Study 2: EU-Approved Cetuximab in Combination with Platinum-based 264 Therapy with 5-Fluorouracil 265 Study 2 used EU-approved cetuximab. Since U.S.-licensed Erbitux provides 266 approximately 22% higher exposure relative to the EU-approved cetuximab, the data 267 provided below may underestimate the incidence and severity of adverse reactions 268 anticipated with Erbitux for this indication. However, the tolerability of the 269 recommended dose is supported by safety data from additional studies of Erbitux [see 270 Clinical Pharmacology (12.3)]. 271 Table 3 contains selected adverse reactions in 434 patients with recurrent locoregional 272 disease or metastatic SCCHN receiving EU-approved cetuximab in combination with 273 platinum-based therapy with 5-FU or platinum-based therapy with 5-FU alone in Study 2. 274 Cetuximab was administered at 400 mg/m2 for the initial dose, followed by 250 mg/m2 275 weekly. Patients received a median of 17 infusions (range 1–89). Table 3: Incidence of Selected Adverse Reactions (10%) in Patients with Recurrent Locoregional Disease or Metastatic SCCHN EU-Approved Cetuximab plus Platinum-based Therapy Platinum-based Therapy with 5-FU with 5-FU Alone System Organ Class (n=219) (n=215) Preferred Term Grades Grades Grades Grades 1–4 3 and 4 1–4 3 and 4 % of Patients Eye Disorders Conjunctivitis 10 0 0 0 Gastrointestinal Disorders Nausea 54 4 47 4 Diarrhea 26 5 16 1 General Disorders and Administration Site Conditions Pyrexia 22 0 13 1 Infusion Reactiona 10 2 <1 0 Infections and Infestations b Infection 44 11 27 8 Metabolism and Nutrition Disorders Anorexia 25 5 14 1 Hypocalcemia 12 4 5 1 263 Study 2: EU-Approved Cetuximab in Combination with Platinum-based 264 Therapy with 5-Fluorouracil 265 Study 2 used EU-approved cetuximab. Since U.S.-licensed Erbitux provides 266 approximately 22% higher exposure relative to the EU-approved cetuximab, the data 267 provided below may underestimate the incidence and severity of adverse reactions 268 anticipated with Erbitux for this indication. However, the tolerability of the 269 recommended dose is supported by safety data from additional studies of Erbitux [see 270 Clinical Pharmacology (12.3)]. 271 Table 3 contains selected adverse reactions in 434 patients with recurrent locoregional 272 disease or metastatic SCCHN receiving EU-approved cetuximab in combination with 273 platinum-based therapy with 5-FU or platinum-based therapy with 5-FU alone in Study 2. 274 Cetuximab was administered at 400 mg/m2 for the initial dose, followed by 250 mg/m2 275 weekly. Patients received a median of 17 infusions (range 1–89). Table 3: Incidence of Selected Adverse Reactions (10%) in Patients with Recurrent Locoregional Disease or Metastatic SCCHN EU-Approved Cetuximab plus Platinum-based Therapy Platinum-based Therapy with 5-FU with 5-FU Alone System Organ Class (n=219) (n=215) Preferred Term Grades Grades Grades Grades 1–4 3 and 4 1–4 3 and 4 % of Patients Eye Disorders Conjunctivitis 10 0 0 0 Gastrointestinal Disorders Nausea 54 4 47 4 Diarrhea 26 5 16 1 General Disorders and Administration Site Conditions Pyrexia 22 0 13 1 Infusion Reactiona 10 2 <1 0 Infections and Infestations b Infection 44 11 27 8 Metabolism and Nutrition Disorders Anorexia 25 5 14 1 Hypocalcemia 12 4 5 1 Hypokalemia 12 7 7 5 14 D: 3270603 Incidence of Selected Adverse Reactions (10%) in Patients ith R t L i l Di M t t ti SCCHN Incidence of Selected Adverse Reactions (10%) in Patients with Recurrent Locoregional Disease or Metastatic SCCHN 14 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. 263 Study 2: EU-Approved Cetuximab in Combination with Platinum-based 264 Therapy with 5-Fluorouracil U.S.-licensed Erbitux provides approximately 289 22% higher exposure to cetuximab relative to the EU-approved cetuximab. The data 290 provided below for Study 4 is consistent in incidence and severity of adverse reactions 291 with those seen for Erbitux in this indication. The tolerability of the recommended dose is 292 supported by safety data from additional studies of Erbitux [see Clinical Pharmacology 293 (12.3)]. 294 Table 4 contains selected adverse reactions in 667 patients with K-Ras mutation-negative 295 (wild-type), EGFR-expressing, metastatic colorectal cancer receiving EU-approved 296 cetuximab plus FOLFIRI or FOLFIRI alone in Study 4 [see Warnings and Precautions 297 (5.8)]. Cetuximab was administered at the recommended dose and schedule (400 mg/m2 298 initial dose, followed by 250 mg/m2 weekly). Patients received a median of 26 infusions 299 (range 1–224). Table 4: Incidence of Selected Adverse Reactions Occurring in 10% of Patients with K-Ras Mutation-negative (Wild-type) and EGFR- expressing, Metastatic Colorectal Cancera EU-Approved Cetuximab plus FOLFIRI FOLFIRI Alone (n=317) (n=350) Body System Preferred Term Grades 1–4b Grades 3 and 4 Grades 1–4 Grades 3 and 4 % of Patients Blood and Lymphatic System Disorders Neutropenia 49 31 42 24 Eye Disorders Conjunctivitis 18 <1 3 0 Gastrointestinal Disorders Diarrhea 66 16 60 10 Stomatitis 31 3 19 1 Dyspepsia 16 0 9 0 General Disorders and Administration Site Conditions Infusion-related Reactionc 14 2 <1 0 Pyrexia 26 1 14 1 263 Study 2: EU-Approved Cetuximab in Combination with Platinum-based 264 Therapy with 5-Fluorouracil For current labeling information, please visit https://www.fda.gov/drugsatfda y pp y For current labeling information, please visit https://www.fda.gov/drugsatfd Table 3: Incidence of Selected Adverse Reactions (10%) in Patients with Recurrent Locoregional Disease or Metastatic SCCHN EU-Approved Cetuximab plus Platinum-based Therapy Platinum-based Therapy with 5-FU with 5-FU Alone System Organ Class (n=219) (n=215) Preferred Term Grades Grades Grades Grades 1–4 3 and 4 1–4 3 and 4 % of Patients Hypomagnesemia 11 5 5 1 Skin and Subcutaneous Tissue Disorders Acneiform Rashc 70 9 2 0 Rash 28 5 2 0 Acne 22 2 0 0 Dermatitis Acneiform 15 2 0 0 Dry Skin 14 0 <1 0 Alopecia 12 0 7 0 a Infusion reaction defined as any event of “anaphylactic reaction”, “hypersensitivity”, “fever and/or chills”, “dyspnea”, or “pyrexia” on the first day of dosing. b Infection – this term excludes sepsis-related events which are presented separately. c Acneiform rash defined as any event described as “acne”, “dermatitis acneiform”, “dry skin”, “exfoliative rash”, “rash”, “rash erythematous”, “rash macular”, “rash papular”, or “rash pustular”. Chemotherapy = cisplatin + 5-fluorouracil or carboplatin + 5-fluorouracil Incidence of Selected Adverse Reactions (10%) in Patients with Recurrent Locoregional Disease or Metastatic SCCHN 276 For cardiac disorders, approximately 9% of subjects in both the EU-approved cetuximab 277 plus chemotherapy and chemotherapy-only treatment arms in Study 2 experienced a 278 cardiac event. The majority of these events occurred in patients who received 279 cisplatin/5-FU, with or without cetuximab as follows: 11% and 12% in patients who 280 received cisplatin/5-FU with or without cetuximab, respectively, and 6% or 4% in 281 patients who received carboplatin/5-FU with or without cetuximab, respectively. In both 282 arms, the incidence of cardiovascular events was higher in the cisplatin with 5-FU 283 containing subgroup. Death attributed to cardiovascular event or sudden death was 284 reported in 3% of the patients in the cetuximab plus platinum-based therapy with 5-FU 285 arm and 2% in the platinum-based chemotherapy with 5-FU alone arm. 15 15 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda For current labeling information, please visit https://www.fda.gov/drugsatfda 286 Colorectal Cancer 287 Study 4: EU-Approved Cetuximab in Combination with FOLFIRI 288 Study 4 used EU-approved cetuximab. 287 Study 4: EU-Approved Cetuximab in Combination with FOLFIRI 288 Study 4 used EU-approved cetuximab. U.S.-licensed Erbitux provides approximately 289 22% higher exposure to cetuximab relative to the EU-approved cetuximab. The data 290 provided below for Study 4 is consistent in incidence and severity of adverse reactions 291 with those seen for Erbitux in this indication. The tolerability of the recommended dose is 292 supported by safety data from additional studies of Erbitux [see Clinical Pharmacology 293 (12.3)]. 294 Table 4 contains selected adverse reactions in 667 patients with K-Ras mutation-negative 295 (wild-type), EGFR-expressing, metastatic colorectal cancer receiving EU-approved 296 cetuximab plus FOLFIRI or FOLFIRI alone in Study 4 [see Warnings and Precautions 297 (5.8)]. Cetuximab was administered at the recommended dose and schedule (400 mg/m2 298 initial dose, followed by 250 mg/m2 weekly). Patients received a median of 26 infusions 299 (range 1–224). Table 4: Incidence of Selected Adverse Reactions Occurring in 10% of Patients with K-Ras Mutation-negative (Wild-type) and EGFR- expressing, Metastatic Colorectal Cancera EU-Approved Cetuximab plus FOLFIRI FOLFIRI Alone (n=317) (n=350) Body System Preferred Term Grades 1–4b Grades 3 and 4 Grades 1–4 Grades 3 and 4 % of Patients Blood and Lymphatic System Disorders Neutropenia 49 31 42 24 Eye Disorders Conjunctivitis 18 <1 3 0 Gastrointestinal Disorders Diarrhea 66 16 60 10 Stomatitis 31 3 19 1 Dyspepsia 16 0 9 0 General Disorders and Administration Site Conditions Infusion-related Reactionc 14 2 <1 0 Pyrexia 26 1 14 1 Incidence of Selected Adverse Reactions Occurring in 10% of Patients with K-Ras Mutation-negative (Wild-type) and EGFR- expressing, Metastatic Colorectal Cancera 16 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. 287 Study 4: EU-Approved Cetuximab in Combination with FOLFIRI y pp y For current labeling information, please visit https://www.fda.gov/drugsatfd Table 4: Incidence of Selected Adverse Reactions Occurring in 10% of Patients with K-Ras Mutation-negative (Wild-type) and EGFR- expressing, Metastatic Colorectal Cancera EU-Approved Cetuximab plus FOLFIRI FOLFIRI Alone (n=317) (n=350) Grades Body System Grades Grades Grades b Preferred Term 1–4 3 and 4 1–4 3 and 4 % of Patients Infections and Infestations Paronychia 20 4 <1 0 Investigations Weight Decreased 15 1 9 1 Metabolism and Nutrition Disorders Anorexia 30 3 23 2 Skin and Subcutaneous Tissue Disorders d Acne-like Rash 86 18 13 <1 Rash 44 9 4 0 Dermatitis Acneiform 26 5 <1 0 Dry Skin 22 0 4 0 Acne 14 2 0 0 Pruritus 14 0 3 0 Palmar-plantar Erythrodysesthesia 19 4 4 <1 Syndrome Skin Fissures 19 2 1 0 a Adverse reactions occurring in at least 10% of Erbitux combination arm with a frequency at least 5% greater than that seen in the FOLFIRI arm. b b Adverse reactions were graded using the NCI CTC, V 2.0. c Infusion related reaction is defined as any event meeting the medical concepts of allergy/anaphylaxis at any time during the clinical study or any event occurring on the first day of dosing and meeting the medical concepts of dyspnea and fever or by the following events using MedDRA preferred terms: “acute myocardial infarction”, “angina pectoris”, “angioedema”, “autonomic seizure”, “blood pressure abnormal”, “blood pressure decreased”, “blood pressure increased”, “cardiac failure”, “cardiopulmonary failure”, “cardiovascular insufficiency”, “clonus”, “convulsion”, “coronary no-reflow phenomenon”, “epilepsy”, “hypertension”, “hypertensive crisis”, “hypertensive emergency”, “hypotension”, “infusion related reaction”, “loss of consciousness”, “myocardial infarction”, “myocardial ischaemia”, “prinzmetal angina”, “shock”, “sudden death”, “syncope”, or “systolic hypertension”. c Infusion related reaction is defined as any event meeting the medical concepts of allergy/anaphylaxis at any time during the clinical study or any event occurring on the first day of dosing and meeting the medical concepts of dyspnea and fever or by the following events using MedDRA preferred terms: “acute myocardial infarction”, “angina pectoris”, “angioedema”, “autonomic seizure”, “blood pressure abnormal”, “blood pressure decreased”, “blood pressure increased”, “cardiac failure”, “cardiopulmonary failure”, “cardiovascular insufficiency”, “clonus”, “convulsion”, “coronary no-reflow phenomenon”, “epilepsy”, “hypertension”, “hypertensive crisis”, “hypertensive emergency”, “hypotension”, “infusion related reaction”, “loss of consciousness”, “myocardial infarction”, “myocardial ischaemia”, “prinzmetal angina”, “shock”, “sudden death”, “syncope”, or “systolic hypertension”. 287 Study 4: EU-Approved Cetuximab in Combination with FOLFIRI d Acne-like rash is defined by the events using MedDRA preferred terms and included “acne”, “acne pustular”, “butterfly rash”, “dermatitis acneiform”, “drug rash with eosinophilia and systemic symptoms”, “dry skin”, “erythema”, “exfoliative rash”, “folliculitis”, “genital rash”, “mucocutaneous rash”, “pruritus”, “rash”, “rash erythematous”, “rash follicular”, “rash generalized”, “rash macular”, “rash maculopapular”, “rash maculovesicular”, “rash morbilliform”, “rash papular”, “rash papulosquamous”, “rash pruritic”, “rash pustular”, “rash rubelliform”, “rash scarlatiniform”, “rash vesicular”, “skin exfoliation”, “skin hyperpigmentation”, “skin plaque”, “telangiectasia”, or “xerosis”. 17 Reference ID: 3270603 300 Erbitux Monotherapy 301 Table 5 contains selected adverse reactions in 242 patients with K-Ras mutation-negative 302 (wild-type), EGFR-expressing, metastatic colorectal cancer who received best supportive 303 care (BSC) alone or with Erbitux in Study 5 [see Warnings and Precautions (5.8)]. 304 Erbitux was administered at the recommended dose and schedule (400 mg/m2 initial 305 dose, followed by 250 mg/m2 weekly). Patients received a median of 17 infusions (range 306 1–51). Table 5: Incidence of Selected Adverse Reactions Occurring in 10% of Patients with K-Ras Mutation-negative (Wild-type), EGFR- expressing, Metastatic Colorectal Cancer Treated with Erbitux Monotherapya Erbitux plus BSC BSC alone (n=118) (n=124) Body System Grades Grades b Preferred Term Grades 1–4 3 and 4 Grades 1–4 3 and 4 % of Patients Dermatology/Skin Rash/Desquamation 95 16 21 1 Dry Skin 57 0 15 0 Pruritus 47 2 11 0 Other-Dermatology 35 0 7 2 Nail Changes 31 0 4 0 Constitutional Symptoms Fatigue 91 31 79 29 Fever 25 3 16 0 Infusion Reactionsc 18 3 0 0 Rigors, Chills 16 1 3 0 Pain Pain-Other 59 18 37 10 Headache 38 2 11 0 Bone Pain 15 4 8 2 Pulmonary Dyspnea 49 16 44 13 Cough 30 2 19 2 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 300 Erbitux Monotherapy 301 Table 5 contains selected adverse reactions in 242 patients with K-Ras mutation-negative 302 (wild-type), EGFR-expressing, metastatic colorectal cancer who received best supportive 303 care (BSC) alone or with Erbitux in Study 5 [see Warnings and Precautions (5.8)]. 304 Erbitux was administered at the recommended dose and schedule (400 mg/m2 initial 305 dose, followed by 250 mg/m2 weekly). Patients received a median of 17 infusions (range 306 1–51). Table 5: Incidence of Selected Adverse Reactions Occurring in 10% of Patients with K-Ras Mutation-negative (Wild-type), EGFR- expressing, Metastatic Colorectal Cancer Treated with Erbitux Monotherapya Erbitux plus BSC BSC alone (n=118) (n=124) Body System Grades Grades b Preferred Term Grades 1–4 3 and 4 Grades 1–4 3 and 4 % of Patients Dermatology/Skin Rash/Desquamation 95 16 21 1 Dry Skin 57 0 15 0 Pruritus 47 2 11 0 Other-Dermatology 35 0 7 2 Nail Changes 31 0 4 0 Constitutional Symptoms Fatigue 91 31 79 29 Fever 25 3 16 0 Infusion Reactionsc 18 3 0 0 Rigors, Chills 16 1 3 0 Pain Pain-Other 59 18 37 10 Headache 38 2 11 0 Bone Pain 15 4 8 2 Pulmonary Dyspnea 49 16 44 13 Cough 30 2 19 2 Incidence of Selected Adverse Reactions Occurring in 10% of Patients with K Ras Mutation negative (Wild type) EGFR 18 Reference ID: 3270603 This label may not be the latest approved by FDA. a Adverse reactions occurring in at least 10% of Erbitux plus BSC arm with a frequency at least 5% greater than that seen in the BSC alone arm. b 300 Erbitux Monotherapy For current labeling information, please visit https://www.fda.gov/drugsatfda Table 5: Incidence of Selected Adverse Reactions Occurring in 10% of Patients with K-Ras Mutation-negative (Wild-type), EGFR- expressing, Metastatic Colorectal Cancer Treated with Erbitux Monotherapya Erbitux plus BSC BSC alone (n=118) (n=124) Body System Grades Grades b Preferred Term Grades 1–4 3 and 4 Grades 1–4 3 and 4 % of Patients Gastrointestinal Nausea 64 6 50 6 Constipation 53 3 38 3 Diarrhea 42 2 23 2 Vomiting 40 5 26 5 Stomatitis 32 1 10 0 Other-Gastrointestinal 22 12 16 5 Dehydration 13 5 3 0 Mouth Dryness 12 0 6 0 Taste Disturbance 10 0 5 0 Infection Infection without neutropenia 38 11 19 5 Musculoskeletal Arthralgia 14 3 6 0 Neurology Neuropathy-sensory 45 1 38 2 Insomnia 27 0 13 0 Confusion 18 6 10 2 Anxiety 14 1 5 1 Depression 14 0 5 0 a Adverse reactions occurring in at least 10% of Erbitux plus BSC arm with a frequency at least 5% greater than that seen in the BSC alone arm. a Adverse reactions occurring in at least 10% of Erbitux plus BSC arm with a frequency at least 5% greater than that seen in the BSC alone arm. b a Adverse reactions occurring in at least 10% of Erbitux plus BSC arm with a frequency at least 5% greater than that seen in the BSC alone arm. b b Adverse reactions were graded using the NCI CTC, V 2.0. b Adverse reactions were graded using the NCI CTC, V 2.0. b Adverse reactions were graded using the NCI CTC, V 2.0. c Infusion reaction is defined as any event (chills, rigors, dyspnea, tachycardia, bronchospasm, chest tightness, swelling, urticaria, hypotension, flushing, rash, hypertension, nausea, angioedema, pain, sweating, tremors, shaking, drug fever, or other hypersensitivity reaction) recorded by the investigator as infusion-related. 19 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda For current labeling information, please visit https://www.fda.gov/drugsatfda 312 6.2 313 As with all therapeutic proteins, there is potential for immunogenicity. Immunogenic 314 responses to cetuximab were assessed using either a double antigen radiometric assay or 315 an ELISA assay. Due to limitations in assay performance and sampling timing, the 316 incidence of antibody development in patients receiving Erbitux has not been adequately 317 determined. Non-neutralizing anti-cetuximab antibodies were detected in 5% (49 of 318 1001) of evaluable patients without apparent effect on the safety or antitumor activity of 319 Erbitux. 313 As with all therapeutic proteins, there is potential for immunogenicity. Immunogenic 314 responses to cetuximab were assessed using either a double antigen radiometric assay or 315 an ELISA assay. Due to limitations in assay performance and sampling timing, the 316 incidence of antibody development in patients receiving Erbitux has not been adequately 317 determined. Non-neutralizing anti-cetuximab antibodies were detected in 5% (49 of 318 1001) of evaluable patients without apparent effect on the safety or antitumor activity of 319 Erbitux. 320 The incidence of antibody formation is highly dependent on the sensitivity and specificity 321 of the assay. Additionally, the observed incidence of antibody (including neutralizing 322 antibody) positivity in an assay may be influenced by several factors including assay 323 methodology, sample handling, timing of sample collection, concomitant medications, 324 and underlying disease. For these reasons, comparison of the incidence of antibodies to 325 Erbitux with the incidence of antibodies to other products may be misleading. 320 The incidence of antibody formation is highly dependent on the sensitivity and specificity 321 of the assay. Additionally, the observed incidence of antibody (including neutralizing 322 antibody) positivity in an assay may be influenced by several factors including assay 323 methodology, sample handling, timing of sample collection, concomitant medications, 324 and underlying disease. For these reasons, comparison of the incidence of antibodies to 325 Erbitux with the incidence of antibodies to other products may be misleading. 307 Erbitux in Combination with Irinotecan 308 The most frequently reported adverse reactions in 354 patients treated with Erbitux plus 309 irinotecan in clinical trials were acneiform rash (88%), asthenia/malaise (73%), diarrhea 310 (72%), and nausea (55%). The most common Grades 3–4 adverse reactions included 311 diarrhea (22%), leukopenia (17%), asthenia/malaise (16%), and acneiform rash (14%). 308 The most frequently reported adverse reactions in 354 patients treated with Erbitux plus 309 irinotecan in clinical trials were acneiform rash (88%), asthenia/malaise (73%), diarrhea 310 (72%), and nausea (55%). The most common Grades 3–4 adverse reactions included 311 diarrhea (22%), leukopenia (17%), asthenia/malaise (16%), and acneiform rash (14%). 308 The most frequently reported adverse reactions in 354 patients treated with Erbitux plus 309 irinotecan in clinical trials were acneiform rash (88%), asthenia/malaise (73%), diarrhea 310 (72%), and nausea (55%). The most common Grades 3–4 adverse reactions included 311 diarrhea (22%), leukopenia (17%), asthenia/malaise (16%), and acneiform rash (14%). 312 6.2 Immunogenicity 313 As with all therapeutic proteins, there is potential for immunogenicity. Immunogenic 314 responses to cetuximab were assessed using either a double antigen radiometric assay or 315 an ELISA assay. Due to limitations in assay performance and sampling timing, the 316 incidence of antibody development in patients receiving Erbitux has not been adequately 317 determined. Non-neutralizing anti-cetuximab antibodies were detected in 5% (49 of 318 1001) of evaluable patients without apparent effect on the safety or antitumor activity of 319 Erbitux. 320 The incidence of antibody formation is highly dependent on the sensitivity and specificity 321 of the assay. Additionally, the observed incidence of antibody (including neutralizing 322 antibody) positivity in an assay may be influenced by several factors including assay 323 methodology, sample handling, timing of sample collection, concomitant medications, 324 and underlying disease. For these reasons, comparison of the incidence of antibodies to 325 Erbitux with the incidence of antibodies to other products may be misleading. 326 6.3 Postmarketing Experience 327 The following adverse reactions have been identified during post-approval use of Erbitux. 328 Because these reactions are reported from a population of uncertain size, it is not always 329 possible to reliably estimate their frequency or establish a causal relationship to drug 330 exposure. 331  Aseptic meningitis 332  Mucosal inflammation 333 7 DRUG INTERACTIONS 334 A drug interaction study was performed in which Erbitux was administered in 335 combination with irinotecan. 307 Erbitux in Combination with Irinotecan There was no evidence of any pharmacokinetic interactions 336 between Erbitux and irinotecan. 326 6.3 327 The following adverse reactions have been identified during post-approval use of Erbitux. 328 Because these reactions are reported from a population of uncertain size, it is not always 329 possible to reliably estimate their frequency or establish a causal relationship to drug 330 exposure. DRUG INTERACTIONS If nursing is interrupted, 360 based on the mean half-life of cetuximab [see Clinical Pharmacology (12.3)], nursing 361 should not be resumed earlier than 60 days following the last dose of Erbitux. 362 8 4 Pediatric Use 340 There are no adequate and well-controlled studies of Erbitux in pregnant women. Based 341 on animal models, EGFR has been implicated in the control of prenatal development and 342 may be essential for normal organogenesis, proliferation, and differentiation in the 343 developing embryo. Human IgG is known to cross the placental barrier; therefore, 344 Erbitux may be transmitted from the mother to the developing fetus, and has the potential 345 to cause fetal harm when administered to pregnant women. Erbitux should be used during 346 pregnancy only if the potential benefit justifies the potential risk to the fetus. 347 Pregnant cynomolgus monkeys were treated weekly with 0.4 to 4 times the recommended 348 human dose of cetuximab (based on body surface area) during the period of 349 organogenesis (gestation day [GD] 20–48). Cetuximab was detected in the amniotic fluid 350 and in the serum of embryos from treated dams at GD 49. No fetal malformations or 351 other teratogenic effects occurred in offspring. However, significant increases in 352 embryolethality and abortions occurred at doses of approximately 1.6 to 4 times the 353 recommended human dose of cetuximab (based on total body surface area). 354 8.3 Nursing Mothers 355 It is not known whether Erbitux is secreted in human milk. IgG antibodies, such as 356 Erbitux, can be excreted in human milk. Because many drugs are excreted in human milk 357 and because of the potential for serious adverse reactions in nursing infants from Erbitux, 358 a decision should be made whether to discontinue nursing or to discontinue the drug, 359 taking into account the importance of the drug to the mother. If nursing is interrupted, 360 based on the mean half-life of cetuximab [see Clinical Pharmacology (12.3)], nursing 361 should not be resumed earlier than 60 days following the last dose of Erbitux. 362 8.4 Pediatric Use 363 The safety and effectiveness of Erbitux in pediatric patients have not been established. 364 The pharmacokinetics of cetuximab, in combination with irinotecan, were evaluated in 365 pediatric patients with refractory solid tumors in an open-label, single-arm, dose-finding 366 study. DRUG INTERACTIONS 334 A drug interaction study was performed in which Erbitux was administered in 335 combination with irinotecan. There was no evidence of any pharmacokinetic interactions 336 between Erbitux and irinotecan. 334 A drug interaction study was performed in which Erbitux was administered in 335 combination with irinotecan. There was no evidence of any pharmacokinetic interactions 336 between Erbitux and irinotecan. 20 Reference ID: 3270603 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 337 8 USE IN SPECIFIC POPULATIONS 338 8.1 Pregnancy 339 Pregnancy Category C 340 There are no adequate and well-controlled studies of Erbitux in pregnant women. Based 341 on animal models, EGFR has been implicated in the control of prenatal development and 342 may be essential for normal organogenesis, proliferation, and differentiation in the 343 developing embryo. Human IgG is known to cross the placental barrier; therefore, 344 Erbitux may be transmitted from the mother to the developing fetus, and has the potential 345 to cause fetal harm when administered to pregnant women. Erbitux should be used during 346 pregnancy only if the potential benefit justifies the potential risk to the fetus. 347 Pregnant cynomolgus monkeys were treated weekly with 0.4 to 4 times the recommended 348 human dose of cetuximab (based on body surface area) during the period of 349 organogenesis (gestation day [GD] 20–48). Cetuximab was detected in the amniotic fluid 350 and in the serum of embryos from treated dams at GD 49. No fetal malformations or 351 other teratogenic effects occurred in offspring. However, significant increases in 352 embryolethality and abortions occurred at doses of approximately 1.6 to 4 times the 353 recommended human dose of cetuximab (based on total body surface area). 354 8.3 Nursing Mothers 355 It is not known whether Erbitux is secreted in human milk. IgG antibodies, such as 356 Erbitux, can be excreted in human milk. Because many drugs are excreted in human milk 357 and because of the potential for serious adverse reactions in nursing infants from Erbitux, 358 a decision should be made whether to discontinue nursing or to discontinue the drug, 359 taking into account the importance of the drug to the mother. DRUG INTERACTIONS Erbitux was administered once-weekly, at doses up to 250 mg/m2, to 27 patients 340 There are no adequate and well-controlled studies of Erbitux in pregnant women. Based 341 on animal models, EGFR has been implicated in the control of prenatal development and 342 may be essential for normal organogenesis, proliferation, and differentiation in the 343 developing embryo. Human IgG is known to cross the placental barrier; therefore, 344 Erbitux may be transmitted from the mother to the developing fetus, and has the potential 345 to cause fetal harm when administered to pregnant women. Erbitux should be used during 346 pregnancy only if the potential benefit justifies the potential risk to the fetus. 347 Pregnant cynomolgus monkeys were treated weekly with 0.4 to 4 times the recommended 348 human dose of cetuximab (based on body surface area) during the period of 349 organogenesis (gestation day [GD] 20–48). Cetuximab was detected in the amniotic fluid 350 and in the serum of embryos from treated dams at GD 49. No fetal malformations or 351 other teratogenic effects occurred in offspring. However, significant increases in 352 embryolethality and abortions occurred at doses of approximately 1.6 to 4 times the 353 recommended human dose of cetuximab (based on total body surface area). 353 recommended human dose of cetuximab (based on total body surface area). 354 8.3 Nursing Mothers 355 It is not known whether Erbitux is secreted in human milk. IgG antibodies, such as 356 Erbitux, can be excreted in human milk. Because many drugs are excreted in human milk 357 and because of the potential for serious adverse reactions in nursing infants from Erbitux, 358 a decision should be made whether to discontinue nursing or to discontinue the drug, 359 taking into account the importance of the drug to the mother. If nursing is interrupted, 360 based on the mean half-life of cetuximab [see Clinical Pharmacology (12.3)], nursing 361 should not be resumed earlier than 60 days following the last dose of Erbitux. 362 8.4 Pediatric Use 363 The safety and effectiveness of Erbitux in pediatric patients have not been established. 364 The pharmacokinetics of cetuximab, in combination with irinotecan, were evaluated in 365 pediatric patients with refractory solid tumors in an open-label, single-arm, dose-finding 366 study. Erbitux was administered once-weekly, at doses up to 250 mg/m2, to 27 patients 354 8.3 355 It is not known whether Erbitux is secreted in human milk. IgG antibodies, such as 356 Erbitux, can be excreted in human milk. Because many drugs are excreted in human milk 357 and because of the potential for serious adverse reactions in nursing infants from Erbitux, 358 a decision should be made whether to discontinue nursing or to discontinue the drug, 359 taking into account the importance of the drug to the mother. If nursing is interrupted, 360 based on the mean half-life of cetuximab [see Clinical Pharmacology (12.3)], nursing 361 should not be resumed earlier than 60 days following the last dose of Erbitux. 363 The safety and effectiveness of Erbitux in pediatric patients have not been established. 364 The pharmacokinetics of cetuximab, in combination with irinotecan, were evaluated in 365 pediatric patients with refractory solid tumors in an open-label, single-arm, dose-finding 366 study. Erbitux was administered once-weekly, at doses up to 250 mg/m2, to 27 patients 363 The safety and effectiveness of Erbitux in pediatric patients have not been established. 364 The pharmacokinetics of cetuximab, in combination with irinotecan, were evaluated in 365 pediatric patients with refractory solid tumors in an open-label, single-arm, dose-finding 366 study. Erbitux was administered once-weekly, at doses up to 250 mg/m2, to 27 patients 21 Reference ID: 3270603 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. y pp y For current labeling information, please visit https://www.fda.gov/drugsatfda 367 ranging from 1 to 12 years old; and in 19 patients ranging from 13 to 18 years old. No 368 new safety signals were identified in pediatric patients. The pharmacokinetic profiles of 369 cetuximab between the two age groups were similar at the 75 and 150 mg/m2 single dose 370 levels. The volume of the distribution appeared to be independent of dose and 371 approximated the vascular space of 2–3 L/m2. Following a single dose of 250 mg/m2, the 372 geometric mean AUC0-inf (CV%) value was 17.7 mgh/mL (34%) in the younger age 373 group (1–12 years, n=9) and 13.4 mgh/mL (38%) in the adolescent group (13–18 years, 374 n=6). The mean half-life of cetuximab was 110 hours (range 69 to 188 hours) for the 375 younger age group, and 82 hours (range 55 to 117 hours) for the adolescent age group. Reference ID: 3270603 376 8.5 377 Of the 1662 patients who received Erbitux with irinotecan, FOLFIRI or Erbitux 378 monotherapy in six studies of advanced colorectal cancer, 588 patients were 65 years of 379 age or older. No overall differences in safety or efficacy were observed between these 380 patients and younger patients. 377 Of the 1662 patients who received Erbitux with irinotecan, FOLFIRI or Erbitux 378 monotherapy in six studies of advanced colorectal cancer, 588 patients were 65 years of 379 age or older. No overall differences in safety or efficacy were observed between these 380 patients and younger patients. 381 Clinical studies of Erbitux conducted in patients with head and neck cancer did not 382 include sufficient number of subjects aged 65 and over to determine whether they 383 respond differently from younger subjects. 381 Clinical studies of Erbitux conducted in patients with head and neck cancer did not 382 include sufficient number of subjects aged 65 and over to determine whether they 381 Clinical studies of Erbitux conducted in patients with head and neck 384 10 385 The maximum single dose of Erbitux administered is 1000 mg/m2 in one patient. No 386 adverse events were reported for this patient. 385 The maximum single dose of Erbitux administered is 1000 mg/m2 in one patient. No 386 adverse events were reported for this patient. 385 The maximum single dose of Erbitux administered is 1000 mg/m2 in one patient. No 386 adverse events were reported for this patient. 401 12.1 Mechanism of Action However, in cells with activating K-Ras somatic mutations, the mutant 415 K-Ras protein is continuously active and appears independent of EGFR regulation. 416 In vitro, cetuximab can mediate antibody-dependent cellular cytotoxicity (ADCC) against 417 certain human tumor types. In vitro assays and in vivo animal studies have shown that 418 cetuximab inhibits the growth and survival of tumor cells that express the EGFR. No 419 anti-tumor effects of cetuximab were observed in human tumor xenografts lacking EGFR 420 expression. The addition of cetuximab to radiation therapy or irinotecan in human tumor 421 xenograft models in mice resulted in an increase in anti-tumor effects compared to 422 radiation therapy or chemotherapy alone. 416 In vitro, cetuximab can mediate antibody-dependent cellular cytotoxicity (ADCC) against 417 certain human tumor types. In vitro assays and in vivo animal studies have shown that 418 cetuximab inhibits the growth and survival of tumor cells that express the EGFR. No 419 anti-tumor effects of cetuximab were observed in human tumor xenografts lacking EGFR 420 expression. The addition of cetuximab to radiation therapy or irinotecan in human tumor 421 xenograft models in mice resulted in an increase in anti-tumor effects compared to 422 radiation therapy or chemotherapy alone. 416 In vitro, cetuximab can mediate antibody-dependent cellular cytotoxicity (ADCC) against 417 certain human tumor types. In vitro assays and in vivo animal studies have shown that 418 cetuximab inhibits the growth and survival of tumor cells that express the EGFR. No 419 anti-tumor effects of cetuximab were observed in human tumor xenografts lacking EGFR 420 expression. The addition of cetuximab to radiation therapy or irinotecan in human tumor 421 xenograft models in mice resulted in an increase in anti-tumor effects compared to 422 radiation therapy or chemotherapy alone. 387 11 DESCRIPTION 388 Erbitux® (cetuximab) is a recombinant, human/mouse chimeric monoclonal antibody that 389 binds specifically to the extracellular domain of the human epidermal growth factor 390 receptor (EGFR). Cetuximab is composed of the Fv regions of a murine anti-EGFR 391 antibody with human IgG1 heavy and kappa light chain constant regions and has an 392 approximate molecular weight of 152 kDa. Cetuximab is produced in mammalian 393 (murine myeloma) cell culture. 394 Erbitux is a sterile, clear, colorless liquid of pH 7.0 to 7.4, which may contain a small 395 amount of easily visible, white, amorphous cetuximab particulates. Erbitux is supplied at 396 a concentration of 2 mg/mL in either 100 mg (50 mL) or 200 mg (100 mL), single-use 388 Erbitux® (cetuximab) is a recombinant, human/mouse chimeric monoclonal antibody that 389 binds specifically to the extracellular domain of the human epidermal growth factor 390 receptor (EGFR). Cetuximab is composed of the Fv regions of a murine anti-EGFR 391 antibody with human IgG1 heavy and kappa light chain constant regions and has an 392 approximate molecular weight of 152 kDa. Cetuximab is produced in mammalian 393 (murine myeloma) cell culture. 394 Erbitux is a sterile, clear, colorless liquid of pH 7.0 to 7.4, which may contain a small 395 amount of easily visible, white, amorphous cetuximab particulates. Erbitux is supplied at 396 a concentration of 2 mg/mL in either 100 mg (50 mL) or 200 mg (100 mL), single-use 394 Erbitux is a sterile, clear, colorless liquid of pH 7.0 to 7.4, which may contain a small 395 amount of easily visible, white, amorphous cetuximab particulates. Erbitux is supplied at 396 a concentration of 2 mg/mL in either 100 mg (50 mL) or 200 mg (100 mL), single-use 394 Erbitux is a sterile, clear, colorless liquid of pH 7.0 to 7.4, which may contain a small 395 amount of easily visible, white, amorphous cetuximab particulates. Erbitux is supplied at 396 a concentration of 2 mg/mL in either 100 mg (50 mL) or 200 mg (100 mL), single-use 22 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 397 vials. 387 11 DESCRIPTION Cetuximab is formulated in a solution with no preservatives, which contains 398 8.48 mg/mL sodium chloride, 1.88 mg/mL sodium phosphate dibasic heptahydrate, 399 0.41 mg/mL sodium phosphate monobasic monohydrate, and Water for Injection, USP. 398 8.48 mg/mL sodium chloride, 1.88 mg/mL sodium phosphate diba 399 0.41 mg/mL sodium phosphate monobasic monohydrate, and Water for Inj 400 12 CLINICAL PHARMACOLOGY 401 12.1 Mechanism of Action 402 The epidermal growth factor receptor (EGFR, HER1, c-ErbB-1) is a transmembrane 403 glycoprotein that is a member of a subfamily of type I receptor tyrosine kinases including 404 EGFR, HER2, HER3, and HER4. The EGFR is constitutively expressed in many normal 405 epithelial tissues, including the skin and hair follicle. Expression of EGFR is also 406 detected in many human cancers including those of the head and neck, colon, and rectum. 407 Cetuximab binds specifically to the EGFR on both normal and tumor cells, and 408 competitively inhibits the binding of epidermal growth factor (EGF) and other ligands, 409 such as transforming growth factor-alpha. In vitro assays and in vivo animal studies have 410 shown that binding of cetuximab to the EGFR blocks phosphorylation and activation of 411 receptor-associated kinases, resulting in inhibition of cell growth, induction of apoptosis, 412 and decreased matrix metalloproteinase and vascular endothelial growth factor 413 production. Signal transduction through the EGFR results in activation of wild-type 414 K-Ras protein. However, in cells with activating K-Ras somatic mutations, the mutant 415 K-Ras protein is continuously active and appears independent of EGFR regulation. 416 In vitro, cetuximab can mediate antibody-dependent cellular cytotoxicity (ADCC) against 417 certain human tumor types. In vitro assays and in vivo animal studies have shown that 418 cetuximab inhibits the growth and survival of tumor cells that express the EGFR. No 419 anti-tumor effects of cetuximab were observed in human tumor xenografts lacking EGFR 420 expression. The addition of cetuximab to radiation therapy or irinotecan in human tumor 421 xenograft models in mice resulted in an increase in anti-tumor effects compared to 422 radiation therapy or chemotherapy alone. 423 12.2 Pharmacodynamics 424 Effects on Electrocardiogram (ECG) 400 12 CLINICAL PHARMACOLOGY 401 12.1 Mechanism of Action 401 12.1 Mechanism of Action 402 The epidermal growth factor receptor (EGFR, HER1, c-ErbB-1) is a transmembrane 403 glycoprotein that is a member of a subfamily of type I receptor tyrosine kinases including 404 EGFR, HER2, HER3, and HER4. The EGFR is constitutively expressed in many normal 405 epithelial tissues, including the skin and hair follicle. Expression of EGFR is also 406 detected in many human cancers including those of the head and neck, colon, and rectum. 407 Cetuximab binds specifically to the EGFR on both normal and tumor cells, and 408 competitively inhibits the binding of epidermal growth factor (EGF) and other ligands, 409 such as transforming growth factor-alpha. In vitro assays and in vivo animal studies have 410 shown that binding of cetuximab to the EGFR blocks phosphorylation and activation of 411 receptor-associated kinases, resulting in inhibition of cell growth, induction of apoptosis, 412 and decreased matrix metalloproteinase and vascular endothelial growth factor 413 production. Signal transduction through the EGFR results in activation of wild-type 414 K-Ras protein. However, in cells with activating K-Ras somatic mutations, the mutant 415 K-Ras protein is continuously active and appears independent of EGFR regulation. 407 Cetuximab binds specifically to the EGFR on both normal and tumor cells, and 408 competitively inhibits the binding of epidermal growth factor (EGF) and other ligands, 409 such as transforming growth factor-alpha. In vitro assays and in vivo animal studies have 410 shown that binding of cetuximab to the EGFR blocks phosphorylation and activation of 411 receptor-associated kinases, resulting in inhibition of cell growth, induction of apoptosis, 412 and decreased matrix metalloproteinase and vascular endothelial growth factor 413 production. Signal transduction through the EGFR results in activation of wild-type 414 K-Ras protein. However, in cells with activating K-Ras somatic mutations, the mutant 415 K-Ras protein is continuously active and appears independent of EGFR regulation. 407 Cetuximab binds specifically to the EGFR on both normal and tumor cells, and 408 competitively inhibits the binding of epidermal growth factor (EGF) and other ligands, 409 such as transforming growth factor-alpha. In vitro assays and in vivo animal studies have 410 shown that binding of cetuximab to the EGFR blocks phosphorylation and activation of 411 receptor-associated kinases, resulting in inhibition of cell growth, induction of apoptosis, 412 and decreased matrix metalloproteinase and vascular endothelial growth factor 413 production. Signal transduction through the EGFR results in activation of wild-type 414 K-Ras protein. 431 12.3 432 Erbitux administered as monotherapy or in combination with concomitant chemotherapy 433 or radiation therapy exhibits nonlinear pharmacokinetics. The area under the 434 concentration time curve (AUC) increased in a greater than dose proportional manner 435 while clearance of cetuximab decreased from 0.08 to 0.02 L/h/m2 as the dose increased 436 from 20 to 200 mg/m2, and at doses >200 mg/m2, it appeared to plateau. The volume of 437 the distribution for cetuximab appeared to be independent of dose and approximated the 438 vascular space of 2–3 L/m2 . 439 Following the recommended dose regimen (400 mg/m2 initial dose; 250 mg/m2 weekly 440 dose), concentrations of cetuximab reached steady-state levels by the third weekly 441 infusion with mean peak and trough concentrations across studies ranging from 168 to 442 235 and 41 to 85 g/mL, respectively. The mean half-life of cetuximab was 443 approximately 112 hours (range 63–230 hours). The pharmacokinetics of cetuximab were 444 similar in patients with SCCHN and those with colorectal cancer. 439 Following the recommended dose regimen (400 mg/m2 initial dose; 250 mg/m2 weekly 440 dose), concentrations of cetuximab reached steady-state levels by the third weekly 441 infusion with mean peak and trough concentrations across studies ranging from 168 to 442 235 and 41 to 85 g/mL, respectively. The mean half-life of cetuximab was 443 approximately 112 hours (range 63–230 hours). The pharmacokinetics of cetuximab were 444 similar in patients with SCCHN and those with colorectal cancer. 439 Following the recommended dose regimen (400 mg/m2 initial dose; 250 mg/m2 weekly 440 dose), concentrations of cetuximab reached steady-state levels by the third weekly 441 infusion with mean peak and trough concentrations across studies ranging from 168 to 442 235 and 41 to 85 g/mL, respectively. The mean half-life of cetuximab was 443 approximately 112 hours (range 63–230 hours). The pharmacokinetics of cetuximab were 444 similar in patients with SCCHN and those with colorectal cancer. 439 Following the recommended dose regimen (400 mg/m2 initial dose; 250 mg/m2 weekly 440 dose), concentrations of cetuximab reached steady-state levels by the third weekly 441 infusion with mean peak and trough concentrations across studies ranging from 168 to 442 235 and 41 to 85 g/mL, respectively. The mean half-life of cetuximab was 443 approximately 112 hours (range 63–230 hours). The pharmacokinetics of cetuximab were 444 similar in patients with SCCHN and those with colorectal cancer. 431 12.3 445 Erbitux had an approximately 22% (90% confidence interval; 6%, 38%) higher systemic 446 exposure relative to the EU-approved cetuximab used in Studies 2 and 4 based on a 447 population pharmacokinetic analysis. [See Clinical Studies (14.1).] 446 exposure relative to the EU approved cetuximab used in Studies 2 and 4 based on a 447 population pharmacokinetic analysis. [See Clinical Studies (14.1).] 447 population pharmacokinetic analysis. [See Clinical Studies (14.1).] 424 Effects on Electrocardiogram (ECG) 445 Erbitux had an approximately 22% (90% confidence interval; 6%, 38%) higher systemic 446 exposure relative to the EU-approved cetuximab used in Studies 2 and 4 based on a 447 population pharmacokinetic analysis. [See Clinical Studies (14.1).] 448 13 NONCLINICAL TOXICOLOGY 449 13.1 Carcinogenesis, Mutagenesis, Impairment of Fertility 450 Long-term animal studies have not been performed to test cetuximab for carcinogenic 451 potential, and no mutagenic or clastogenic potential of cetuximab was observed in the 452 Salmonella-Escherichia coli (Ames) assay or in the in vivo rat micronucleus test. 453 Menstrual cyclicity was impaired in female cynomolgus monkeys receiving weekly doses 454 of 0.4 to 4 times the human dose of cetuximab (based on total body surface area). 455 Cetuximab-treated animals exhibited increased incidences of irregular or absent cycles, 428 large changes in the mean QT interval of >20 ms from baseline were detected in the trial 429 based on the Fridericia correction method. A small increase in the mean QTc interval of 430 <10 ms cannot be excluded because of the limitations in the trial design. 424 Effects on Electrocardiogram (ECG) 425 The effect of cetuximab on QT interval was evaluated in an open-label, single-arm, 426 monotherapy trial in 37 subjects with advanced malignancies who received an initial dose 427 of 400 mg/m2, followed by weekly infusions of 250 mg/m2 for a total of 5 weeks. No 5 e e ect o cetu ab o Q te va was eva uated a ope abe , s g e a , 426 monotherapy trial in 37 subjects with advanced malignancies who received an initial dose 2 2 427 of 400 mg/m2, followed by weekly infusions of 250 mg/m2 for a total of 5 weeks. No 23 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. y pp y For current labeling information, please visit https://www.fda.gov/drugsatfda 428 large changes in the mean QT interval of >20 ms from baseline were detected in the trial 429 based on the Fridericia correction method. A small increase in the mean QTc interval of 430 <10 ms cannot be excluded because of the limitations in the trial design. 431 12.3 Pharmacokinetics 432 Erbitux administered as monotherapy or in combination with concomitant chemotherapy 433 or radiation therapy exhibits nonlinear pharmacokinetics. The area under the 434 concentration time curve (AUC) increased in a greater than dose proportional manner 435 while clearance of cetuximab decreased from 0.08 to 0.02 L/h/m2 as the dose increased 436 from 20 to 200 mg/m2, and at doses >200 mg/m2, it appeared to plateau. The volume of 437 the distribution for cetuximab appeared to be independent of dose and approximated the 438 vascular space of 2–3 L/m2 . 439 Following the recommended dose regimen (400 mg/m2 initial dose; 250 mg/m2 weekly 440 dose), concentrations of cetuximab reached steady-state levels by the third weekly 441 infusion with mean peak and trough concentrations across studies ranging from 168 to 442 235 and 41 to 85 g/mL, respectively. The mean half-life of cetuximab was 443 approximately 112 hours (range 63–230 hours). The pharmacokinetics of cetuximab were 444 similar in patients with SCCHN and those with colorectal cancer. 449 13.1 Carcinogenesis, Mutagenesis, Impairment of Fertility At the highest dose level, the epithelial mucosa of the nasal 467 passage, esophagus, and tongue were similarly affected, and degenerative changes in the 468 renal tubular epithelium occurred. Deaths due to sepsis were observed in 50% (5/10) of 469 the animals at the highest dose level beginning after approximately 13 weeks of 470 treatment. 459 (ie, serum testosterone levels and analysis of sperm counts, viability, and motility) as 460 compared to control male monkeys. It is not known if cetuximab can impair fertility in 461 humans. 14 472 Studies 2 and 4 were conducted outside the U.S. using an EU-approved cetuximab as the 473 clinical trial material. Erbitux provides approximately 22% higher exposure relative to 474 the EU-approved cetuximab used in Studies 2 and 4; these pharmacokinetic data, together 475 with the results of Studies 2, 4, and other clinical trial data establish the efficacy of 476 Erbitux at the recommended dose in SCCHN and mCRC [see Clinical Pharmacology 477 (12.3)]. 472 Studies 2 and 4 were conducted outside the U.S. using an EU-approved cetuximab as the 473 clinical trial material. Erbitux provides approximately 22% higher exposure relative to 474 the EU-approved cetuximab used in Studies 2 and 4; these pharmacokinetic data, together 475 with the results of Studies 2, 4, and other clinical trial data establish the efficacy of 476 Erbitux at the recommended dose in SCCHN and mCRC [see Clinical Pharmacology 477 (12 3)] 462 13.2 463 In cynomolgus monkeys, cetuximab, when administered at doses of approximately 0.4 to 464 4 times the weekly human exposure (based on total body surface area), resulted in 465 dermatologic findings, including inflammation at the injection site and desquamation of 466 the external integument. At the highest dose level, the epithelial mucosa of the nasal 467 passage, esophagus, and tongue were similarly affected, and degenerative changes in the 468 renal tubular epithelium occurred. Deaths due to sepsis were observed in 50% (5/10) of 469 the animals at the highest dose level beginning after approximately 13 weeks of 470 treatment. Reference ID: 3270603 449 13.1 Carcinogenesis, Mutagenesis, Impairment of Fertility 450 Long-term animal studies have not been performed to test cetuximab for carcinogenic 451 potential, and no mutagenic or clastogenic potential of cetuximab was observed in the 452 Salmonella-Escherichia coli (Ames) assay or in the in vivo rat micronucleus test. 453 Menstrual cyclicity was impaired in female cynomolgus monkeys receiving weekly doses 454 of 0.4 to 4 times the human dose of cetuximab (based on total body surface area). 455 Cetuximab-treated animals exhibited increased incidences of irregular or absent cycles, 456 as compared to control animals. These effects were initially noted beginning week 25 of 457 cetuximab treatment and continued through the 6-week recovery period. In this same 458 study, there were no effects of cetuximab treatment on measured male fertility parameters 450 Long-term animal studies have not been performed to test cetuximab for carcinogenic 451 potential, and no mutagenic or clastogenic potential of cetuximab was observed in the 452 Salmonella-Escherichia coli (Ames) assay or in the in vivo rat micronucleus test. 453 Menstrual cyclicity was impaired in female cynomolgus monkeys receiving weekly doses 454 of 0.4 to 4 times the human dose of cetuximab (based on total body surface area). 455 Cetuximab-treated animals exhibited increased incidences of irregular or absent cycles, 456 as compared to control animals. These effects were initially noted beginning week 25 of 457 cetuximab treatment and continued through the 6-week recovery period. In this same 458 study, there were no effects of cetuximab treatment on measured male fertility parameters 24 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda y pp y abeling information, please visit https://www.fda.gov/drugsatfda 459 (ie, serum testosterone levels and analysis of sperm counts, viability, and motility) as 460 compared to control male monkeys. It is not known if cetuximab can impair fertility in 461 humans. 462 13.2 Animal Pharmacology and/or Toxicology 463 In cynomolgus monkeys, cetuximab, when administered at doses of approximately 0.4 to 464 4 times the weekly human exposure (based on total body surface area), resulted in 465 dermatologic findings, including inflammation at the injection site and desquamation of 466 the external integument. 478 479 14.1 Squamous Cell Carcinoma of the Head and Neck (SCCHN) 501 Patients with no prior therapy for recurrent locoregional disease or metastatic SCCHN 502 were randomized (1:1) to receive EU-approved cetuximab plus cisplatin or carboplatin 503 and 5-FU, or cisplatin or carboplatin and 5-FU alone. Choice of cisplatin or carboplatin 504 was at the discretion of the treating physician. Stratification factors were 491 Of the 424 randomized patients, the median age was 57 years, 80% were male, 83% were 492 Caucasian, and 90% had baseline Karnofsky performance status 80. There were 493 258 patients enrolled in U.S. sites (61%). Sixty percent of patients had oropharyngeal, 494 25% laryngeal, and 15% hypopharyngeal primary tumors; 28% had AJCC T4 tumor 495 stage. Fifty-six percent of the patients received radiation therapy with concomitant boost, 496 26% received once-daily regimen, and 18% twice-daily regimen. 491 Of the 424 randomized patients, the median age was 57 years, 80% were male, 83% were 492 Caucasian, and 90% had baseline Karnofsky performance status 80. There were 493 258 patients enrolled in U.S. sites (61%). Sixty percent of patients had oropharyngeal, 494 25% laryngeal, and 15% hypopharyngeal primary tumors; 28% had AJCC T4 tumor 495 stage. Fifty-six percent of the patients received radiation therapy with concomitant boost, 496 26% received once-daily regimen, and 18% twice-daily regimen. 497 The main outcome measure of this trial was duration of locoregional control. Overall 498 survival was also assessed. Results are presented in Table 6. Table 6: Study 1: Clinical Efficacy in Locoregionally Advanced SCCHN Erbitux + Radiation (n=211) Radiation Alone (n=213) Hazard Ratio (95% CIa) Stratified Log-rank p-value Locoregional Control Median duration (months) 24.4 14.9 0.68 (0.52–0.89) 0.005 Overall Survival Median duration (months) 49.0 29.3 0.74 (0.57–0.97) 0.03 a CI = confidence interval 499 Study 2 was an open-label, randomized, multicenter, controlled trial of 442 patients with 500 recurrent locoregional disease or metastatic SCCHN. Table 6: Study 1: Clinical Efficacy in Locoregionally Advanced Study 1: Clinical Efficacy in Locoregionally Advanced SCCHN 499 Study 2 was an open-label, randomized, multicenter, controlled trial of 442 patients with 500 recurrent locoregional disease or metastatic SCCHN. 501 Patients with no prior therapy for recurrent locoregional disease or metastatic SCCHN 502 were randomized (1:1) to receive EU-approved cetuximab plus cisplatin or carboplatin 503 and 5-FU, or cisplatin or carboplatin and 5-FU alone. Choice of cisplatin or carboplatin 504 was at the discretion of the treating physician. 478 479 14.1 Squamous Cell Carcinoma of the Head and Neck (SCCHN) 480 Study 1 was a randomized, multicenter, controlled trial of 424 patients with locally or 481 regionally advanced SCCHN. Patients with Stage III/IV SCCHN of the oropharynx, 482 hypopharynx, or larynx with no prior therapy were randomized (1:1) to receive either 483 Erbitux plus radiation therapy or radiation therapy alone. Stratification factors were 484 Karnofsky performance status (60–80 versus 90–100), nodal stage (N0 versus N+), tumor 485 stage (T1–3 versus T4 using American Joint Committee on Cancer 1998 staging criteria), 486 and radiation therapy fractionation (concomitant boost versus once-daily versus twice 487 daily). Radiation therapy was administered for 6–7 weeks as once-daily, twice-daily, or 488 concomitant boost. Erbitux was administered as a 400 mg/m2 initial dose beginning one 489 week prior to initiation of radiation therapy, followed by 250 mg/m2 weekly administered 490 1 hour prior to radiation therapy for the duration of radiation therapy (6–7 weeks). 25 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 491 Of the 424 randomized patients, the median age was 57 years, 80% were male, 83% were 492 Caucasian, and 90% had baseline Karnofsky performance status 80. There were 493 258 patients enrolled in U.S. sites (61%). Sixty percent of patients had oropharyngeal, 494 25% laryngeal, and 15% hypopharyngeal primary tumors; 28% had AJCC T4 tumor 495 stage. Fifty-six percent of the patients received radiation therapy with concomitant boost, 496 26% received once-daily regimen, and 18% twice-daily regimen. 497 The main outcome measure of this trial was duration of locoregional control. Overall 498 survival was also assessed. Results are presented in Table 6. Table 6: Study 1: Clinical Efficacy in Locoregionally Advanced SCCHN Erbitux + Radiation (n=211) Radiation Alone (n=213) Hazard Ratio (95% CIa) Stratified Log-rank p-value Locoregional Control Median duration (months) 24.4 14.9 0.68 (0.52–0.89) 0.005 Overall Survival Median duration (months) 49.0 29.3 0.74 (0.57–0.97) 0.03 a CI = confidence interval 499 Study 2 was an open-label, randomized, multicenter, controlled trial of 442 patients with 500 recurrent locoregional disease or metastatic SCCHN. 478 479 14.1 Squamous Cell Carcinoma of the Head and Neck (SCCHN) Table 7: Study 2: Clinical Efficacy in Recurrent Locoregional Disease or Metastatic SCCHN EU-Approved Cetuximab + Platinum-based Platinum-based Stratified Hazard Ratio Therapy + 5-FU Therapy + 5-FU Log-rank a (n=222) (n=220) (95% CI ) p-value Overall Survival Median duration (months) 10.1 7.4 0.80 (0.64, 0.98) 0.034 Progression-free Survival Median duration (months) 5.5 3.3 0.57 (0.46, 0.72) <0.0001 EU-Approved Cetuximab + Platinum-based Therapy + 5-FU (n=222) Platinum-based Therapy + 5-FU (n=220) Odds Ratio (95% CIa) CMHb test p-value Objective Response Rate 35.6% 19.5% 2.33 (1.50, 3.60) 0.0001 a CI = confidence interval b CMH = Cochran-Mantel-Haenszel 517 Of the 442 randomized patients, the median age was 57 years, 90% were male, 98% were 518 Caucasian, and 88% had baseline Karnofsky performance status 80. Thirty-four percent 519 of patients had oropharyngeal, 25% laryngeal, 20% oral cavity, and 14% hypopharyngeal 520 primary tumors. Fifty-three percent of patients had recurrent locoregional disease only 521 and 47% had metastatic disease. Fifty-eight percent had AJCC Stage IV disease and 522 21% had Stage III disease. Sixty-four percent of patients received cisplatin therapy and 523 34% received carboplatin as initial therapy. Approximately fifteen percent of the patients 524 in the cisplatin alone arm switched to carboplatin during the treatment period. 525 The main outcome measure of this trial was overall survival. Results are presented in 526 Table 7 and Figure 1. Table 7: Study 2: Clinical Efficacy in Recurrent Locoregional Disease or Metastatic SCCHN EU-Approved Cetuximab + Platinum-based Platinum-based Stratified Hazard Ratio Therapy + 5-FU Therapy + 5-FU Log-rank a (n=222) (n=220) (95% CI ) p-value Overall Survival Median duration (months) 10.1 7.4 0.80 (0.64, 0.98) 0.034 Progression-free Survival Median duration (months) 5.5 3.3 0.57 (0.46, 0.72) <0.0001 EU-Approved Cetuximab + Platinum-based Therapy + 5-FU (n=222) Platinum-based Therapy + 5-FU (n=220) Odds Ratio (95% CIa) CMHb test p-value Objective Response Rate 35.6% 19.5% 2.33 (1.50, 3.60) 0.0001 a CI = confidence interval b CMH = Cochran-Mantel-Haenszel 517 Of the 442 randomized patients, the median age was 57 years, 90% were male, 98% were 518 Caucasian, and 88% had baseline Karnofsky performance status 80. Thirty-four percent 519 of patients had oropharyngeal, 25% laryngeal, 20% oral cavity, and 14% hypopharyngeal 520 primary tumors. Fifty-three percent of patients had recurrent locoregional disease only 521 and 47% had metastatic disease. Fifty-eight percent had AJCC Stage IV disease and 522 21% had Stage III disease. 478 479 14.1 Squamous Cell Carcinoma of the Head and Neck (SCCHN) Stratification factors were 505 Karnofsky performance status (<80 versus 80) and previous chemotherapy. Cisplatin 506 (100 mg/m2 , Day 1) or carboplatin (AUC 5, Day 1) plus intravenous 5-FU 507 (1000 mg/m2/day, Days 1–4) were administered every 3 weeks (1 cycle) for a maximum 508 of 6 cycles in the absence of disease progression or unacceptable toxicity. Cetuximab was 509 administered at a 400 mg/m2 initial dose, followed by a 250 mg/m2 weekly dose in 510 combination with chemotherapy. Patients demonstrating at least stable disease on 511 cetuximab in combination with chemotherapy were to continue cetuximab monotherapy 512 at 250 mg/m2 weekly, in the absence of disease progression or unacceptable toxicity after 513 completion of 6 planned courses of platinum-based therapy. For patients where treatment 514 was delayed because of the toxic effects of chemotherapy, weekly cetuximab was 515 continued. If chemotherapy was discontinued for toxicity, cetuximab could be continued 516 as monotherapy until disease progression or unacceptable toxicity. 26 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. y pp y For current labeling information, please visit https://www.fda.gov/drugsatfda 517 Of the 442 randomized patients, the median age was 57 years, 90% were male, 98% were 518 Caucasian, and 88% had baseline Karnofsky performance status 80. Thirty-four percent 519 of patients had oropharyngeal, 25% laryngeal, 20% oral cavity, and 14% hypopharyngeal 520 primary tumors. Fifty-three percent of patients had recurrent locoregional disease only 521 and 47% had metastatic disease. Fifty-eight percent had AJCC Stage IV disease and 522 21% had Stage III disease. Sixty-four percent of patients received cisplatin therapy and 523 34% received carboplatin as initial therapy. Approximately fifteen percent of the patients 524 in the cisplatin alone arm switched to carboplatin during the treatment period. 525 The main outcome measure of this trial was overall survival. Results are presented in 526 Table 7 and Figure 1. 478 479 14.1 Squamous Cell Carcinoma of the Head and Neck (SCCHN) Sixty-four percent of patients received cisplatin therapy and 523 34% received carboplatin as initial therapy. Approximately fifteen percent of the patients 524 in the cisplatin alone arm switched to carboplatin during the treatment period. Table 7: Study 2: Clinical Efficacy in Recurrent Locoregional Disease or Metastatic SCCHN EU-Approved Cetuximab + Platinum-based Platinum-based Stratified Hazard Ratio Therapy + 5-FU Therapy + 5-FU Log-rank a (n=222) (n=220) (95% CI ) p-value Overall Survival Median duration (months) 10.1 7.4 0.80 (0.64, 0.98) 0.034 Progression-free Survival Median duration (months) 5.5 3.3 0.57 (0.46, 0.72) <0.0001 EU-Approved Cetuximab + Platinum-based Therapy + 5-FU (n=222) Platinum-based Therapy + 5-FU (n=220) Odds Ratio (95% CIa) CMHb test p-value Objective Response Rate 35.6% 19.5% 2.33 (1.50, 3.60) 0.0001 a CI = confidence interval b CMH = Cochran-Mantel-Haenszel Study 2: Clinical Efficacy in Recurrent Locoregional Disease or Metastatic SCCHN 27 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 527 Figure 1: Kaplan-Meier Curve for Overall Survival in Patients with 528 Recurrent Locoregional Disease or Metastatic Squamous Cell 529 Carcinoma of the Head and Neck This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 527 Figure 1: Kaplan-Meier Curve for Overall Survival in Patients with 528 Recurrent Locoregional Disease or Metastatic Squamous Cell 529 Carcinoma of the Head and Neck graph 531 CT = Platinum-based therapy with 5-FU 532 CET = EU-approved cetuximab 533 In exploratory subgroup analyses of Study 2 by initial platinum therapy (cisplatin or 534 carboplatin), for patients (N=284) receiving cetuximab plus cisplatin with 5-FU 535 compared to cisplatin with 5-FU alone, the difference in median overall survival was 536 3.3 months (10.6 versus 7.3 months, respectively; HR 0.71; 95% CI 0.54, 0.93). The 537 difference in median progression-free survival was 2.1 months (5.6 versus 3.5 months, 538 respectively; HR 0.55; 95% CI 0.41, 0.73). The objective response rate was 39% and 539 23%, respectively (OR 2.18; 95% CI 1.29, 3.69). For patients (N=149) receiving 540 cetuximab plus carboplatin with 5-FU compared to carboplatin with 5-FU alone, the 541 difference in median overall survival was 1.4 months (9.7 versus 8.3 months; HR 0.99; 542 95% CI 0.69, 1.43). The difference in median progression-free survival was 1.7 months 543 (4.8 versus 3.1 months, respectively; HR 0.61; 95% CI 0.42, 0.89). 478 479 14.1 Squamous Cell Carcinoma of the Head and Neck (SCCHN) The objective 544 response rate was 30% and 15%, respectively (OR 2.45; 95% CI 1.10, 5.46). y pp y For current labeling information, please visit https://www.fda.gov/drugsatfda y pp y For current labeling information, please visit https://www.fda.gov/drugsatfda 527 Figure 1: Kaplan-Meier Curve for Overall Survival in Patients with 528 Recurrent Locoregional Disease or Metastatic Squamous Cell 529 Carcinoma of the Head and Neck 527 Figure 1: Kaplan-Meier Curve for Overall Survival in Patients with 528 Recurrent Locoregional Disease or Metastatic Squamous Cell 529 Carcinoma of the Head and Neck graph 531 CT = Platinum-based therapy with 5-FU 532 CET = EU-approved cetuximab graph 531 CT = Platinum-based therapy with 5-FU 532 CET = EU-approved cetuximab 533 In exploratory subgroup analyses of Study 2 by initial platinum therapy (cisplatin or 534 carboplatin), for patients (N=284) receiving cetuximab plus cisplatin with 5-FU 535 compared to cisplatin with 5-FU alone, the difference in median overall survival was 536 3.3 months (10.6 versus 7.3 months, respectively; HR 0.71; 95% CI 0.54, 0.93). The 537 difference in median progression-free survival was 2.1 months (5.6 versus 3.5 months, 538 respectively; HR 0.55; 95% CI 0.41, 0.73). The objective response rate was 39% and 539 23%, respectively (OR 2.18; 95% CI 1.29, 3.69). For patients (N=149) receiving 540 cetuximab plus carboplatin with 5-FU compared to carboplatin with 5-FU alone, the 541 difference in median overall survival was 1.4 months (9.7 versus 8.3 months; HR 0.99; 542 95% CI 0.69, 1.43). The difference in median progression-free survival was 1.7 months 543 (4.8 versus 3.1 months, respectively; HR 0.61; 95% CI 0.42, 0.89). The objective 544 response rate was 30% and 15%, respectively (OR 2.45; 95% CI 1.10, 5.46). 533 In exploratory subgroup analyses of Study 2 by initial platinum therapy (cisplatin or 534 carboplatin), for patients (N=284) receiving cetuximab plus cisplatin with 5-FU 535 compared to cisplatin with 5-FU alone, the difference in median overall survival was 536 3.3 months (10.6 versus 7.3 months, respectively; HR 0.71; 95% CI 0.54, 0.93). The 537 difference in median progression-free survival was 2.1 months (5.6 versus 3.5 months, 538 respectively; HR 0.55; 95% CI 0.41, 0.73). The objective response rate was 39% and 539 23%, respectively (OR 2.18; 95% CI 1.29, 3.69). 478 479 14.1 Squamous Cell Carcinoma of the Head and Neck (SCCHN) For patients (N=149) receiving 540 cetuximab plus carboplatin with 5-FU compared to carboplatin with 5-FU alone, the 541 difference in median overall survival was 1.4 months (9.7 versus 8.3 months; HR 0.99; 542 95% CI 0.69, 1.43). The difference in median progression-free survival was 1.7 months 543 (4.8 versus 3.1 months, respectively; HR 0.61; 95% CI 0.42, 0.89). The objective 544 response rate was 30% and 15%, respectively (OR 2.45; 95% CI 1.10, 5.46). 545 Study 3 was a single-arm, multicenter clinical trial in 103 patients with recurrent or 546 metastatic SCCHN. All patients had documented disease progression within 30 days of a 547 platinum-based chemotherapy regimen. Patients received a 20-mg test dose of Erbitux on 28 Reference ID: 3270603 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda current labeling information, please visit https://www.fda.gov/drugsatfda 548 Day 1, followed by a 400 mg/m2 initial dose, and 250 mg/m2 weekly until disease 549 progression or unacceptable toxicity. 550 The median age was 57 years, 82% were male, 100% Caucasian, and 62% had a 551 Karnofsky performance status of 80. 552 The objective response rate was 13% (95% confidence interval 7%–21%). Median 553 duration of response was 5.8 months (range 1.2–5.8 months). 552 The objective response rate was 13% (95% confidence interval 7%–21%). Median 553 duration of response was 5.8 months (range 1.2–5.8 months). 553 duration of response was 5.8 months (range 1.2–5.8 months). 554 14.2 Colorectal Cancer 555 Erbitux Clinical Trials in K-Ras Mutation-negative (Wild-type), EGFR 556 expressing, Metastatic Colorectal Cancer 557 Study 4 was a randomized, open-label, multicenter, study of 1217 patients with EGFR 558 expressing, metastatic colorectal cancer. Patients were randomized (1:1) to receive either 559 EU-approved cetuximab in combination with FOLFIRI or FOLFIRI alone as first-line 560 treatment. Stratification factors were Eastern Cooperative Oncology Group (ECOG) 561 performance status (0 and 1 versus 2) and region (sites in Western Europe versus Eastern 562 Europe versus other). 563 FOLFIRI regimen included 14-day cycles of irinotecan (180 mg/m2 administered 564 intravenously on Day 1), folinic acid (400 mg/m2 [racemic] or 200 mg/m2 [L-form] 565 administered intravenously on Day 1), and 5-FU (400 mg/m2 bolus on Day 1 followed by 566 2400 mg/m2 as a 46-hour continuous infusion). 478 479 14.1 Squamous Cell Carcinoma of the Head and Neck (SCCHN) Cetuximab was administered as a 567 400 mg/m2 initial dose on Day 1, Week 1, followed by 250 mg/m2 weekly administered 568 1 hour prior to chemotherapy. Study treatment continued until disease progression or 569 unacceptable toxicity occurred. 570 Of the 1217 randomized patients, the median age was 61 years, 60% were male, 86% 571 were Caucasian, and 96% had a baseline ECOG performance status 0–1, 60% had 572 primary tumor localized in colon, 84% had 1–2 metastatic sites and 20% had received 573 prior adjuvant and/or neoadjuvant chemotherapy. Demographics and baseline 574 characteristics were similar between study arms. 575 K-Ras mutation status was available for 1079/1217 (89%) of the patients: 676 (63%) 576 patients had K-Ras mutation-negative (wild-type) tumors and 403 (37%) patients had 577 K-Ras mutation-positive tumors where testing assessed for the following somatic Reference ID: 3270603 555 Erbitux Clinical Trials in K-Ras Mutation-negative (Wild-type), EGFR 556 expressing, Metastatic Colorectal Cancer 557 Study 4 was a randomized, open-label, multicenter, study of 1217 patients with EGFR 558 expressing, metastatic colorectal cancer. Patients were randomized (1:1) to receive either 559 EU-approved cetuximab in combination with FOLFIRI or FOLFIRI alone as first-line 560 treatment. Stratification factors were Eastern Cooperative Oncology Group (ECOG) 561 performance status (0 and 1 versus 2) and region (sites in Western Europe versus Eastern 562 Europe versus other). 557 Study 4 was a randomized, open-label, multicenter, study of 1217 patients with EGFR 558 expressing, metastatic colorectal cancer. Patients were randomized (1:1) to receive either 559 EU-approved cetuximab in combination with FOLFIRI or FOLFIRI alone as first-line 560 treatment. Stratification factors were Eastern Cooperative Oncology Group (ECOG) 561 performance status (0 and 1 versus 2) and region (sites in Western Europe versus Eastern 562 Europe versus other). 563 FOLFIRI regimen included 14-day cycles of irinotecan (180 mg/m2 administered 564 intravenously on Day 1), folinic acid (400 mg/m2 [racemic] or 200 mg/m2 [L-form] 565 administered intravenously on Day 1), and 5-FU (400 mg/m2 bolus on Day 1 followed by 566 2400 mg/m2 as a 46-hour continuous infusion). Cetuximab was administered as a 567 400 mg/m2 initial dose on Day 1, Week 1, followed by 250 mg/m2 weekly administered 568 1 hour prior to chemotherapy. Study treatment continued until disease progression or 569 unacceptable toxicity occurred. 570 Of the 1217 randomized patients, the median age was 61 years, 60% were male, 86% 571 were Caucasian, and 96% had a baseline ECOG performance status 0–1, 60% had 572 primary tumor localized in colon, 84% had 1–2 metastatic sites and 20% had received 573 prior adjuvant and/or neoadjuvant chemotherapy. Demographics and baseline 574 characteristics were similar between study arms. 570 Of the 1217 randomized patients, the median age was 61 years, 60% were male, 86% 571 were Caucasian, and 96% had a baseline ECOG performance status 0–1, 60% had 572 primary tumor localized in colon, 84% had 1–2 metastatic sites and 20% had received 573 prior adjuvant and/or neoadjuvant chemotherapy. Demographics and baseline 574 characteristics were similar between study arms. 555 Erbitux Clinical Trials in K-Ras Mutation-negative (Wild-type), EGFR 556 expressing, Metastatic Colorectal Cancer 575 K-Ras mutation status was available for 1079/1217 (89%) of the patients: 676 (63%) 576 patients had K-Ras mutation-negative (wild-type) tumors and 403 (37%) patients had 577 K-Ras mutation-positive tumors where testing assessed for the following somatic 575 K-Ras mutation status was available for 1079/1217 (89%) of the patients: 676 (63%) 576 patients had K-Ras mutation-negative (wild-type) tumors and 403 (37%) patients had 577 K-Ras mutation-positive tumors where testing assessed for the following somatic 29 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 578 mutations in codons 12 and 13 (exon 2): G12A, G12D, G12R, G12C, G12S, G12V, 579 G13D [see Warnings and Precautions (5.7)]. 580 Baseline characteristics and demographics in the K-Ras mutation-negative (wild-type) 581 subset were similar to that seen in the overall population [see Warnings and Precautions 582 (5.7)]. 583 The main outcome measure of this trial was progression-free survival assessed by an 584 independent review committee (IRC). Overall survival and response rate were also 585 assessed. A statistically significant improvement in PFS was observed for the cetuximab 586 plus FOLFIRI arm compared with the FOLFIRI arm (median PFS 8.9 vs. 8.1 months, 587 HR 0.85 [95% CI 0.74, 0.99], p-value=0.036). Overall survival was not significantly 588 different at the planned, final analysis based on 838 events [HR=0.93, 95% CI (0.8, 1.1), 589 p-value 0.327]. 590 Results of the planned PFS and ORR analysis in all randomized patients and post-hoc 591 PFS and ORR analysis in subgroups of patients defined by K-Ras mutation status, and 592 post-hoc analysis of updated OS based on additional follow-up (1000 events) in all 593 randomized patients and in subgroups of patients defined by K-Ras mutation status are 594 presented in Table 8 and Figure 2. The treatment effect in the all-randomized population 595 for PFS was driven by treatment effects limited to patients who have K-Ras mutation 596 negative (wild-type) tumors. There is no evidence of effectiveness in the subgroup of 597 patients with K-Ras mutation-positive tumors. 30 30 Reference ID: 3270603 This label may not be the latest approved by FDA. 555 Erbitux Clinical Trials in K-Ras Mutation-negative (Wild-type), EGFR 556 expressing, Metastatic Colorectal Cancer For current labeling information, please visit https://www.fda.gov/drugsatfda Table 8: Clinical Efficacy in First-line EGFR-expressing, Metastatic Colorectal Cancer (All Randomized and K-Ras Status) K-Ras Mutation-negative All Randomized (Wild-type) K-Ras Mutation-positive EU- EU- EU- Approved Approved Approved Cetuximab Cetuximab Cetuximab plus plus plus FOLFIRI FOLFIRI FOLFIRI FOLFIRI FOLFIRI FOLFIRI (n=608) (n=609) (n=320) (n=356) (n=216) (n=187) Progression-Free Survival Number of Events (%) 343 (56) 371 (61) 165 (52) 214 (60) 138 (64) 112 (60) Median (months) 8.9 8.1 9.5 8.1 7.5 8.2 (95% CI) (8.0, 9.4) (7.6, 8.8) (8.9, 11.1) (7.4, 9.2) (6.7, 8.7) (7.4, 9.2) HR (95% CI) 0.85 (0.74, 0.99) 0.70 (0.57, 0.86) 1.13 (0.88, 1.46) a p-value 0.0358 b Overall Survival Number of Events (%) 491 (81) 509 (84) 244 (76) 292 (82) 189 (88) 159 (85) Median (months) 19.6 18.5 23.5 19.5 16.0 16.7 (95% CI) (18, 21) (17, 20) (21, 26) (17, 21) (15, 18) (15, 19) HR (95% CI) 0.88 (0.78, 1.0) 0.80 (0.67, 0.94) 1.04 (0.84, 1.29) Objective Response Rate ORR (95% CI) 46% (42, 50) 38% (34, 42) 57% (51, 62) 39% (34, 44) 31% (25, 38) 35% (28, 43) a Based on the Stratified Log-rank test. b Post-hoc updated OS analysis, results based on an additional 162 events. Clinical Efficacy in First-line EGFR-expressing, Metastatic Colorectal Cancer (All Randomized and K-Ras Status) 31 31 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 600 Figure 2: Kaplan-Meier Curve for Overall Survival in the K-Ras 601 Mutation-negative (Wild-type) Population in Study 4 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. y pp y For current labeling information, please visit https://www.fda.gov/drugsatfda 600 Figure 2: Kaplan-Meier Curve for Overall Survival in the K-Ras 601 Mutation-negative (Wild-type) Population in Study 4 graph 600 Figure 2: Kaplan-Meier Curve for Overall Survival in the K-Ras 601 Mutation-negative (Wild-type) Population in Study 4 603 Study 5 was a multicenter, open-label, randomized, clinical trial conducted in 604 572 patients with EGFR-expressing, previously treated, recurrent mCRC. Patients were 605 randomized (1:1) to receive either Erbitux plus best supportive care (BSC) or BSC alone. 606 Erbitux was administered as a 400 mg/m2 initial dose, followed by 250 mg/m2 weekly 607 until disease progression or unacceptable toxicity. 555 Erbitux Clinical Trials in K-Ras Mutation-negative (Wild-type), EGFR 556 expressing, Metastatic Colorectal Cancer 603 Study 5 was a multicenter, open label, randomized, clinical trial conducted in 604 572 patients with EGFR-expressing, previously treated, recurrent mCRC. Patients were 605 randomized (1:1) to receive either Erbitux plus best supportive care (BSC) or BSC alone. 606 Erbitux was administered as a 400 mg/m2 initial dose, followed by 250 mg/m2 weekly 607 until disease progression or unacceptable toxicity. 608 Of the 572 randomized patients, the median age was 63 years, 64% were male, 89% were 609 Caucasian, and 77% had baseline ECOG performance status of 0–1. Demographics and 610 baseline characteristics were similar between study arms. All patients were to have 611 received and progressed on prior therapy including an irinotecan-containing regimen and 612 an oxaliplatin-containing regimen. 613 K-Ras status was available for 453/572 (79%) of the patients: 245 (54%) patients had 614 K-Ras mutation-negative (wild-type) tumors and 208 (46%) patients had K-Ras mutation 615 positive tumors where testing assessed for the following somatic mutations in codons 616 12 and 13 (exon 2): G12A, G12D, G12R, G12C, G12S, G12V, G13D [see Warnings and 617 Precautions (5.7)]. 618 The main outcome measure of the study was overall survival. Results are presented in 619 Table 9 and Figure 3. 608 Of the 572 randomized patients, the median age was 63 years, 64% were male, 89% were 609 Caucasian, and 77% had baseline ECOG performance status of 0–1. Demographics and 610 baseline characteristics were similar between study arms. All patients were to have 611 received and progressed on prior therapy including an irinotecan-containing regimen and 612 an oxaliplatin-containing regimen. 608 Of the 572 randomized patients, the median age was 63 years, 64% were male, 89% were 609 Caucasian, and 77% had baseline ECOG performance status of 0–1. Demographics and 610 baseline characteristics were similar between study arms. All patients were to have 611 received and progressed on prior therapy including an irinotecan-containing regimen and 612 an oxaliplatin-containing regimen. 608 Of the 572 randomized patients, the median age was 63 years, 64% were male, 89% were 609 Caucasian, and 77% had baseline ECOG performance status of 0–1. Demographics and 610 baseline characteristics were similar between study arms. All patients were to have 611 received and progressed on prior therapy including an irinotecan-containing regimen and 612 an oxaliplatin-containing regimen. 555 Erbitux Clinical Trials in K-Ras Mutation-negative (Wild-type), EGFR 556 expressing, Metastatic Colorectal Cancer 613 K-Ras status was available for 453/572 (79%) of the patients: 245 (54%) patients had 614 K-Ras mutation-negative (wild-type) tumors and 208 (46%) patients had K-Ras mutation 615 positive tumors where testing assessed for the following somatic mutations in codons 616 12 and 13 (exon 2): G12A, G12D, G12R, G12C, G12S, G12V, G13D [see Warnings and 617 Precautions (5.7)]. 618 The main outcome measure of the study was overall survival. Results are presented in 619 Table 9 and Figure 3. 618 The main outcome measure of the study was overall survival. Results are presented in 619 Table 9 and Figure 3. 618 The main outcome measure of the study was overall survival. Results are presented in 619 Table 9 and Figure 3. 32 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda y pp y For current labeling information, please visit https://www.fda.gov/drugsatfda 4.6 Table 9: Overall Survival in Previously Treated EGFR-expressing, Metastatic Colorectal Cancer (All Randomized and K-Ras Status) K-Ras Mutation-negative All Randomized (Wild-type) K-Ras Mutation-positive Erbitux Erbitux Erbitux plus BSC BSC plus BSC BSC plus BSC BSC (N=287) (N=285) (N=117) (N=128) (N=108) (N=100) Median (months) 6.1 4.6 8.6 5.0 4.8 (95% CI) (5.4, 6.7) (4.2, 4.9) (7.0, 10.3) (4.3, 5.7) (3.9, 5.6) (3.6, 4.9) HR 0.77 0.63 0.91 (95% CI) (0.64, 0.92) (0.47, 0.84) (0.67, 1.24) p-valuea 0.0046 a 620 Based on the Stratified Log-rank test. 621 Figure 3: Kaplan-Meier Curve for Overall Survival in Patients with 622 K-Ras Mutation-negative (Wild-type) Metastatic Colorectal 623 Cancer in Study 5 4.6 Table 9: Overall Survival in Previously Treated EGFR-expressing, Metastatic Colorectal Cancer (All Randomized and K-Ras Status) K-Ras Mutation-negative All Randomized (Wild-type) K-Ras Mutation-positive Erbitux Erbitux Erbitux plus BSC BSC plus BSC BSC plus BSC BSC (N=287) (N=285) (N=117) (N=128) (N=108) (N=100) Median (months) 6.1 4.6 8.6 5.0 4.8 (95% CI) (5.4, 6.7) (4.2, 4.9) (7.0, 10.3) (4.3, 5.7) (3.9, 5.6) (3.6, 4.9) HR 0.77 0.63 0.91 (95% CI) (0.64, 0.92) (0.47, 0.84) (0.67, 1.24) p-valuea 0.0046 a 620 Based on the Stratified Log-rank test. 555 Erbitux Clinical Trials in K-Ras Mutation-negative (Wild-type), EGFR 556 expressing, Metastatic Colorectal Cancer 621 Figure 3: Kaplan-Meier Curve for Overall Survival in Patients with 622 K-Ras Mutation-negative (Wild-type) Metastatic Colorectal 623 Cancer in Study 5 4.6 Table 9: Overall Survival in Previously Treated EGFR-expressing, Metastatic Colorectal Cancer (All Randomized and K-Ras Status) K-Ras Mutation-negative All Randomized (Wild-type) K-Ras Mutation-positive Erbitux Erbitux Erbitux plus BSC BSC plus BSC BSC plus BSC BSC (N=287) (N=285) (N=117) (N=128) (N=108) (N=100) Median (months) 6.1 4.6 8.6 5.0 4.8 (95% CI) (5.4, 6.7) (4.2, 4.9) (7.0, 10.3) (4.3, 5.7) (3.9, 5.6) (3.6, 4.9) HR 0.77 0.63 0.91 (95% CI) (0.64, 0.92) (0.47, 0.84) (0.67, 1.24) p-valuea 0.0046 a 620 Based on the Stratified Log-rank test. le 9: Overall Survival in Previously Treated EGFR-expressing, Overall Survival in Previously Treated EGFR-expressing, 620 621 Figure 3: Kaplan-Meier Curve for Overall Survival in Patients with 622 K-Ras Mutation-negative (Wild-type) Metastatic Colorectal 623 Cancer in Study 5 graph 625 Study 6 was a multicenter clinical trial conducted in 329 patients with EGFR-expressing 621 Figure 3: Kaplan-Meier Curve for Overall Survival in Patients with 622 K-Ras Mutation-negative (Wild-type) Metastatic Colorectal 623 Cancer in Study 5 graph 625 Study 6 was a multicenter, clinical trial conducted in 329 patients with EGFR-expressing 626 recurrent mCRC. Tumor specimens were not available for testing for K-Ras mutation 627 status. Patients were randomized (2:1) to receive either Erbitux plus irinotecan 628 (218 patients) or Erbitux monotherapy (111 patients). Erbitux was administered as a 629 400 mg/m2 initial dose, followed by 250 mg/m2 weekly until disease progression or 621 Figure 3: Kaplan-Meier Curve for Overall Survival in Patients with 622 K-Ras Mutation-negative (Wild-type) Metastatic Colorectal 623 Cancer in Study 5 625 Study 6 was a multicenter, clinical trial conducted in 329 patients with EGFR-expressing 626 recurrent mCRC. Tumor specimens were not available for testing for K-Ras mutation 627 status. Patients were randomized (2:1) to receive either Erbitux plus irinotecan 628 (218 patients) or Erbitux monotherapy (111 patients). Erbitux was administered as a 629 400 mg/m2 initial dose, followed by 250 mg/m2 weekly until disease progression or 33 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. y pp y For current labeling information, please visit https://www.fda.gov/drugsatfda 630 unacceptable toxicity. 555 Erbitux Clinical Trials in K-Ras Mutation-negative (Wild-type), EGFR 556 expressing, Metastatic Colorectal Cancer In the Erbitux plus irinotecan arm, irinotecan was added to Erbitux 631 using the same dose and schedule for irinotecan as the patient had previously failed. 632 Acceptable irinotecan schedules were 350 mg/m2 every 3 weeks, 180 mg/m2 every 633 2 weeks, or 125 mg/m2 weekly times four doses every 6 weeks. Of the 329 patients, the 634 median age was 59 years, 63% were male, 98% were Caucasian, and 88% had baseline 635 Karnofsky performance status 80. Approximately two-thirds had previously failed 636 oxaliplatin treatment. 637 The efficacy of Erbitux plus irinotecan or Erbitux monotherapy, based on durable 638 objective responses, was evaluated in all randomized patients and in two pre-specified 639 subpopulations: irinotecan refractory patients, and irinotecan and oxaliplatin failures. In 640 patients receiving Erbitux plus irinotecan, the objective response rate was 641 23% (95% confidence interval 18%–29%), median duration of response was 5.7 months, 642 and median time to progression was 4.1 months. In patients receiving Erbitux 643 monotherapy, the objective response rate was 11% (95% confidence interval 6%–18%), 644 median duration of response was 4.2 months, and median time to progression was 645 1.5 months. Similar response rates were observed in the pre-defined subsets in both the 646 combination arm and monotherapy arm of the study. 16 647 16 HOW SUPPLIED/STORAGE AND HANDLING 648 Erbitux® (cetuximab) is supplied at a concentration of 2 mg/mL as a 100 mg/50 mL, 649 single-use vial or as a 200 mg/100 mL, single-use vial as a sterile, injectable liquid 650 containing no preservatives. 651 NDC 66733-948-23 100 mg/50 mL, single-use vial, individually packaged in a carton 652 NDC 66733-958-23 200 mg/100 mL, single-use vial, individually packaged in a carton 653 Store vials under refrigeration at 2 C to 8 C (36 F to 46 F). Do not freeze. Increased 654 particulate formation may occur at temperatures at or below 0 C. This product contains 655 no preservatives. Preparations of Erbitux in infusion containers are chemically and 656 physically stable for up to 12 hours at 2 C to 8 C (36 F to 46 F) and up to 8 hours at 657 controlled room temperature (20 C to 25 C; 68 F to 77 F). Discard any remaining 658 solution in the infusion container after 8 hours at controlled room temperature or after 659 12 hours at 2 C to 8 C. Discard any unused portion of the vial. 648 Erbitux® (cetuximab) is supplied at a concentration of 2 mg/mL as a 100 mg/50 mL, 649 single-use vial or as a 200 mg/100 mL, single-use vial as a sterile, injectable liquid 650 containing no preservatives. 648 Erbitux® (cetuximab) is supplied at a concentration of 2 mg/mL as a 100 mg/50 mL, 649 single-use vial or as a 200 mg/100 mL, single-use vial as a sterile, injectable liquid 650 containing no preservatives. 653 Store vials under refrigeration at 2 C to 8 C (36 F to 46 F). Do not freeze. Increased 654 particulate formation may occur at temperatures at or below 0 C. This product contains 655 no preservatives. Preparations of Erbitux in infusion containers are chemically and 656 physically stable for up to 12 hours at 2 C to 8 C (36 F to 46 F) and up to 8 hours at 657 controlled room temperature (20 C to 25 C; 68 F to 77 F). Discard any remaining 658 solution in the infusion container after 8 hours at controlled room temperature or after 659 12 hours at 2 C to 8 C. Discard any unused portion of the vial. 653 Store vials under refrigeration at 2 C to 8 C (36 F to 46 F). Do not freeze. 16 Increased 654 particulate formation may occur at temperatures at or below 0 C. This product contains 655 no preservatives. Preparations of Erbitux in infusion containers are chemically and 656 physically stable for up to 12 hours at 2 C to 8 C (36 F to 46 F) and up to 8 hours at 657 controlled room temperature (20 C to 25 C; 68 F to 77 F). Discard any remaining 658 solution in the infusion container after 8 hours at controlled room temperature or after 659 12 hours at 2 C to 8 C. Discard any unused portion of the vial. 34 Reference ID: 3270603 Reference ID: 3270603 This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda This label may not be the latest approved by FDA. For current labeling information, please visit https://www.fda.gov/drugsatfda 671 Erbitux® is a registered trademark of ImClone LLC a wholly-owned subsidiary of 672 Eli Lilly and Company. 673 Manufactured by ImClone LLC a wholly-owned subsidiary of Eli Lilly and Company, 674 Branchburg, NJ 08876 USA 675 Distributed and marketed by Bristol-Myers Squibb Company, Princeton, NJ 08543 USA 676 Co-marketed by Eli Lilly and Company, Indianapolis, IN 46285 USA company logo 677 Copyright  2004–2013 ImClone LLC a wholly-owned subsidiary of Eli Lilly and 678 Company, and Bristol-Myers Squibb Company. All rights reserved. 679 1236886B3 Rev March 2013 671 Erbitux® is a registered trademark of ImClone LLC a wholly-owned subsidiary of 672 Eli Lilly and Company. 671 Erbitux® is a registered trademark of ImClone LLC a wholly-owned subsidiary of 672 Eli Lilly and Company. 673 Manufactured by ImClone LLC a wholly-owned subsidiary of Eli Lilly and Company, 674 Branchburg, NJ 08876 USA 673 Manufactured by ImClone LLC a wholly-owned subsidiary of Eli Lilly and Company, 6 hb 6 675 Distributed and marketed by Bristol-Myers Squibb Company, Princeton, 675 Distributed and marketed by Bristol-Myers Squibb Company, Princeton, NJ 08543 USA 676 Co-marketed by Eli Lilly and Company, Indianapolis, IN 46285 USA 675 Distributed and marketed by Bristol-Myers Squibb Company, Princeton, NJ 08543 USA 676 Co-marketed by Eli Lilly and Company, Indianapolis, IN 46285 USA company logo 676 Co-marketed by Eli Lilly and Company, Indianapolis, IN 46285 USA company logo 660 17 PATIENT COUNSELING INFORMATION 661 Advise patients: 661 Advise patients: 662  To report signs and symptoms of infusion reactions such as fever, chills, or breathing 663 problems. 664  Of the potential risks of using Erbitux during pregnancy or nursing and of the need 665 to use adequate contraception in both males and females during and for 6 months 666 following the last dose of Erbitux therapy. 667  That nursing is not recommended during, and for 2 months following the last dose of 668 Erbitux therapy. 669  To limit sun exposure (use sunscreen, wear hats) while receiving and for 2 months 670 following the last dose of Erbitux. y pp y For current labeling information, please visit https://www.fda.gov/drugsatfda 677 Copyright  2004–2013 ImClone LLC a wholly-owned subsidiary of Eli Lilly and 678 Company, and Bristol-Myers Squibb Company. All rights reserved. 677 Copyright  2004–2013 ImClone LLC a wholly-owned subsidiary of Eli Lilly and 678 C d B i l M S ibb C All i h d 679 1236886B3 Rev March 2 679 1236886B3 Rev March 201 Rev March 2013 35 Reference ID: 3270603
https://openalex.org/W2136184404	https://journalofinequalitiesandapplications.springeropen.com/counter/pdf/10.1186/1029-242X-2014-365	English	null	Best proximity points of implicit relation type modified α 3 -proximal contractions	Journal of inequalities and applications	2,014	cc-by	9,371	R ES EARCH Open Access ©2014 Zabihi and Razani; 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. Abstract In this paper, we introduce the concept of an α3-proximal admissible mappings and establish the existence of best proximity point theorems for implicit relation type modiﬁed α3-proximal contractions. As applications of our theorems, we derive some new best proximity point results for implicit relation type contractions whenever the range space is endowed with a graph or with a partial order. The obtained results generalize, extend, and modify some best proximity point results in the literature. Several interesting consequences of our theorems are also provided. MSC: 46N40; 47H10; 54H25; 46T99 Keywords: ﬁxed point; best proximity point; α3-proximal admissible mapping; implicit relation type α3-proximal contractions; metric space endowed with graph Farzaneh Zabihi and Abdolrahman Razani* Correspondence: razani@ipm.ir Department of Mathematics, Science and Research Branch, Islamic Azad University, Tehran, Iran Correspondence: razani@ipm.ir Department of Mathematics, Science and Research Branch, Islamic Azad University, Tehran, Iran Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Example Let F(t,t,t,t,t,t) = t– ψ max t,t,t, t+ t  – Lmin{t,t,t,t}, where L ≥and ψ ∈Ψ . Then F ∈F. where L ≥and ψ ∈Ψ . Then F ∈F. 1 Introduction In nonlinear functional analysis, one of the most signiﬁcant research areas is ﬁxed point theory. On the other hand, ﬁxed point theory has an application in distinct branches of mathematics and also in diﬀerent sciences, such as engineering, computer science, eco- nomics, etc. In , Banach proved that every contraction in a complete metric space has a unique ﬁxed point. Following this celebrated result, many authors have generalized, improved, and extended this result in the context of diﬀerent abstract spaces for various operators. On the other hand, several classical ﬁxed point theorems and common ﬁxed point the- orems have been recently uniﬁed by considering general contractive conditions expressed by an implicit relation (see Popa [, ]). Following Popa’s approach, many results on ﬁxed point, common ﬁxed points, and coincidence points have been obtained, in various am- bient spaces (see [–], and references therein). On the other hand, Samet et al. [] in- troduced and studied α-ψ-contractive mappings in complete metric spaces and provided applications of the results to ordinary diﬀerential equations. More recently, Salimi et al. [] modiﬁed the notions of α-ψ-contractive and α-admissible mappings and established ﬁxed point theorems to modify the results in []. For more details and applications of this line of research, we refer the reader to some related papers [–] and references therein. In this paper, we introduce the concept of an α-proximal admissible mappings and es- tablish the existence of best proximity point theorems for implicit relation type modi- ﬁed α-proximal contractions. As applications of our theorems, we derive some new best proximity point results for implicit relation type contractions whenever the range space Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 2 of 19 is endowed with a graph or with a partial order. The obtained results generalize, extend, and modify some best proximity point results in the literature. 2 Main results Let A and B be two nonempty subsets of metric space (X,d) and T : A →B be a nonself mapping. We say that x∗is a best proximity of T if d x∗,Tx∗ = d(A,B), where d(A,B) = inf d(x,y) : x ∈A,y ∈B . We deﬁne Aand Bas follows: A= x ∈A : d(x,y) = d(A,B) for some y ∈B and and B= y ∈B : d(x,y) = d(A,B) for some x ∈A . B= y ∈B : d(x,y) = d(A,B) for some x ∈A . We denote by Ψ the set of all nondecreasing functions ψ : [,+∞) →[,+∞) such that ∞ n=ψn(t) < +∞for all t > , where ψn is the nth iterate of ψ. Let F be the set of all continuous functions F : R + →R satisfying the following asser- tions: (F) if F(u,v,v,u,u + v,) ≤, where u,v > , then u ≤ψ(v); (F) if F(u,v,v,u,u + v,) ≤, where u,v > , then u ≤ψ(v); (F) F(t,...,t) is decreasing in t; (F) if F(u,v,,u + v,u,v) ≤, where u,v ≥, then u ≤ψ(v); (F) F(u,u,,,u,u) > for all u > . (F) F(u,u,,,u,u) > for all u > . (F) F(u,u,,,u,u) > for all u > . Example Let F(t,t,t,t,t,t) = t– at– b[+ t]t + t – c[t+ t] – d[t+ t], where a + b + c + d < . Then F ∈F. Deﬁnition Let A, B be two nonempty subsets of a metric space (X,d) and α : A × A → [,+∞) be a function. We say that a nonself mapping T : A →B is α-proximal admissible Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 Page 3 of 19 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 3 of 19 if, for all x,x,u,u∈A, ⎧ ⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎩ α(x,x) ≥, α(x,x) ≥, α(x,x) ≥, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ ⎧ ⎪⎨ ⎪⎩ α(u,u) ≥, α(u,u) ≥, α(u,u) ≥. if, for all x,x,u,u∈A, ⎧ ⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎩ α(x,x) ≥, α(x,x) ≥, α(x,x) ≥, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ ⎧ ⎪⎨ ⎪⎩ α(u,u) ≥, α(u,u) ≥, α(u,u) ≥. Deﬁnition Let A and B be nonempty subsets of a metric space (X,d) and α : A × A → [,∞) be a function. Then T : A →B is said to be an implicit relation type modiﬁed α-proximal contraction, if for all x,y,u,v ∈A, Deﬁnition Let A and B be nonempty subsets of a metric space (X,d) and α : A × A → [,∞) be a function. Then T : A →B is said to be an implicit relation type modiﬁed α-proximal contraction, if for all x,y,u,v ∈A, ⎧ ⎪⎨ ⎪⎩ α(x,y) ≥, d(u,Tx) = d(A,B), d(v,Ty) = d(A,B) ⎧ ⎪⎨ ⎪⎩ α(x,y) ≥, d(u,Tx) = d(A,B), d(v,Ty) = d(A,B) ⇒ F d(u,v),d(x,y),d(x,u),d(y,v),d(x,v),d(y,u) ≤L – α(x,x)α(y,y) , (.) ⇒ F d(u,v),d(x,y),d(x,u),d(y,v),d(x,v),d(y,u) ≤L – α(x,x)α(y,y) , (.) where L ≥and F ∈F. where L ≥and F ∈F. where L ≥and F ∈F. where L ≥and F ∈F. where L ≥and F ∈F. where L ≥and F ∈F. Deﬁnition Let (X,d) be a metric space and A and B be two nonempty subsets of X. Then B is said to be approximatively compact with respect to A if every sequence {yn} in B, satisfying the condition d(x,yn) →d(x,B) for some x in A, has a convergent subsequence. Theorem Let A, B be two nonempty subsets of a metric space (X,d) such that A is com- plete and Ais nonempty. Example Let Assume that T : A →B is a continuous implicit relation type modiﬁed α-proximal contraction such that the following conditions hold: (i) T is an α-proximal admissible mapping and (i) T is an α-proximal admissible mapping and T(A) ⊆B, (ii) there exist x,x∈Asuch that d(x,Tx) = d(A,B), α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥. Then T has a best proximity point. Further, the best proximity point is unique if (iii) for every x,y ∈A with d(x,Tx) = d(A,B) = d(y,Ty), we have α(x,y) ≥, α(x,x) ≥, and α(y,y) ≥. Proof By (ii) there exist x,x∈Asuch that d(x,Tx) = d(A,B), α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥. d(x,Tx) = d(A,B), α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥. On the other hand, T(A) ⊆B, then there exists x∈Asuch that On the other hand, T(A) ⊆B, then there exists x∈Asuch that d(x,Tx) = d(A,B). Page 4 of 19 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Now, since T is α-proximal admissible, we have α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥. Hence, d(x,Tx) = d(A,B), α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥. Since T(A) ⊆B, there exists x∈Asuch that d(xTx) = d(A B) Now, since T is α-proximal admissible, we have Hence, d(x,Tx) = d(A,B), α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥. d(x,Tx) = d(A,B), Since T(A) ⊆B, there exists x∈Asuch that Since T(A) ⊆B, there exists x∈Asuch that d(x,Tx) = d(A,B). Then we have Then we have d(x,Tx) = d(A,B), d(x,Tx) = d(A,B), α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥. Again, since T is α-proximal admissible, we obtain α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥. Also, there exists x∈Asuch that Also, there exists x∈Asuch that d(x,Tx) = d(A,B), and hence d(x,Tx) = d(A,B), d(x,Tx) = d(A,B), α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥. By continuing this process, we construct a sequence {xn} such that α(xn,xn) ≥, α(xn–,xn–) ≥ and ⎧ ⎪⎨ ⎪⎩ α(xn–,xn) ≥, d(xn,Txn–) = d(A,B), d(xn+,Txn) = d(A,B) (.) (.) for all n ∈N. Now, from (.) with u = xn, v = xn+, x = xn–, and y = xn, we get F d(xn,xn+),d(xn–,xn),d(xn–,xn),d(xn,xn+),d(xn–,xn+),d(xn,xn) ≤L – α(xn–,xn–)α(xn,xn) . ≤L – α(xn–,xn–)α(xn,xn) . On the other hand from (.) we obtain On the other hand from (.) we obtain On the other hand from (.) we obtain α(xn–,xn–)α(xn,xn) ≥. α(xn–,xn–)α(xn,xn) ≥. Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 5 of 19 That is, – α(xn–,xn–)α(xn,xn) ≤for all n ∈N. Therefore, That is, – α(xn–,xn–)α(xn,xn) ≤for all n ∈N. Therefore, F d(xn,xn+),d(xn–,xn),d(xn–,xn),d(xn,xn+),d(xn–,xn+),d(xn,xn) ≤L – α(xn–,xn–)α(xn,xn) ≤. Now, since F is decreasing in t Now, since F is decreasing in t F d(xn,xn+),d(xn–,xn),d(xn–,xn),d(xn,xn+),d(xn,xn+) + d(xn–,xn), ≤, and so from (F) we get d(xn,xn+) ≤ψ d(xn–,xn) . By induction, we have By induction, we have d(xn,xn+) ≤ψn d(x,x) . Fix ϵ > , there exists N ∈N such that Fix ϵ > , there exists N ∈N such that N ψn d(x,x) < ϵ for all n ∈N. n≥N ψn d(x,x) < ϵ for all n ∈N. Let m,n ∈N with m > n ≥N. Then by the triangular inequality, we get Let m,n ∈N with m > n ≥N. Then by the triangular inequality, we get d(xn,xm) ≤ m– k=n d(xk,xk+) ≤ n≥N ψn d(x,x) < ϵ. Consequently limm,n,→+∞d(xn,xm) = . Hence {xn} is a Cauchy sequence. Since A is com- plete, there is z ∈A such that xn →z. Since T is continuous, Txn →Tz as n →∞. Hence, d(A,B) = lim n→∞d(xn+,Txn) = d(z,Tz). d(A,B) = lim n→∞d(xn+,Txn) = d(z,Tz). Thus z is the desired best proximity point of T. Let x,y ∈A be two best proximity point of T such that x ̸= y. That is, d(x,Tx) = d(A,B) = d(y,Ty). From (iii), we get α(x,y) ≥, α(x,x) ≥, and α(y,y) ≥. So by (.) we derive F d(x,y),d(x,y),d(x,x),d(y,y),d(y,x),d(x,y) ≤L – α(x,x)α(y,y) ≤, which implies which implies F d(x,y),d(x,y),,,d(y,x),d(x,y) ≤, □ ch is a contradiction to (F). Hence, T has a unique best proximity point. □ which is a contradiction to (F). Hence, T has a unique best proximity point. □ Theorem Let A, B be two nonempty subsets of a metric space (X,d) such that A is com- plete, B is approximatively compact with respect to A, and Ais nonempty. Assume that T : A →B is an implicit relation type modiﬁed α-proximal contraction such that the fol- lowing conditions hold: Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 6 of 19 d(x,Tx) = d(A,B), α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥, (iii) if {xn} is a sequence in X such that α(xn,xn+) ≥for all n ∈N ∪{} with xn →x as n →∞, then α(xn,x) ≥and α(x,x) ≥. Then T has a best proximity point. Now, since F is decreasing in t Further, the best proximity point is unique if (iv) for every x,y ∈A, where d(x,Tx) = d(A,B) = d(y,Ty), we have α(x,y) ≥, α(x,x) ≥, and α(y,y) ≥. (iii) if {xn} is a sequence in X such that α(xn,xn+) ≥for all n ∈N ∪{} with xn →x as n →∞, then α(xn,x) ≥and α(x,x) ≥. Then T has a best proximity point. Further, the best proximity point is unique if (iv) for every x,y ∈A, where d(x,Tx) = d(A,B) = d(y,Ty), we have α(x,y) ≥, α(x,x) ≥, and α(y,y) ≥. Then T has a best proximity point. Further, the best proximity point is unique if (iv) for every x,y ∈A, where d(x,Tx) = d(A,B) = d(y,Ty), we have α(x,y) ≥, α(x,x) ≥, and α(y,y) ≥. Proof Following the proof of Theorem , there exist a Cauchy sequence {xn} ⊆A and z ∈A such that (.) holds and xn →z as n →+∞. On the other hand, for all n ∈N, we can write Proof Following the proof of Theorem , there exist a Cauchy sequence {xn} ⊆A and z ∈A such that (.) holds and xn →z as n →+∞. On the other hand, for all n ∈N, we can write d(z,B) ≤d(z,Txn) ≤d(z,xn+) + d(xn+,Txn) = d(z,xn+) + d(A,B). ≤d(z,xn+) + d(xn+,Txn) = d(z,xn+) + d(A,B). Taking the limit as n →+∞in the above inequality, we get Taking the limit as n →+∞in the above inequality, we get lim n→+∞d(z,Txn) = d(z,B) = d(A,B). (.) (.) lim n→+∞d(z,Txn) = d(z,B) = d(A,B). Since B is approximatively compact with respect to A, the sequence {Txn} has a subse- quence {Txnk} that converges to some y∗∈B. Hence, d z,y∗ = lim n→∞d(xnk+,Txnk) = d(A,B) d z,y∗ = lim n→∞d(xnk+,Txnk) = d(A,B) and so z ∈A. Now, since T(A) ⊆B, we have d(w,Tz) = d(A,B) for some w ∈A. By (iii) and (.), we have α(xn,z) ≥, α(z,z) ≥, and d(xn+,Txn) = d(A,B) for all n ∈N ∪{}. Also, since T is an implicit relation type α-proximal contraction, we get F d(xn+,w),d(xn,z),d(xn,xn+),d(z,w),d(xn,w),d(z,xn+) ≤. F d(xn+,w),d(xn,z),d(xn,xn+),d(z,w),d(xn,w),d(z,xn+) ≤. Taking the limit as n →+∞in the above inequality and applying continuity of F, we have F d(z,w),,,d(z,w),d(z,w), ≤. Now, if we take u = d(z,w) and v = , then we have F(u,v,,u + v,u,v) ≤ Assume that T : A →B is a nonself mapping satisfying the following conditions: (i) T is an α-proximal admissible mapping and T(A) ⊆B, (ii) there exist x,x∈Asuch that T : A →B is a nonself mapping satisfying the following conditions: (i) T is an α-proximal admissible mapping and T(A) ⊆B, f pp g fy g f g (i) T is an α-proximal admissible mapping and T(A) ⊆B, T is an α-proximal admissible mapping and T(A) ⊆B, (ii) there exist x,x∈Asuch that there exist x,x∈Asuch that d(x,Tx) = d(A,B), α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥, (iii) if {xn} is a sequence in X such that α(xn,xn+) ≥for all n ∈N ∪{} with xn →x as n →∞, then α(xn,x) ≥and α(x,x) ≥, (iii) if {xn} is a sequence in X such that α(xn,xn+) ≥for all n ∈N ∪{} with xn →x as n →∞, then α(xn,x) ≥and α(x,x) ≥, (iv) there exist nonnegative real numbers a, b, c, d with a + b + c + d < , such that for all x,x,u,u∈A, (iv) there exist nonnegative real numbers a, b, c, d with a + b + c + d < , such that for all x,x,u,u∈A, ⎧ ⎪⎨ ⎪⎩ α(x,x) ≥, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⎧ ⎪⎨ ⎪⎩ α(x,x) ≥, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ d(u,u) + Lα(x,x)α(x,x) ≤ad(x,x) + b[+ d(x,u)]d(x,u) + d(x,x) + c d(x,u) + d(x,u) + c d(x,u) + d(x,u) where L ≥. Then T has a best proximity point. Further, the best proximity point is unique if (v) for every x,y ∈A, where d(x,Tx) = d(A,B) = d(y,Ty), we have α(x,y) ≥, α(x,x) ≥, and α(y,y) ≥. If in Corollary we take b = c = d = , then we have the following corollary. If in Corollary we take b = c = d = , then we have the following corollary. Corollary Let A, B be two nonempty subsets of a metric space (X,d) such that A is com- plete, B is approximatively compact with respect to A, and Ais nonempty. F(u,v,,u + v,u,v) ≤ F(u,v,,u + v,u,v) ≤ and so from (F) we get u ≤ψ(v). That is, d(z,w) ≤ψ() = . Thus, z = w. Hence z is a best proximity point of T. Uniqueness follows similarly to the proof of Theorem . □ and so from (F) we get u ≤ψ(v). That is, d(z,w) ≤ψ() = . Thus, z = w. Hence z is a best proximity point of T. Uniqueness follows similarly to the proof of Theorem . □ Using Example and Theorem we obtain the following corollary. Using Example and Theorem we obtain the following corollary. Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 7 of 19 Corollary Let A, B be two nonempty subsets of a metric space (X,d) such that A is com- plete, B is approximatively compact with respect to A, and Ais nonempty. Assume that T : A →B is a nonself mapping satisfying the following conditions: Corollary Let A, B be two nonempty subsets of a metric space (X,d) such that A is com- plete, B is approximatively compact with respect to A, and Ais nonempty. Assume that T : A →B is a nonself mapping satisfying the following conditions: Corollary Let A, B be two nonempty subsets of a metric space (X,d) such that A is com- plete, B is approximatively compact with respect to A, and Ais nonempty. F(u,v,,u + v,u,v) ≤ Assume that T : A →B is a nonself mapping satisfying the following conditions: (i) T is an αproximal admissible mapping and T(A ) ⊆B (ii) there exist x,x∈Asuch that d(x,Tx) = d(A,B), α(x,x) ≥, α(x,x) ≥ and α(x,x) ≥, (iii) if {xn} is a sequence in X such that α(xn,xn+) ≥for all n ∈N ∪{} with xn →x as n →∞, then α(xn,x) ≥and α(x,x) ≥, (iii) if {xn} is a sequence in X such that α(xn,xn+) ≥for all n ∈N ∪{} with xn →x as n →∞, then α(xn,x) ≥and α(x,x) ≥, (iv) there exists a nonnegative real number a with a < , such that for all x,x,u,u∈A, ⎧ ⎪⎨ ⎪⎩ α(x,x) ≥, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ d(u,u) + Lα(x,x)α(x,x) ≤ad(x,x) + L, ⎧ ⎪⎨ ⎪⎩ α(x,x) ≥, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ d(u,u) + Lα(x,x)α(x,x) ≤ad(x,x) + L, where L ≥. where L ≥. Then T has a best proximity point. Further, the best proximity point is unique if Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 Page 8 of 19 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 Page 8 of 19 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 8 of 19 (v) for every x,y ∈A, where d(x,Tx) = d(A,B) = d(y,Ty), we have α(x,y) ≥, α(x,x) ≥, and α(y,y) ≥. (v) for every x,y ∈A, where d(x,Tx) = d(A,B) = d(y,Ty), we have α(x,y) ≥, α(x,x) ≥, and α(y,y) ≥. (v) for every x,y ∈A, where d(x,Tx) = d(A,B) = d(y,Ty), we have α(x,y) ≥, α(x,x) ≥, and α(y,y) ≥. Example Let X = R be endowed with the usual metric d(x,y) = \|x – y\|, for all x,y ∈X. Consider A = (–∞,–], B = [,+∞) and deﬁne T : A →B by Tx = ⎧ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩ , if x ∈(–∞,–), , if x ∈[–,–), , if x ∈[–,–), , if x ∈[–,–), , if x ∈[–,–), , if x ∈[–,–), , if x ∈[–,–), , if x ∈[–,–]. Tx = ⎧ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩ , if x ∈(–∞,–), , if x ∈[–,–), , if x ∈[–,–), , if x ∈[–,–), , if x ∈[–,–), , if x ∈[–,–), , if x ∈[–,–), , if x ∈[–,–]. Deﬁne α : X × X →[,+∞) by α(x,y) = , if x,y ∈[–,–],  , otherwise. Clearly, B is approximatively compact with respect to A and d(A,B) = . Then A= {–} and B= {}. Clearly, T(A) ⊆B, d(–,T(–)) = d(A,B) = , and α(–,–) ≥. A Assume ⎧ ⎪⎨ ⎪⎩ α(x,x) ≥, d(u,Tx) = d(A,B) = , d(u,Tx) = d(A,B) = , then then ⎧ ⎪⎨ ⎪⎩ x,x∈[–,–], d(u,Tx) = , d(u,Tx) = . ⎧ ⎪⎨ ⎪⎩ x,x∈[–,–], d(u,Tx) = , d(u,Tx) = . Therefore, u= u= –, that is, α(u,u) ≥, α(u,u) ≥, and α(u,u) ≥. Further, Therefore, u= u= –, that is, α(u,u) ≥, α(u,u) ≥, and α(u,u) ≥. Further, d(u,u) ≤ad(x,x) + b[+ d(x,u)]d(x,u) + d(x,x) + c d(x,u) + d(x,u) + d d(x,u) + d(x,u) + L – α(x,x)α(x,x) , that is, T is an α-proximal admissible mapping and condition (iv) of Corollary holds true. where L ≥. Moreover, if {xn} is a sequence such that α(xn,xn+) ≥for all n ∈N∪{} and xn →x as n →+∞, then {xn} ⊆[–,–] and hence x ∈[–,–]. Consequently, α(x,x) ≥and α(xn,x) ≥for all n ∈N ∪{}. Therefore all the conditions of Corollary hold for this example and T has a best proximity point. Here z = –is the best proximity point of T. Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 9 of 19 If in Corollary we take α(x,y) = , then we have the following corollary. Corollary (Theorem .of []) Let A and B be nonempty closed subsets of a complete metric space (X,d) such that B is approximatively compact with respect to A. Assume that a+b+c+d < . Let Aand Bbe nonempty and T : A →B be a nonself mapping satisfying the following assertions: Corollary (Theorem .of []) Let A and B be nonempty closed subsets of a complete metric space (X,d) such that B is approximatively compact with respect to A. Assume that a+b+c+d < . Let Aand Bbe nonempty and T : A →B be a nonself mapping satisfying the following assertions: (ii) d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ d(u,u) ≤ad(x,x) + b[+ d(x,u)]d(x,u) + d(x,x) + c d(x,u) + d(x,u) + d d(x,u) + d(x,u) . ⇒ d(u,u) ≤ad(x,x) + b[+ d(x,u)]d(x,u) + d(x,x) Then there exists z ∈A such that d(z,Tz) = d(A,B). d(z,Tz) = d(A,B). By taking α(x,y) = in Theorem , we deduce the following corollary. Corollary Let A, B be two nonempty subsets of a metric space (X,d) such that A is com- plete, B is approximatively compact with respect to A, and Ais nonempty. Assume that T : A →B is a nonself mapping such that TA⊆Band for all x,y,u,v ∈A, d(u,Tx) = d(A,B), d(v,Ty) = d(A,B) ⇒ F d(u,v),d(x,y),d(x,u),d(y,v),d(y,u),d(x,v) ≤, where F ∈F. Then T has a unique best proximity point. where F ∈F. Then T has a unique best proximity point. Using Example and Corollary , we deduce the following result. Corollary Let A, B be two nonempty subsets of a metric space (X,d) such that A is com- plete, B is approximatively compact with respect to A, and Ais nonempty. where L ≥. Assume that T : A →B is a nonself mapping such that TA⊆Band, for all x,y,u,v ∈A, d(u,Tx) = d(A,B), d(v,Ty) = d(A,B) ⇒ d(u,v) ≤ψ max d(x,y),d(x,u),d(y,v), d(y,u) + d(x,v)  + Lmin d(x,u),d(y,v),d(y,u),d(x,v) , d(u,Tx) = d(A,B), d(v,Ty) = d(A,B) d(u,Tx) = d(A,B), d(u,Tx) = d(A,B), ⇒ d(u,v) ≤ψ max d(x,y),d(x,u),d(y,v), d(y,u) + d(x,v)  + Lmin d(x,u),d(y,v),d(y,u),d(x,v) , where ψ ∈Ψ . Then T has a unique best proximity point. Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 10 of 19 3 Some results in metric spaces endowed with a graph Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 Page 11 of 19 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 htt // j l fi liti d li ti / t t/2014/1/365 Page 11 of 19 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Firstly, we prove that T is an α-proximal admissible mapping. To this aim, assume Firstly, we prove that T is an α-proximal admissible mapping. To this aim, assume ⎧ ⎪⎨ ⎪⎩ α(x,y) ≥, d(u,Tx) = d(A,B), d(v,Ty) = d(A,B). Therefore, we have ⎧ ⎪⎨ ⎪⎩ (x,y) ∈E(G), d(u,Tx) = d(A,B), d(v,Ty) = d(A,B). ⎧ ⎪⎨ ⎪⎩ (x,y) ∈E(G), d(u,Tx) = d(A,B), d(v,Ty) = d(A,B). Since T is an implicit relation type G-proximal contraction, we get (u,v) ∈E(G). Also, since Δ ⊆E(G), (u,u),(v,v) ∈E(G). That is, α(u,v) ≥, α(u,u) ≥, α(v,v) ≥, and Since T is an implicit relation type G-proximal contraction, we get (u,v) ∈E(G). Also, since Δ ⊆E(G), (u,u),(v,v) ∈E(G). That is, α(u,v) ≥, α(u,u) ≥, α(v,v) ≥, and F d(u,v),d(x,y),d(x,u),d(y,v),d(y,u),d(x,v) ≤= L – α(x,x)α(y,y) when L = . Thus T is an α-proximal admissible mapping with T(A) ⊆Band con- tinuous implicit relation type G-proximal contraction. From (ii) there exist x,x∈A such that d(x,Tx) = d(A,B) and (x,x) ∈E(G), that is, d(x,Tx) = d(A,B), α(x,x) ≥, α(x,x) ≥, and α(x,x) ≥. Hence, all the conditions of Theorem are satisﬁed and T has a best proximity point. □ Similarly, by using Theorem , we can prove the following theorem. Theorem Let A, B be two nonempty closed subsets of a metric space (X,d) endowed with a graph G. Assume that A is complete, B is approximatively compact with respect to A, and Ais nonempty. Also suppose that T : A →B is an implicit relation type G-proximal contraction mapping such that the following conditions hold: (i) T(A) ⊆B, (i) T(A) ⊆B, (ii) there exist elements x,x∈Asuch that (ii) there exist elements x,x∈Asuch that d(x,Tx) = d(A,B) and (x,x) ∈E(G), d(x,Tx) = d(A,B) and (x,x) ∈E(G), (iii) if {xn} is a sequence in X such that (xn,xn+) ∈E(G) for all n ∈N ∪{} and xn →x as n →+∞, then (xn,x) ∈E(G) for all n ∈N ∪{}. Then T has a best proximity point. 3 Some results in metric spaces endowed with a graph p g p Consistent with Jachymski [], let (X,d) be a metric space and Δ denotes the diagonal of the Cartesian product X × X. Consider a directed graph G such that the set V(G) of its vertices coincides with X, and the set E(G) of its edges contains all loops, i.e., E(G) ⊇Δ. We assume G has no parallel edges, so we can identify G with the pair (V(G),E(G)). More- over, we may treat G as a weighted graph (see []) by assigning to each edge the distance between its vertices. If x and y are vertices in a graph G, then a path in G from x to y of length N (N ∈N) is a sequence {xi}N i=of N + vertices such that x= x, xN = y and (xn–,xn) ∈E(G) for i = ,...,N. A graph G is connected if there is a path between any two vertices. G is weakly connected if ˜G is connected (see for details [, , ]). In , Espínola and Kirk [] established an important combination of ﬁxed point theory and graph theory. Deﬁnition Let A, B be two nonempty closed subsets of a metric space (X,d) endowed with a graph G. Then T : A →B is said to be an implicit relation type G-proximal con- traction, if, for all x,y,u,v ∈A, ⎧ ⎪⎨ ⎪⎩ (x,y) ∈E(G), d(u,Tx) = d(A,B), d(v,Ty) = d(A,B) ⇒ (u,v) ∈E(G) and ⎧ ⎪⎨ ⎪⎩ (x,y) ∈E(G), d(u,Tx) = d(A,B), d(v,Ty) = d(A,B) ⇒ F d(u,v),d(x,y),d(x,u),d(y,v),d(y,u),d(x,v) ≤, where F ∈F. where F ∈F. Theorem Let A, B be two nonempty closed subsets of a metric space (X,d) endowed with a graph G. Assume that A is complete, Ais nonempty, and T : A →B is a continuous implicit relation type G-proximal contraction such that the following conditions hold: (i) T(A) ⊆B, (i) T(A) ⊆B, (ii) there exist elements x,x∈Asuch that (ii) there exist elements x,x∈Asuch that d(x,Tx) = d(A,B) and (x,x) ∈E(G). Then T has a best proximity point. Further, the best proximity point is unique if, for every x,y ∈A such that d(x,Tx) = d(A,B) = d(y,Ty), we have (x,y) ∈E(G). Proof Deﬁne α : X × X →[,+∞) by α(x,y) = , if (x,y) ∈E(G),  , otherwise. α(x,y) = y  , otherwise. (i) T(A) ⊆B, 3 Some results in metric spaces endowed with a graph ⇒ d(u,u) ≤ad(x,x) + b[+ d(x,u)]d(x,u) + d(x,x) + c d(x,u) + d(x,u) + d d(x,u) + d(x,u) . Then T has a best proximity point. Further, the best proximity point is unique if, for every x,y ∈A such that d(x,Tx) = d(A,B) = d(y,Ty), we have (x,y) ∈E(G). Corollary Let A, B be two nonempty closed subsets of a metric space (X,d) endowed with a graph G. Assume that A is complete, B is approximatively compact with respect to A, and Ais nonempty. Also, suppose that T : A →B satisﬁes the following conditions: (i) T(A) ⊆B, (ii) there exist elements x,x∈Asuch that (ii) there exist elements x,x∈Asuch that d(x,Tx) = d(A,B) and (x,x) ∈E(G), d(x,Tx) = d(A,B) and (x,x) ∈E(G), d(x,Tx) = d(A,B) and (x,x) ∈E(G), (iii) if {xn} is a sequence in X such that (xn,xn+) ∈E(G) for all n ∈N ∪{} and xn →x as n →+∞, then (xn,x) ∈E(G) for all n ∈N ∪{}, (iii) if {xn} is a sequence in X such that (xn,xn+) ∈E(G) for all n ∈N ∪{} and xn →x as n →+∞, then (xn,x) ∈E(G) for all n ∈N ∪{}, (iii) if {xn} is a sequence in X such that (xn,xn+) ∈E(G) for all n ∈N ∪{} and xn →x as n →+∞, then (xn,x) ∈E(G) for all n ∈N ∪{}, (iv) for x,x,u,u∈A, ⎧ ⎪⎨ ⎪⎩ (x,x) ∈E(G), d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ d(u,u) ≤ψ max d(x,x),d(x,u),d(x,u), d(x,u) + d(x,u)  + Lmin d(x,u),d(x,u),d(x,u),d(x,u) , ⎧ ⎪⎨ ⎪⎩ (x,x) ∈E(G), d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ d(u,u) ≤ψ max d(x,x),d(x,u),d(x,u), d(x,u) + d(x,u)  ⇒ d(u,u) ≤ψ max d(x,x),d(x,u),d(x,u), d(x,u) + d(x,u)  + Lmin d(x,u),d(x,u),d(x,u),d(x,u) , + Lmin d(x,u),d(x,u),d(x,u),d(x,u) , where ψ ∈Ψ . where ψ ∈Ψ . ψ Then T has a best proximity point. Further, the best proximity point is unique if, for every x,y ∈A such that d(x,Tx) = d(A,B) = d(y,Ty), we have (x,y) ∈E(G). 3 Some results in metric spaces endowed with a graph Further, the best proximity point is unique if, for every x,y ∈A such that d(x,Tx) = d(A,B) = d(y,Ty), we have (x,y) ∈E(G). (iii) if {xn} is a sequence in X such that (xn,xn+) ∈E(G) for all n ∈N ∪{} and xn →x as n →+∞, then (xn,x) ∈E(G) for all n ∈N ∪{}. Then T has a best proximity point. Further, the best proximity point is unique if, for every x,y ∈A such that d(x,Tx) = d(A,B) = d(y,Ty), we have (x,y) ∈E(G). Corollary Let A, B be two nonempty closed subsets of a metric space (X,d) endowed with a graph G. Assume that A is complete, B is approximatively compact with respect to A, and Ais nonempty. Assume a + b + c + d < . Also, suppose that T : A →B satisﬁes the following conditions: (i) T(A) ⊆B, (ii) there exist elements x,x∈Asuch that (ii) there exist elements x,x∈Asuch that d(x,Tx) = d(A,B) and (x,x) ∈E(G), d(x,Tx) = d(A,B) and (x,x) ∈E(G), Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 Page 12 of 19 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 Page 12 of 19 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 12 of 19 (iii) if {xn} is a sequence in X such that (xn,xn+) ∈E(G) for all n ∈N ∪{} and xn →x as n →+∞, then (xn,x) ∈E(G) for all n ∈N ∪{}, (i ) f A ⎧ ⎪⎨ ⎪⎩ (x,x) ∈E(G), d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⎧ ⎪⎨ ⎪⎩ (x,x) ∈E(G), d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ d(u,u) ≤ad(x,x) + b[+ d(x,u)]d(x,u) + d(x,x) + c d(x,u) + d(x,u) + d d(x,u) + d(x,u) . ⎩( , ) ( , ) ⇒ d(u,u) ≤ad(x,x) + b[+ d(x,u)]d(x,u) + d(x,x) + c d(x,u) + d(x,u) + d d(x,u) + d(x,u) . ⇒ d(u,u) ≤ad(x,x) + b[+ d(x,u)]d(x,u) + d(x,x) + c d(x,u) + d(x,u) + d d(x,u) + d(x,u) . ⇒ d(u,u) ≤ad(x,x) + b[+ d(x,u)]d(x,u) + d(x,x) + c d(x,u) + d(x,u) + d d(x,u) + d(x,u) . 4 Some results in metric spaces endowed with a partially ordered The study of existence of ﬁxed points in partially ordered sets has been established by Ran and Reurings [] with applications to matrix equations. Agarwal et al. [], Ćirić et al. [], and Hussain et al. [, ] obtained some new ﬁxed point results for nonlinear contractions in partially ordered Banach and metric spaces with some applications. In this Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 13 of 19 section, as an application of our results we derive some new best proximity point results whenever the range space is endowed with a partial order. section, as an application of our results we derive some new best proximity point results whenever the range space is endowed with a partial order. Deﬁnition [] Let (X,d,⪯) be a partially ordered metric space. We say that a nonself mapping T : A →B is proximally ordered-preserving if and only if, for all x,x,u,u∈A, ⎧ ⎪⎨ ⎪⎩ x⪯x, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ u⪯u. ⎧ ⎪⎨ ⎪⎩ x⪯x, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ u⪯u. Theorem Let A, B be two nonempty closed subsets of a partially ordered metric space (X,d,⪯) such that A is complete, B is approximatively compact with respect to A, and A is nonempty. Assume that T : A →B satisﬁes the following conditions: Theorem Let A, B be two nonempty closed subsets of a partially ordered metric space (X,d,⪯) such that A is complete, B is approximatively compact with respect to A, and A is nonempty. Assume that T : A →B satisﬁes the following conditions: (i) T is continuous and proximally ordered-preserving such that T(A) ⊆B, (ii) there exist elements x,x∈Asuch that (i) T is continuous and proximally ordered-preserving such that T(A) ⊆B, (ii) th i t l t ∈A h th t (i) T is continuous and proximally ordered-preserving such that T(A) ⊆B, (ii) there exist elements x,x∈Asuch that p y p g (ii) there exist elements x,x∈Asuch that d(x,Tx) = d(A,B) and x⪯x, (iii) for all x,y,u,v ∈A, (iii) for all x,y,u,v ∈A, ⎧ ⎪⎨ ⎪⎩ x ⪯y, d(u,Tx) = d(A,B), d(y,Ty) = d(A,B) (.) ⇒ F d(u,v),d(x,y),d(x,u),d(y,v),d(y,u),d(x,v) ≤. (.) Then T has a best proximity point. Then T has a best proximity point. Then T has a best proximity point. Proof Deﬁne α : A × A →[,+∞) by Proof Deﬁne α : A × A →[,+∞) by α(x,y) = , if x ⪯y,  , otherwise. α(x,y) = , if x ⪯y,  , otherwise. Firstly, we prove that T is an α-proximal admissible mapping. To this aim, assume Firstly, we prove that T is an α-proximal admissible mapping. To this aim, assume ⎧ ⎪⎨ ⎪⎩ α(x,y) ≥, d(u,Tx) = d(A,B), d(v,Ty) = d(A,B). d(u,Tx) = d(A,B), Therefore, we have Therefore, we have Therefore, we have ⎧ ⎪⎨ ⎪⎩ x ⪯y, d(u,Tx) = d(A,B), d(v,Ty) = d(A,B). ⎧ ⎪⎨ ⎪⎩ x ⪯y, d(u,Tx) = d(A,B), d(v,Ty) = d(A,B). ⎧ ⎪⎨ ⎪⎩ x ⪯y, d(u,Tx) = d(A,B), d(v,Ty) = d(A,B). ⎨ ⎪⎩d(v,Ty) = d(A,B). Now, since T is proximally ordered-preserving, then u ⪯v, that is, α(u,v) ≥. Further, by (ii) we have d(x,Tx) = d(A,B) and α(x,x) ≥. d(x,Tx) = d(A,B) and α(x,x) ≥. Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 14 of 19 Moreover, from (iii) we get ⎧ ⎪⎨ ⎪⎩ α(x,y) ≥, d(u,Tx) = d(A,B), d(y,Ty) = d(A,B) ⇒ F d(u,v),d(x,y),d(x,u),d(y,v),d(y,u),d(x,v) ≤. Thus all the conditions of Theorem hold (when L = ) and T has a best proximity point. □ Theorem Let A, B be two nonempty closed subsets of a partially ordered metric space (X,d,⪯) such that A is complete, B is approximatively compact with respect to A, and A is nonempty. Assume that T : A →B satisﬁes the following conditions: (i) T is proximally ordered-preserving such that T(A) ⊆B, (ii) there exist elements x,x∈Asuch that (ii) there exist elements x,x∈Asuch that d(x,Tx) = d(A,B) and x⪯x, d(x,Tx) = d(A,B) and x⪯x, (iii) for all x,y,u,v ∈A, (iii) for all x,y,u,v ∈A, ⎧ ⎪⎨ ⎪⎩ x ⪯y, d(u,Tx) = d(A,B), d(y,Ty) = d(A,B) ⎧ ⎪⎨ ⎪⎩ x ⪯y, d(u,Tx) = d(A,B), d(y,Ty) = d(A,B) ⎧ ⎪⎨ ⎪⎩ x ⪯y, d(u,Tx) = d(A,B), d(y,Ty) = d(A,B) ⇒ F d(u,v),d(x,y),d(x,u),d(y,v),d(y,u),d(x,v) ≤, (.) (.) (iv) if {xn} is an increasing sequence in A converging to x ∈A, then xn ⪯x for all n ∈N. Then T has a best proximity point. Corollary Let A, B be two nonempty closed subsets of a partially ordered metric space (X,d,⪯) such that A is complete, B is approximatively compact with respect to A, and A is nonempty. Then T has a best proximity point. Also, suppose that T : A →B satisﬁes the following conditions: ( ) ( ) (ii) there exist elements x,x∈Asuch that (ii) there exist elements x,x∈Asuch that d(x,Tx) = d(A,B) and x⪯x, (iii) if {xn} is a sequence in X such that xn ⪯xn+for all n ∈N ∪{} and xn →x as n →+∞, then xn ⪯x for all n ∈N ∪{}, (iv) for x,x,u,u∈A, (iii) if {xn} is a sequence in X such that xn ⪯xn+for all n ∈N ∪{} and xn →x as n →+∞, then xn ⪯x for all n ∈N ∪{}, (iv) for x,x,u,u∈A, ⎧ ⎪⎨ ⎪⎩ x⪯x, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⎧ ⎪⎨ ⎪⎩ x⪯x, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ d(u,u) ≤ψ max d(x,x),d(x,u),d(x,u), d(x,u) + d(x,u)  + Lmin d(x,u),d(x,u),d(x,u),d(x,u) , ⇒ d(u,u) ≤ψ max d(x,x),d(x,u),d(x,u), d(x,u) + d(x,u)  + Lmin d(x,u),d(x,u),d(x,u),d(x,u) , where ψ ∈Ψ . where ψ ∈Ψ . ψ Then T has a best proximity point. Further, the best proximity point is unique if, for every x,y ∈A such that d(x,Tx) = d(A,B) = d(y,Ty), we have x ⪯y. Then T has a best proximity point. Assume a+b+c+d < . Also, suppose that T : A →B satisﬁes the following conditions: (i) T(A) ⊆B, (i) T(A) ⊆B, (i) T(A) ⊆B, (ii) there exist elements x,x∈Asuch that d(x,Tx) = d(A,B) and x⪯x, d(x,Tx) = d(A,B) and x⪯x, (iii) if {xn} is a sequence in X such that xn ⪯xn+for all n ∈N ∪{} and xn →x as n →+∞, then xn ⪯x for all n ∈N ∪{}, (iv) for x,x,u,u∈A, (iii) if {xn} is a sequence in X such that xn ⪯xn+for all n ∈N ∪{} and xn →x as n →+∞, then xn ⪯x for all n ∈N ∪{}, (iv) for x,x,u,u∈A, (iv) for x,x,u,u∈A, ⎧ ⎪⎨ ⎪⎩ x⪯x, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ d(u,u) ≤ad(x,x) + b[+ d(x,u)]d(x,u) + d(x,x) + c d(x,u) + d(x,u) + d d(x,u) + d(x,u) . ⎧ ⎪⎨ ⎪⎩ x⪯x, d(u,Tx) = d(A,B), d(u,Tx) = d(A,B) ⇒ d(u,u) ≤ad(x,x) + b[+ d(x,u)]d(x,u) + d(x,x) b[+ d(x,u)]d(x,u) + d(x,x) + c d(x,u) + d(x,u) + d d(x,u) + d(x,u) . Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 15 of 19 Then T has a best proximity point. Further, the best proximity point is unique if, for every x,y ∈A such that d(x,Tx) = d(A,B) = d(y,Ty), we have x ⪯y. Corollary Let A, B be two nonempty closed subsets of a partially ordered metric space (X,d,⪯) such that A is complete, B is approximatively compact with respect to A, and A is nonempty. 5 Application to ﬁxed point theory 5.1 Implicit relation type modiﬁed α-contraction Then T has a ﬁxed point. Theorem Let (X,d) be a complete metric space. Assume that T : X →X is a self- mapping and the following conditions hold: (i) T is α-admissible, (ii) there exists xin X such that α(x,x) ≥and α(x,Tx) ≥, (iii) T is an implicit relation type modiﬁed α-contraction, 5.1 Implicit relation type modiﬁed α-contraction Deﬁnition [] Let T be a self-mapping on X and α : X × X →[,+∞) be a function. We say that T is an α-admissible mapping if Deﬁnition [] Let T be a self-mapping on X and α : X × X →[,+∞) be a function. Deﬁnition [] Let T be a self-mapping on X and α : X × X →[,+∞) be a function. We say that T is an α-admissible mapping if Deﬁnition [] Let T be a self-mapping on X and α : X × X →[,+∞) be a function. We say that T is an α-admissible mapping if We say that T is an α-admissible mapping if We say that T is an α-admissible mapping if x,y ∈X, α(x,y) ≥ ⇒ α(Tx,Ty) ≥. Remark Note that every α-admissible mappings are α-proximal admissible mappings when A = B = X. Deﬁnition Let (X,d) be a metric space and α : A × A →[,∞) be a function. Then T : X →X is said to be an implicit relation type α-contraction, if for all x,y ∈X with α(x,y) ≥, we have F d(Tx,Ty),d(x,y),d(x,Tx),d(y,Ty),d(y,Tx),d(x,Ty) ≤L – α(x,x)α(y,y) , (.) F d(Tx,Ty),d(x,y),d(x,Tx),d(y,Ty),d(y,Tx),d(x,Ty) ≤L – α(x,x)α(y,y) , F d(Tx,Ty),d(x,y),d(x,Tx),d(y,Ty),d(y,Tx),d(x,Ty) F d(Tx,Ty),d(x,y),d(x,Tx),d(y,Ty),d(y,Tx),d(x,Ty) (.) ≤L – α(x,x)α(y,y) , (.) where L ≥and F ∈F. where L ≥and F ∈F. Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Page 16 of 19 Theorem Let (X,d) be a complete metric space. Assume that T : X →X is a continuous self-mapping satisfying the following conditions: (i) T is α-admissible, (ii) there exists xin X such that α(x,x) ≥and α(x,Tx) ≥, (ii) there exists xin X such that α(x,x) ≥and α(x,Tx) ≥, (iii) T is an implicit relation type modiﬁed α-contraction. (iii) T is an implicit relation type modiﬁed α-contraction. Then T has a ﬁxed point. 5.2 Implicit relation type G-contraction Assume that T : X →X is a self-mapping satisfying the following conditions: (i) there exists xin X such that (x,Tx) ∈E(G), (ii) T is an implicit relation type G-contraction, (iii) if {xn} is a sequence in X such that (xn,xn+) ∈E(G) and xn →x as n →+∞, then (xn,x) ∈E(G) for all n ∈N. Then T has a ﬁxed point. Theorem Let (X,d) be a complete metric space endowed with a graph G. Assume that T : X →X is a self-mapping satisfying the following conditions: (i) there exists xin X such that (x,Tx) ∈E(G), (ii) T is an implicit relation type G-contraction, (iii) if {xn} is a sequence in X such that (xn,xn+) ∈E(G) and xn →x as n →+∞, then (xn,x) ∈E(G) for all n ∈N. Then T has a ﬁxed point Then T has a ﬁxed point. Using Example and Theorem , we deduce the following result. Corollary Let (X,d) be a complete metric space. Assume that T : X →X is a self- mapping and the following conditions hold: Corollary Let (X,d) be a complete metric space. Assume that T : X →X is a self- mapping and the following conditions hold: (i) T is α-admissible, (ii) there exists xin X such that α(x,x) ≥and α(x,Tx) ≥, (ii) there exists xin X such that α(x,x) ≥and α(x,Tx) ≥, (iii) for all x,y ∈X with α(x,y) ≥we have d(Tx,Ty) + Lα(x,x)α(y,y) ≤ad(x,y) + b[+ d(x,Tx)]d(y,Ty) + d(x,y) + c d(x,Tx) + d(y,Ty) + d d(y,Tx) + d(x,Ty) + L, d(Tx,Ty) + Lα(x,x)α(y,y) ≤ad(x,y) + b[+ d(x,Tx)]d(y,Ty) + d(x,y) + c d(x,Tx) + d(y,Ty) + d d(y,Tx) + d(x,Ty) + L, (iv) if {xn} is a sequence in X such that α(xn,xn+) ≥and xn →x as n →+∞, then α(x,x) ≥and α(xn,x) ≥for all n ∈N. Then T has a ﬁxed point. ﬁ p Corollary Let (X,d) be a complete metric space. Assume that T : X →X is a self- mapping and the following conditions hold: (i) T is α-admissible, (ii) there exists xin X such that α(x,x) ≥and α(x,Tx) ≥, (iii) for all x,y ∈X with α(x,y) ≥we have d(Tx,Ty) + Lα(x,x)α(y,y) ≤ad(x,y) + L, where ≤a < and L ≥, (iv) if {xn} is a sequence in X such that α(xn,xn+) ≥and xn →x as n →+∞, then α(x,x) ≥and α(xn,x) ≥for all n ∈N. Then T has a ﬁxed point. Corollary Let (X,d) be a complete metric space. Assume that T : X →X is a self- mapping and the following conditions hold: (i) T is α-admissible, (ii) there exists xin X such that α(x,x) ≥and α(x,Tx) ≥, (iii) for all x,y ∈X with α(x,y) ≥we have (iii) for all x,y ∈X with α(x,y) ≥we have where ≤a < and L ≥, (iv) if {xn} is a sequence in X such that α(xn,xn+) ≥and xn →x as n →+∞, then α(x,x) ≥and α(xn,x) ≥for all n ∈N. Then T has a ﬁxed point. Page 17 of 19 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 5.2 Implicit relation type G-contraction 5.2 Implicit relation type G-contraction Deﬁnition [] We say that a mapping T : X →X is a Banach G-contraction or simply G-contraction if T preserves edges of G, i.e., 5.2 Implicit relation type G-contraction Deﬁnition [] We say that a mapping T : X →X is a Banach G-contraction or simply G-contraction if T preserves edges of G, i.e., Deﬁnition [] We say that a mapping T : X →X is a Banach G-contraction or simply G-contraction if T preserves edges of G, i.e., ∀x,y ∈X (x,y) ∈E(G) ⇒ T(x),T(y) ∈E(G) and T decreases weights of edges of G in the following way: and T decreases weights of edges of G in the following way: ∃α ∈(,),∀x,y ∈X (x,y) ∈E(G) ⇒d T(x),T(y) ≤αd(x,y) . ∃α ∈(,),∀x,y ∈X (x,y) ∈E(G) ⇒d T(x),T(y) ≤αd(x,y) . Deﬁnition [] A mapping T : X →X is called G-continuous, if for given x ∈X and sequence {xn} xn →x as n →∞ and (xn,xn+) ∈E(G) for all n ∈N imply Txn →Tx. Deﬁnition Let (X,d) be a metric space endowed with a graph G. Then T : X →X is said to be an implicit relation type G-contraction, if, for all x,y ∈X, Deﬁnition Let (X,d) be a metric space endowed with a graph G. Then T : X →X is said to be an implicit relation type G-contraction, if, for all x,y ∈X, (x,y) ∈E(G) ⇒ (Tx,Ty) ∈E(G) and and (x,y) ∈E(G) ⇒ F d(Tx,Ty),d(x,y),d(x,Tx),d(y,Ty),d(y,Tx),d(x,Ty) ≤, (x,y) ∈E(G) ⇒ F d(Tx,Ty),d(x,y),d(x,Tx),d(y,Ty),d(y,Tx),d(x,Ty) ≤, where F ∈F. where F ∈F. Theorem Let (X,d) be a complete metric space endowed with a graph G. Assume that T : X →X is a continuous self-mapping satisfying the following conditions: (i) there exists xin X such that (x,Tx) ∈E(G), (ii) T is an implicit relation type G-contraction. Then T has a ﬁxed point. Theorem Let (X,d) be a complete metric space endowed with a graph G. Assume that T : X →X is a self-mapping satisfying the following conditions: Theorem Let (X,d) be a complete metric space endowed with a graph G. b[+ d(x,Tx)]d(y,Ty) + d(x,y) Then T has a ﬁxed point. Then T has a ﬁxed point. Then T has a ﬁxed point. Corollary Let (X,d,⪯) be complete metric space. Assume that T : X →X is a self- mapping that satisﬁes the following conditions: (i) there exist element x∈X such that x⪯Tx, (ii) if {xn} is an increasing sequence in X such that xn →x as n →+∞, then xn ⪯x for all n ∈N ∪{}, (ii) if {xn} is an increasing sequence in X such that xn →x as n →+∞, then xn ⪯x for all n ∈N ∪{}, d(Tx,Ty) ≤ψ max d(x,y),d(x,Tx),d(y,Ty), d(y,Tx) + d(x,Ty)  + Lmin d(x,Tx),d(y,Ty),d(y,Tx),d(x,Ty) , d(Tx,Ty) ≤ψ max d(x,y),d(x,Tx),d(y,Ty), d(y,Tx) + d(x,Ty)  + Lmin d(x,Tx),d(y,Ty),d(y,Tx),d(x,Ty) , where ψ ∈Ψ . Then T has a ﬁxed point. where ψ ∈Ψ . Then T has a ﬁxed point. Competing interests Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. Authors’ contributions All authors contributed equally to the writing of this paper. All authors read and approved the ﬁnal manuscript. 5.3 Implicit relation type ordered contraction y Theorem ([], Theorem .) Let (X,d,⪯) be a partially ordered complete metric space. Assume that T : X →X is a self-mapping that satisﬁes the following conditions: (i) there exists xin X such that x⪯Tx, (ii) for all x,y ∈X with x ⪯y we have F d(Tx,Ty),d(x,y),d(x,Tx),d(y,Ty),d(y,Tx),d(x,Ty) ≤, where F ∈F, Page 18 of 19 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 Zabihi and Razani Journal of Inequalities and Applications 2014, 2014:365 http://www.journalofinequalitiesandapplications.com/content/2014/1/365 (iii) either T is continuous or if {xn} is an increasing sequence in X such that xn →x as n →+∞, then xn ⪯x for all n ∈N. Then T has a ﬁxed point. Corollary Let (X,d,⪯) be complete metric space. Assume a + b + c + d < . Also, suppose that T : X →X is a self-mapping that satisﬁes the following conditions: (i) there exists an element x∈X such that x⪯Tx, (ii) if {xn} is an increasing sequence in X such that xn →x as n →+∞, then xn ⪯x for all n ∈N ∪{}, (ii) if {xn} is an increasing sequence in X such that xn →x as n →+∞, then xn ⪯x for all n ∈N ∪{}, (iii) for x,y ∈X with x ⪯y, Authors’ contributions ll h b d Authors’ contributions All authors contributed equally to the writing of this paper. All authors read and approved the ﬁnal manuscript. Received: 29 June 2014 Accepted: 9 September 2014 Published: 24 Sep 2014 Received: 29 June 2014 Accepted: 9 September 2014 Published: 24 Sep 2014 References References 1. Popa, V: A general coincidence theorem for compatible multivalued mappings satisfying an implicit relation. Demonstr. Math. 33(1), 159-164 (2000) References 1. Popa, V: A general coincidence theorem for compatible multivalued mappings satisfying an implicit relation. Demonstr. 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https://openalex.org/W3039875026	https://scholarlypublications.universiteitleiden.nl/access/item%3A2969677/view	English	null	Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening	International journal of molecular sciences	2,020	cc-by	6,303	Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC- Derived Endothelium for Anti-Angiogenic Drug Screening Duinen, V. van; Stam, W.; Mulder, E.; Famili, F.; Reijerkerk, A.; Vulto, P.; ... ; Zonneveld, A.J. van Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC- Derived Endothelium for Anti-Angiogenic Drug Screening Duinen, V. van; Stam, W.; Mulder, E.; Famili, F.; Reijerkerk, A.; Vulto, P.; ... ; Zonneveld, A.J. van Received: 12 May 2020; Accepted: 2 July 2020; Published: 7 July 2020 Abstract: To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both signiﬁcantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantiﬁed for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds. Keywords: angiogenesis; drug screening; microﬂuidics; iPSC; endothelial cells Citation Citation Duinen, V. van, Stam, W., Mulder, E., Famili, F., Reijerkerk, A., Vulto, P., … Zonneveld, A. J. van. (2020). Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening. International Journal Of Molecular Sciences, 21(13), 4804. doi:10.3390/ijms21134804 Citation Duinen, V. van, Stam, W., Mulder, E., Famili, F., Reijerkerk, A., Vulto, P., … Zonneveld, A. J. van. (2020). Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening. International Journal Of Molecular Sciences, 21(13), 4804. doi:10.3390/ijms21134804 Version: Publisher's Version License: Creative Commons CC BY 4.0 license Downloaded from: https://hdl.handle.net/1887/138157 License: Note: To cite this publication please use the final published version (if applicable). Note: To cite this publication please use the final published version (if applica International Journal of Molecular Sciences International Journal of Molecular Sciences Robust and Scalable Angiogenesis Assay of 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening Vincent van Duinen 1,2,* , Wendy Stam 1, Eva Mulder 1, Farbod Famili 3, Arie Reijerkerk 3, Paul Vulto 4, Thomas Hankemeier 2 and Anton Jan van Zonneveld 1,* 1 Einthoven Laboratory for Vascular and Regenerative Medicine, Department of Internal Medicine (Nephrology), Leiden University Medical Center, 2333ZA Leiden, The Netherlands; w.stam@lumc.nl (W.S.); e.mulder01@gmail.com (E.M.) g 2 Department of Analytical BioSciences and Metabolomics, Division of Systems Biomedicine and Pharmacology, Leiden University, 2333CC Leiden, The Netherlands; hankemeier@lacdr.leidenuniv.nl 3 Ncardia, 2333 BD Leiden, The Netherlands; f.famili@ncardia.com (F.F.); a.reijerkerk@ncardia.com (A.R.) 4 Mimetas, 2333 CA Leiden, The Netherlands; p.vulto@mimetas.com 2 Department of Analytical BioSciences and Metabolomics, Division of Systems Biomedicine and Pharmacology, Leiden University, 2333CC Leiden, The Netherlands; hankemeier@lacdr.leidenuniv.nl 3 Ncardia, 2333 BD Leiden, The Netherlands; f.famili@ncardia.com (F.F.); a.reijerkerk@ncardia.com (A.R.) 4 Mimetas, 2333 CA Leiden, The Netherlands; p.vulto@mimetas.com p * Correspondence: vvanduinen@lumc.nl (V.v.D.); a.j.van_zonneveld@lumc.nl (A.J.v.Z.) 1. Introduction Angiogenesis, the growth of new blood vessels from pre-existing vasculature, plays a fundamental role in both health and disease [1]. For the discovery of new drug targets that target the angiogenesis process, drug research heavily relies on in vitro models. However, currently used in vitro models have limited translatability to the in vivo situation [2,3]. To meet the demands of pre-clinal vascular drug research, improved in vitro models of angiogenesis are required: assays that are amenable to high-throughput screening, with a scalable and robust endothelial cell (EC) source in a more physiologically relevant cellular micro-environment [4,5]. p y g y Within the last decade, signiﬁcant progress has been made to increase the translational value of in vitro models of angiogenesis. For example, ECs embedded in three-dimensional scaﬀolds such as ﬁbrin and collagen gels show an increased physiologically relevant phenotype, including the presence Int. J. Mol. Sci. 2020, 21, 4804; doi:10.3390/ijms21134804 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 4804 Int. J. Mol. Sci. 2020, 21, 4804 2 of 10 of tip and stalk cells. The tip cells are able to degrade the extracellular matrix, while the stalk cells form lumen [6,7]. However, important cues from the cellular microenvironment, such as biomolecular gradients and ﬂow, are still lacking. The use of microﬂuidic cell culture platforms can further increase the physiological relevancy of in vitro models, as perfusion of culture media induces shear stress in the ECs and allows spatial- temporal control over biomolecular gradients [8]. This has resulted in angiogenic sprouting models with increased physiological relevancy over traditional 2D and 3D cell culture methods. For example, it enables the formation of gradients in combination with a 3D scaﬀold, the stratiﬁcation of cells to force polarization and the possibility to apply luminal perfusion [9–15]. However, most platforms lack the required throughput and scalability in order to be amenable to drug research in general, and drug screening in particular [16]. Furthermore, many of these platforms require the end-user to microfabricate their devices prior to use [17]. This not only requires manufacturing equipment and technical knowledge; it also limits the level of quality control and negatively aﬀects the reproducibility and standardization [18]. To date, primary human ECs such as human umbilical vein endothelial cells (HUVECs) remain the most widely used cell source to model angiogenesis in vitro [4]. 1. Introduction However, while HUVECs have the advantage of being widely available, being robust in their performance and being expandable up to a certain level, the performance of these primary endothelial cells can widely vary from donor to donor, limiting their use for high throughput assays over longer times. ECs derived from induced pluripotent stem cells (iPSC) are a promising alternative. Since iPSCs are self-renewing, they can be expanded in nearly unlimited quantities, and yield endothelial cells with phenotypic properties highly like primary endothelial cells. In addition, iPSCs are amenable to precise gene editing, allowing the introduction of a ﬂuorescent phenotypic reporter that facilitates high throughput imaging. Together, these properties make iPSC-EC a highly favorable source for in vitro screening models assays of endothelial function [19]. Recently, we introduced a platform technology that comprises 40 microﬂuidic chips patterned underneath a microtiter plate [13,20]. We showed formation of a small EC vessel structures with perfused lumen, grown against an extracellular matrix. In this model, like primary ECs, iPSC-ECs, showed important aspects of angiogenic sprouting, including the diﬀerentiation into tip cells that display their characteristic ﬁlopodia and the trailing stalk cells forming lumen [13]. Both directional and repetitive sprouting was observed, which was an indication that iPSC-ECs can sense the imposed vascular endothelial growth factor (VEGF) gradient and suggested that this could be used to screen for anti-angiogenic properties. Here, we investigate whether this platform is suitable for anti-angiogenic screening. We compared the angiogenic response of iPSC-ECs with HUVEC and study the eﬀect of sunitinib and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), two anti-angiogenic compounds that have been shown to reduce angiogenesis in vitro as well as in vivo [21]. Sunitinib is a clinically available tyrosine kinase inhibitor that targets the vascular endothelial growth factor receptor 2 (VEGFR2) and platelet-derived growth factor receptor beta (PDGFRβ). As a glycolysis inhibitor, 3PO targets 6-phosphofructo-2-kinase/fructose-2,6-bisphsphatase isozyme 3 (PFKFB3). We calculated the signal window, Z’ and assay variability window for several metrics to quantify the assay performance and assess its suitability for drug screening. 2. Results The platform consists of 40 individually addressable microﬂuidic units (Figure 1a,b), in which 40 perfused microvessels are cultured against a patterned collagen-I gel. First, we validated the usage of iPSC-ECs in our angiogenesis model [20]. Brieﬂy, collagen-1 gel precursor is patterned inside the chips by a surface tension technique named phaseguiding [22]. After polymerization, ﬁbronectin coating was added in the adjacent channel. Cells were seeded in this coated channel and after they were adhered, additional culture media was added to the wells addressing the lumen. The device was Int. J. Mol. Sci. 2020, 21, 4804 3 of 10 placed on an interval rocker platform set at a 7 degrees inclination angle and an 8 min interval to induce ﬂow through passive levelling between the reservoirs, and thus sustain gradients of angiogenic factors (Figure 1c). Formation of conﬂuent iPSC-EC microvessels was realized after 2 days (Figure 1d), after which angiogenesis was triggered by a gradient of angiogenic growth factors (50 ng/mL VEGF + 500 mM, Sphingosine-1-Phosphate (S1P) + 2 µg/mL phorbol 12-myristate 13-acetate (PMA) for another two days. This shows that iPSC-ECs form angiogenic sprouts including tip cells as well as their characteristic ﬁlopodia and stalk cells that form perfusable lumen (Figure 1e). Cells were stained for nucleus and F-actin and images were subsequently used for automatic segmentation and quantiﬁcation of the angiogenic sprouting (Figure 1f). The sprouting area, number of nuclei within the sprouts and sprouting distance were quantiﬁed using built-in image analysis protocols of the software of the high-content microscope. Since max projections were used for quantiﬁcation, branches and nodes are not meaningful and thus not included in the quantiﬁed parameters. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 9 (Figure 1d), after which angiogenesis was triggered by a gradient of angiogenic growth factors (50 ng/mL VEGF + 500 mM, Sphingosine-1-Phosphate (S1P) + 2 µg/mL phorbol 12-myristate 13-acetate (PMA) for another two days. This shows that iPSC-ECs form angiogenic sprouts including tip cells as well as their characteristic filopodia and stalk cells that form perfusable lumen (Figure 1e). Cells were stained for nucleus and F-actin and images were subsequently used for automatic segmentation and quantification of the angiogenic sprouting (Figure 1f). The sprouting area, number of nuclei within the sprouts and sprouting distance were quantified using built-in image analysis protocols of the software of the high-content microscope. 2. Results On the right 1 of 40 microfluidic units that are integrated underneath the 384-well plate is depicted. (b) Schematic overview of a single microfluidic unit/chip. The microfluidic channels are separated by ridges (‘phaseguides’), which enable the patterning of hydrogels in the central channel (‘gel channel’) while there is still contact with the adjacent channels (‘perfusion channels’). (c) Method to culture a microvessel within a microfluidic device and induce gradient driven angiogenic sprouting. (d) Microvessel 2 days after seeding iPSC-ECs as single cells. Cells form a monolayer against the patterned collagen-1 gel stained for F-actin (yellow) and nucleus (blue). (e) Gradient driven sprouting angiogenesis of iPSC-ECs after 2 days of stimulation with angiogenic growth factors. (f) Automated segmentation of vessel-like structures within the collagen-1 gel. Reproduced from van Duinen, V.; et al. Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In vitro. J. Vis. Exp. 2019 [13]. Figure 1. Angiogenesis assay of perfused induced pluripotent stem cell–endothelial cell (iPSC-EC microvessels. (a) Bottom of the microﬂuidic cell culture device. On the right 1 of 40 microﬂuidic units that are integrated underneath the 384-well plate is depicted. (b) Schematic overview of a single microﬂuidic unit/chip. The microﬂuidic channels are separated by ridges (‘phaseguides’), which enable the patterning of hydrogels in the central channel (‘gel channel’) while there is still contact with the adjacent channels (‘perfusion channels’). (c) Method to culture a microvessel within a microﬂuidic device and induce gradient driven angiogenic sprouting. (d) Microvessel 2 days after seeding iPSC-ECs as single cells. Cells form a monolayer against the patterned collagen-1 gel stained for F-actin (yellow) and nucleus (blue). (e) Gradient driven sprouting angiogenesis of iPSC-ECs after 2 days of stimulation with angiogenic growth factors. (f) Automated segmentation of vessel-like structures within the collagen-1 gel. Reproduced from van Duinen, V.; et al. Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In vitro. J. Vis. Exp. 2019 [13]. Next, we optimized the concentration for sunitinib inhibition (Figure 2a, Figure S1), and show that sunitinib inhibits sprouting from concentrations >10 nM, while angiogenesis is completely inhibited at concentrations >50 nM. 2. Results Since max projections were used for quantification, branches and nodes are not meaningful and thus not included in the quantified parameters. Figure 1. Angiogenesis assay of perfused induced pluripotent stem cell–endothelial cell (iPSC-EC microvessels. (a) Bottom of the microfluidic cell culture device. On the right 1 of 40 microfluidic units that are integrated underneath the 384-well plate is depicted. (b) Schematic overview of a single microfluidic unit/chip. The microfluidic channels are separated by ridges (‘phaseguides’), which enable the patterning of hydrogels in the central channel (‘gel channel’) while there is still contact with the adjacent channels (‘perfusion channels’). (c) Method to culture a microvessel within a microfluidic device and induce gradient driven angiogenic sprouting. (d) Microvessel 2 days after seeding iPSC-ECs as single cells. Cells form a monolayer against the patterned collagen-1 gel stained for F-actin (yellow) and nucleus (blue). (e) Gradient driven sprouting angiogenesis of iPSC-ECs after 2 days of stimulation with angiogenic growth factors. (f) Automated segmentation of vessel-like structures within the collagen-1 gel. Reproduced from van Duinen, V.; et al. Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In vitro. J. Vis. Exp. 2019 [13]. Figure 1. Angiogenesis assay of perfused induced pluripotent stem cell–endothelial cell (iPSC-EC microvessels. (a) Bottom of the microﬂuidic cell culture device. On the right 1 of 40 microﬂuidic units that are integrated underneath the 384-well plate is depicted. (b) Schematic overview of a single microﬂuidic unit/chip. The microﬂuidic channels are separated by ridges (‘phaseguides’), which enable the patterning of hydrogels in the central channel (‘gel channel’) while there is still contact with the adjacent channels (‘perfusion channels’). (c) Method to culture a microvessel within a microﬂuidic device and induce gradient driven angiogenic sprouting. (d) Microvessel 2 days after seeding iPSC-ECs as single cells. Cells form a monolayer against the patterned collagen-1 gel stained for F-actin (yellow) and nucleus (blue). (e) Gradient driven sprouting angiogenesis of iPSC-ECs after 2 days of stimulation with angiogenic growth factors. (f) Automated segmentation of vessel-like structures within the collagen-1 gel. Reproduced from van Duinen, V.; et al. Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In vitro. J. Vis. Exp. 2019 [13]. Figure 1. Angiogenesis assay of perfused induced pluripotent stem cell–endothelial cell (iPSC-EC microvessels. (a) Bottom of the microfluidic cell culture device. 2. Results To characterize the signal window (SW), Z-factor (Z′) and the coefficient of variation (CV) for each parameter and select the most optimal quantification parameter (sprouting distance, nuclei in vessels and total vessel area), 0 nM of sunitinib was selected as the maximum signal (n = 15) and 50 nM of sunitinib as the minimum signal (n = 14) (Figure 2b,c). This shows that while all parameters have an acceptable Z-factor ≥0.4 and signal window >2, only sprouting distance showed an acceptable coefficient of variation of ≤20% (Table 1). Next, we optimized the concentration for sunitinib inhibition (Figure 2a, Figure S1), and show that sunitinib inhibits sprouting from concentrations >10 nM, while angiogenesis is completely inhibited at concentrations >50 nM. To characterize the signal window (SW), Z-factor (Z′) and the coeﬃcient of variation (CV) for each parameter and select the most optimal quantiﬁcation parameter (sprouting distance, nuclei in vessels and total vessel area), 0 nM of sunitinib was selected as the maximum signal (n = 15) and 50 nM of sunitinib as the minimum signal (n = 14) (Figure 2b,c). This shows that while all parameters have an acceptable Z-factor ≥0.4 and signal window >2, only sprouting distance showed an acceptable coeﬃcient of variation of ≤20% (Table 1). 4 of 10 of 9 Int. J. Mol. Sci. 2020, 21, 4804 t. J. Mol. Sci. 2020, 21, x FOR PEE Figure 2. Concentration optimization for inhibition of angiogenic sprouting of iPSC-ECs using sunitinib. (a) Representative images of a concentration range of sunitinib. (b) Quantification of the vessel area, nuclei density and sprouting distance of maximal inhibition (50 nM) and no inhibition (control, 0 nM). Figure 2. Concentration optimization for inhibition of angiogenic sprouting of iPSC-ECs using sunitinib. (a) Representative images of a concentration range of sunitinib. (b) Quantiﬁcation of the vessel area, nuclei density and sprouting distance of maximal inhibition (50 nM) and no inhibition (control, 0 nM). gure 2. Concentration optimization for inhibition of angiogenic sprouting of iPSC-ECs using unitinib. (a) Representative images of a concentration range of sunitinib. (b) Quantification of the essel area, nuclei density and sprouting distance of maximal inhibition (50 nM) and no inhibition ontrol, 0 nM). Figure 2. Concentration optimization for inhibition of angiogenic sprouting of iPSC-ECs using sunitinib. (a) Representative images of a concentration range of sunitinib. 2. Results (b) Quantiﬁcation of the vessel area, nuclei density and sprouting distance of maximal inhibition (50 nM) and no inhibition (control, 0 nM). Int. J. Mol. Sci. 2020, 21, 4804 5 of 10 Table 1. Assay performance characteristics of the quantiﬁed parameters for iPSC-ECs. The CV at maximum (CVmax) and minimum signal (CVmin) are derived from 0 nM and 50 nM respectively. Recommended values as found in Iversen et al. [23]. Total Area Distance Nuclei Reference Values [23] Signal window 11.70 14.76 11.68 >1 acceptable Z-factor 0.78 0.75 0.77 >0.5 excellent Assay variability ratio 0.84 0.94 0.88 <0.6 recommended CVmax (%) 25 13 24 <20 acceptable CVmin (%) 70 20 71 <20 acceptable Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 o Finally, we compared the inhibition of sprouting of iPSC-ECs with primary HUVECs using sunitinib (Figure 3a,b) and 3PO (Figure 3c,d). This showed that 50% of sprouting length reduction (IC50) is achieved at sunitinib concentrations of around 20 nM (95% conﬁdence interval (CI): 12.9–28.0) for iPSC-ECs and 66 nM (95% CI 43.70–88.58) for HUVECs. Interestingly, HUVECs still showed single cell migration into the collagen-I at high concentrations of sunitinib, whereas the sprouting of iPSC-ECs was completely blocked. Additionally, 3PO showed a signiﬁcant but partial inhibition of sprouting at a concentration of 10 µM, which was similar in iPSC-ECs and HUVECs. Finally, we compared the inhibition of sprouting of iPSC-ECs with primary HUVECs using sunitinib (Figure 3a,b) and 3PO (Figure 3c,d). This showed that 50% of sprouting length reduction (IC50) is achieved at sunitinib concentrations of around 20 nM (95% confidence interval (CI): 12.9– 28.0) for iPSC-ECs and 66 nM (95% CI 43.70–88.58) for HUVECs. Interestingly, HUVECs still showed single cell migration into the collagen-I at high concentrations of sunitinib, whereas the sprouting of iPSC-ECs was completely blocked. Additionally, 3PO showed a significant but partial inhibition of sprouting at a concentration of 10 µM, which was similar in iPSC-ECs and HUVECs. Figure 3. Quantification of inhibition in angiogenic sprouting of iPSC-EC and HUVEC microvessels. (a,b) Sunitinib inhibited angiogenic sprouting of both iPSC-EC and HUVEC microvessels. While sprouting of iPSC-ECs was completely inhibited at 50 nM, HUVECs still showed limited migration and sprout formation (N = 2). (c,d) Inhibition with 3PO shows a significant reduction in sprouting at 10 µM of both IPSC-EC and HUVEC microvessels (N = 2). Figure 3. 3. Discussion We described a phenotypic angiogenesis inhibition assay which consists of 40 individually addressable, perfused micro vessels in a standardized microﬂuidic cell culture platform that includes physiologically relevant cues such as a three-dimensional hydrogel, ﬂow and angiogenic gradients. The assay is shown to be robust and reproducible, showing the potential to be integrated within the drug-screening infrastructure and enables the study of the anti-angiogenic eﬀect of compounds. The parameters quantiﬁed in this study (sprout area, nuclei in vessels and sprout length) all showed acceptable Z’ for complex phenotype assays [23]. The quantiﬁcation of sprout length had the lowest CV and was the most robust parameter. Although total sprouting area is an interesting phenotypic readout to study, as this describes the lumen development and functioning tip and stalk cells [24], we observed diﬀerences in sprouting density. Probably, these diﬀerences in sprout density are caused by diﬀerences in initial seeding densities: proper sprout development requires DLL4-Notch signaling that is only expressed in conﬂuent monolayers [25,26]. We performed our analysis on 2D max-projection images, which reduces the spatial information to the beneﬁt of throughput and analysis time. This approach allows the quantiﬁcation of sprouting length and sprouting density. However, when number of sprouts and/or directionality of sprouts is of interest, one might give preference over 3D analysis. We validated the usage of iPSC-ECs by directly comparing with primary ECs and tested two angiogenic inhibitors: sunitinib and 3PO. Sunitinib inhibited the sprouting of both HUVECs and iPSC-ECs at nanomolar levels, which shows that the angiogenic sprouting of iPSC-ECs is mediated through VEGFR2 signaling. Interestingly, sunitinib completely inhibited the iPSC-EC sprout formation at concentrations ≥50 nM, while HUVECs still showed single cells invading into the collagen-1 matrix at this concentration. This suggests that either the iPSC-ECs are more sensitive to VEGF or less sensitive to other angiogenic factors present (e.g., PMA, S1P, basic ﬁbroblast growth factor). Interestingly, our data show that 3PO inhibits the angiogenic sprouting of iPSC-ECs, which suggests that, like HUVECs, they undergo the same metabolic switch to use glycolysis as the main energy source during angiogenic sprouting [21]. While it has been shown that some endothelial cells diﬀerentiated from iPSCs have lower activity of glycolysis [27], the question remains how the culture conditions dictate the cells phenotype, and whether this eﬀect could be attributed to the speciﬁc cues added by our culture platform. 3. Discussion For example, it has been shown that both tip cells and non-tip cells use glycolysis as well as mitochondrial respiration for energy production, and that this balance depends on microenvironmental circumstances [28]. 2. Results Quantiﬁcation of inhibition in angiogenic sprouting of iPSC-EC and HUVEC microvessels. (a,b) Sunitinib inhibited angiogenic sprouting of both iPSC-EC and HUVEC microvessels. While sprouting of iPSC-ECs was completely inhibited at 50 nM, HUVECs still showed limited migration and sprout formation (N = 2). (c,d) Inhibition with 3PO shows a signiﬁcant reduction in sprouting at 10 µM of both IPSC-EC and HUVEC microvessels (N = 2). Figure 3. Quantification of inhibition in angiogenic sprouting of iPSC-EC and HUVEC microvessels. (a,b) Sunitinib inhibited angiogenic sprouting of both iPSC-EC and HUVEC microvessels. While sprouting of iPSC-ECs was completely inhibited at 50 nM, HUVECs still showed limited migration and sprout formation (N = 2). (c,d) Inhibition with 3PO shows a significant reduction in sprouting at 10 µM of both IPSC-EC and HUVEC microvessels (N = 2). Figure 3. Quantiﬁcation of inhibition in angiogenic sprouting of iPSC-EC and HUVEC microvessels. (a,b) Sunitinib inhibited angiogenic sprouting of both iPSC-EC and HUVEC microvessels. While sprouting of iPSC-ECs was completely inhibited at 50 nM, HUVECs still showed limited migration and sprout formation (N = 2). (c,d) Inhibition with 3PO shows a signiﬁcant reduction in sprouting at 10 µM of both IPSC-EC and HUVEC microvessels (N = 2). Int. J. Mol. Sci. 2020, 21, 4804 6 of 10 4. Materials and Methods 4.1. Device Preparation and Cell Culture in Microﬂuidic Channels 4.1. Device Preparation and Cell Culture in Microﬂuidic Channels The protocol to culture endothelial cells as microvessels is described previously [13,20], with a few adaptions. HUVEC (in-house isolated with approval from the Medical Ethical Committee of the Leiden University Medical Center, Leiden, The Netherlands to use for research purposes) or iPSC-EC (NCardia, Leiden, The Netherlands) were thawed from liquid nitrogen and upon thawing resuspended in human endothelial serum free medium (HE-SFM, 11111044, Thermo Scientiﬁc, USA), centrifugated at 100 g for 5 min and resuspended to yield a concentration of 1·107 cells/mL. For every microﬂuidic unit, 1 µL of cell suspension was seeded in a channel pre-coated with 10 µg/mL ﬁbronectin (F4759-1MG, Sigma-Aldrich, The Netherlands) in Dulbecco’s phosphate-buﬀered saline (dPBS, 14190-094, Thermo Scientiﬁc, USA), adjacent to a patterned collagen-1 gel (3447-020-01, R&D systems, UK). The ECs were cultured for 2 days in medium supplemented with 30 ng/mL vascular endothelial growth factor-165 (450-32-10, Peprotech, USA) and 20 ng/mL bFGF (100-18b Peprotech, USA) (further referred to as vessel culture medium) to form conﬂuent microvessels. Int. J. Mol. Sci. 2020, 21, 4804 7 of 10 7 of 10 4.4. Sprouting Quantiﬁcation The sprouting was quantiﬁed using a custom module developed in Molecular Devices MetaXpress software (MetaMorph v6.5.2.351), which segments the max projection of the angiogenic sprouts into vessels and nuclei within the vessels to extract the total vessel area, the total vessel length and the y-position of the nuclei. We quantiﬁed the average migration distance per site by extracting the average Y-position of the 10 furthest nuclei in µm minus 400 µm (based on the average absolute y-position in the image where the monolayer grows against the gel for the negative controls). For the quantiﬁcation of the area, only areas containing nuclei were used. 4.2. Inhibition of Angiogenic Sprouting An angiogenic sprouting mixture was prepared by supplementing HE-SFM with 50 ng/mL VEGF, 2 ng/mL phorbol 12-myristate 13-acetate (PMA, 10-2165, Focus Biomolecules, USA) and 500 nM sphingosine-1-phosphate (S1P, S-2000-1 mg, Echelon Biosciences, USA). A total of 10 mM sunitinib stock solution in DMSO was ﬁrst diluted to 25 µM in basal medium, which was then serially diluted with 0.001% DMSO used as control. A stock solution in DMSO of 150 mM of 3PO was serially diluted, with 0.007% DMSO used as control. The angiogenic sprouting mixture with inhibitors was added to the bottom perfusion inlet well and outlet well to induce angiogenic sprouting, and a vessel culture medium containing the inhibitor was added to the gel inlet and outlet and the top perfusion inlet and outlet well. 4.3. Fixation, Staining and Imaging The cell culture medium was aspirated and 25 µL of 4% paraformaldehyde (PFA) in phosphate-buﬀered saline (J61899, Alfa Aesar, USA) was added to all the perfusion inlet and outlet wells. The device was placed under a slight angle to induce ﬂow (e.g., by placing one side of the plate on a lid) and incubated for 10 min at room temperature. After ﬁxation, the PFA was aspirated from the wells and the microﬂuidic chips were washed twice with 50 µL Hank’s balanced salt solution (HBSS, 14025-050, Thermo Scienﬁtic, USA) in all the perfusion inlets and outlets, followed by a permeabilization step of 0.2% Triton-X100 (VWR 28817295) and a second wash step with HBSS. Nuclei were stained using 1:2000 Hoechst 33258 (H3569, Life Technologies, USA) and F-actin using 1:200 Phalloidin-TRITC (P1951 Sigma-Aldrich, The Netherlands) in HBSS with 25 µL for every perfusion inlet and outlet well. After adding the staining, the plate was placed under a slight angle and incubated at room temperature for 30 min in the dark, followed by two wash steps with HBSS. Images were acquired using a confocal microscope (ImageXpress Confocal Micro, Molecular Devices, USA) using a 60 µm pinhole spinning disk and 10X Plan Apo objective. Images were acquired in the DAPI and TRITC channels with shading and background subtraction. Imaging depth was set at 16 bits, binning at 1, and imaging resolution at 2048 × 2048 (0.677 µm/pixel). Autofocus was set at a 120 µm oﬀset from channel bottom. 80 Z-steps were acquired with 1 µm Z-step interval and a total of 2 sites with 10% overlap acquired per well. Max projections were stitched in FIJI/ImageJ v1.53n using the pairwise stitching plugin [29] with the ‘linear blending’ fusion method. where SW = signal window, Z′ = Z-factor. g We used a recommended acceptance criterion for Z-factor ≥0.4, signal window ≥2, coeﬃcient of variation (CV) ≤20% as acceptance criterion for the Max-signal, and SDmin ≤SDmax as acceptance criteria for the Min-signal. g We used a recommended acceptance c 5. Conclusions We have demonstrated that iPSC-ECs are an eﬀective alternative to primary endothelial cells when used in a physiologically relevant in vitro angiogenesis inhibition screening assay. The combination of a standardized microﬂuidic 3D cell culture platform with a scalable and more standardized cell source is a major step in the standardization of physiologically relevant in vitro angiogenesis assays, as it oﬀers the required robustness, compatibility and scalability to be integrated within the drug-screening infrastructure. Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/13/4804/s1, Figure S1: Concentration optimization study for sunitinib in a single assay. Inhibition of angiogenic sprouting of iPSC-ECs using various concentrations of sunitinib. Forty microﬂuidic units are distributed over eight conditions per ﬁve replicates and show that sprouting is reproducible within a single plate. Author Contributions: V.v.D. wrote the manuscript and performed the experiments together with W.S. and E.M.; F.F. helped with data analysis and, together with A.R. assisted in experimental design; P.V., T.H. and A.J.v.Z. supervised the project. All authors have read and agreed to the published version of the manuscript. Funding: This study was ﬁnancially supported by the RECONNECT CVON Groot consortium, which is funded by the Dutch Heart Foundation. A.R. and T.H., A.J.v.Z. were supported by a ZonMW MKMD grants (114022501) and Health-Holland Organ-on-Chip Showcases (HHOOCS) Program LSH19008. Funding: This study was ﬁnancially supported by the RECONNECT CVON Groot consortium, which is funded by the Dutch Heart Foundation. A.R. and T.H., A.J.v.Z. were supported by a ZonMW MKMD grants (114022501) and Health-Holland Organ-on-Chip Showcases (HHOOCS) Program LSH19008. Conﬂicts of Interest: P. Vulto and T. Hankemeier are shareholder of Mimetas BV. F. Famili and A. Reijerkerk are employees of Ncardia BV. V. van Duinen, W. Stam, E. Mulder and A.J. van Zonneveld declare no conﬂict of interest. The funders had no role in the design of the study, in the collection, analyses or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. Conﬂicts of Interest: P. Vulto and T. Hankemeier are shareholder of Mimetas BV. F. Famili and A. Reijerkerk are employees of Ncardia BV. V. van Duinen, W. Stam, E. Mulder and A.J. van Zonneveld declare no conﬂict of interest. The funders had no role in the design of the study, in the collection, analyses or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. 4.5. Assay Performance Quantiﬁcation and Plate Acceptance Criteria The assays signal window (SW) and Z-factor (Z′) are deﬁned as follows: AVGmax and SDmax are the mean and standard deviation of the top (maximum) signal, respectively. Similarly, AVGmin and SDmin are the mean and standard deviation of the bottom (minimum) signal, respectively, and n is the number of measurements. Then: SW = AVGmax −3 SDmax √n − AVGmin + 3 SDmin √n SDmax/ √n 8 of 10 Int. J. Mol. Sci. 2020, 21, 4804 Z′ = AVGmax −3 SDmax √n − AVGmin + 3 SDmin √n AVGmax −AVGmin References 1. Wong, B.W.; Marsch, E.; Treps, L.; Baes, M.; Carmeliet, P. Endothelial cell metabolism in health and disease: Impact of hypoxia. EMBO J. 2017, 36, 2187–2203. [CrossRef] [PubMed] 2. 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Pagano, G.; Ventre, M.; Iannone, M.; Greco, F.; Maﬀettone, P.L.; Netti, P. Optimizing design and fabrication of microﬂuidic devices for cell cultures: An eﬀective approach to control cell microenvironment in three dimensions. Biomicroﬂuidics 2014, 8, 046503. [CrossRef] 18. Berthier, E.; Young, E.W.K.; Beebe, D. Engineers are from PDMS-land, Biologists are from Polystyrenia. Lab Chip 2012, 12, 1224. [CrossRef] [PubMed] 19. Passier, R.; Orlova, V.V.; Mummery, C.L. Complex Tissue and Disease Modeling using hiPSCs. Cell Stem Cell 2016, 18, 309–321. [CrossRef] [PubMed] 0. Van Duinen, V.; Zhu, D.; Ramakers, C.; Van Zonneveld, A.J.; Vulto, P.; Hankemeier, T. Perfused 3D angiog sprouting in a high-throughput in vitro platform. 28. Yetkin-Arik, B.; Vogels, I.M.C.; Neyazi, N.; Van Duinen, V.; Houtkooper, R.H.; Van Noorden, C.J.F.; Klaassen, I.; Schlingemann, R.O. Endothelial tip cells in vitro are less glycolytic and have a more ﬂexible response to metabolic stress than non-tip cells. Sci. Rep. 2019, 9, 10414. [CrossRef] References Angiogenesis 2018, 22, 157–165. [CrossRef] [PubMed] 21. Schoors, S.; De Bock, K.; Cantelmo, A.R.; Georgiadou, M.; Ghesquière, B.; Cauwenberghs, S.; Kuchnio, A.; Wong, B.W.; Quaegebeur, A.; Goveia, J.; et al. Partial and Transient Reduction of Glycolysis by PFKFB3 Blockade Reduces Pathological Angiogenesis. Cell Metab. 2014, 19, 37–48. [CrossRef] [PubMed] 22. Vulto, P.; Podszun, S.; Meyer, P.A.; Hermann, C.; Manz, A.; Urban, G.A. Phaseguides: A paradigm shift in microﬂuidic priming and emptying. Lab Chip 2011, 11, 1596. [CrossRef] 23. Iversen, P.W.; Eastwood, B.J.; Sittampalam, G.S.; Cox, K.L. A Comparison of Assay Performance Measures in Screening Assays: Signal Window, Z’ Factor, and Assay Variability Ratio. J. Biomol. Screen. 2006, 11, 247–252. [CrossRef] 24. Arima, S.; Nishiyama, K.; Ko, T.; Arima, Y.; Hakozaki, Y.; Sugihara, K.; Koseki, H.; Uchijima, Y.; Kurihara, Y.; Kurihara, H. Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement. Development 2011, 138, 4763–4776. [CrossRef] [PubMed] 5. Sharghi-Namini, S.; Tan, E.; Ong, L.L.; Ge, R.; Asada, H.H. Dll4-containing exosomes induce capillary sp retraction in a 3D microenvironment. Sci. Rep. 2014, 4, 4031. [CrossRef] 26. Bentley, K.; Mariggi, G.; Gerhardt, H.; Bates, P.A. Tipping the Balance: Robustness of Tip Cell Selection, Migration and Fusion in Angiogenesis. PLoS Comput. Biol. 2009, 5, e1000549. [CrossRef] 27. Tiemeier, G.L.; De Koning, R.; Wang, G.; Kostidis, S.; Rietjens, R.G.J.; Sol, W.M.P.J.; Dumas, S.J.; Giera, M.; Berg, C.W.V.D.; Eikenboom, J.C.J.; et al. Lowering the increased intracellular pH of human-induced pluripotent stem cell-derived endothelial cells induces formation of mature Weibel-Palade bodies. Stem Cells Transl. Med. 2020, 9, 758–772. [CrossRef] Int. J. Mol. Sci. 2020, 21, 4804 10 of 10 28. Yetkin-Arik, B.; Vogels, I.M.C.; Neyazi, N.; Van Duinen, V.; Houtkooper, R.H.; Van Noorden, C.J.F.; Klaassen, I.; Schlingemann, R.O. Endothelial tip cells in vitro are less glycolytic and have a more ﬂexible response to metabolic stress than non-tip cells. Sci. Rep. 2019, 9, 10414. [CrossRef] © 2020 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/W3137799305	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0249458&type=printable	English	null	Correction: Honey bees preferentially consume freshly-stored pollen	PloS one	2,021	cc-by	255	Mark J. Carroll, Nicholas Brown, Craig Goodall, Alex M. Downs, Timothy H. Sheenan, Kirk E. Anderson The fourth author’s name appears incorrectly. The correct name is: Alex M. Downs. Reference 1. Carroll MJ, Brown N, Goodall C, Downs AM, Sheenan TH, Anderson KE (2017) Honey bees preferen- tially consume freshly-stored pollen. PLoS ONE 12(4): e0175933. https://doi.org/10.1371/journal.pone. 0175933 PMID: 28430801 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Carroll MJ, Brown N, Goodall C, Downs AM, Sheenan TH, Anderson KE (2021) Correction: Honey bees preferentially consume freshly-stored pollen. PLoS ONE 16(3): e0249458. https://doi.org/ 10.1371/journal.pone.0249458 Published: March 25, 2021 Copyright: © 2021 Carroll 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. 1. Carroll MJ, Brown N, Goodall C, Downs AM, Sheenan TH, Anderson KE (2017) Honey bees preferen- tially consume freshly-stored pollen. PLoS ONE 12(4): e0175933. https://doi.org/10.1371/journal.pone. 0175933 PMID: 28430801 OPEN ACCESS Citation: Carroll MJ, Brown N, Goodall C, Downs AM, Sheenan TH, Anderson KE (2021) Correction: Honey bees preferentially consume freshly-stored pollen. PLoS ONE 16(3): e0249458. https://doi.org/ 10.1371/journal.pone.0249458 Correction: Honey bees preferentially consume freshly-stored pollen Mark J. Carroll, Nicholas Brown, Craig Goodall, Alex M. Downs, Timothy H. Sheenan, Kirk E. Anderson PLOS ONE PLOS ONE PLOS ONE \| https://doi.org/10.1371/journal.pone.0249458 March 25, 2021 Published: March 25, 2021 1 / 1 PLOS ONE \| https://doi.org/10.1371/journal.pone.0249458 March 25, 2021 PLOS ONE \| https://doi.org/10.1371/journal.pone.0249458 March 25, 2021
https://openalex.org/W4361773614	https://amu.hal.science/hal-02195476/document	English	null	From insects to robots and back	HAL (Le Centre pour la Communication Scientifique Directe)	2,008	cc-by	814	From insects to robots and back Nicolas Franceschini, Franck Ruﬀier, Julien Serres To cite this version: Nicolas Franceschini, Franck Ruﬀier, Julien Serres. From insects to robots and back. 2nd International Conference on Invertebrate Vision (ICIV), Aug 2008, Bäckaskog, Sweden. hal-02195476 From insects to robots and back To cite this version: Nicolas Franceschini, Franck Ruﬀier, Julien Serres. From insects to robots and back. 2nd International Conference on Invertebrate Vision (ICIV), Aug 2008, Bäckaskog, Sweden. hal-02195476 Distributed under a Creative Commons Attribution 4.0 International License [1] Ruffier, F., Franceschini, N., Robotics and Autonomous Systems 50 (2005) 177-194 [2] Franceschini, N., Ruffier, F., Serres, J., Current Biology 17 (2007) 329-335 [3] Srinivasan M.V., Zhang, S., Chahl, J.S., Barth, E., and Venkatesh, S., Biol. Cyb. 83 (2000) 171-183 [4] Srinivasan, M.V., Zhang, S., Lehrer ,M., and Collett, T., J. Exp. Biol., 199 (1996) 237-244 [5] Serres, J., Ruffier, F., Franceschini, N., Autonomous Robots (2008, in press) [6] Pudas, M. et al., Sensors and Actuators A133, (2007) 88-95 [7] Franceschini, N. et al., In: Facets of vision, D. Stavenga and R. Hardie, Eds. Springer (1989) 360-390 From insects to robots and back When insects are flying forward, the image of the environment sweeps backward across their viewfield and forms an “optic flow” (OF) that depends on both the groundspeed and the distance to the ground (or to the lateral obstacles). Several studies have shown that insects are able to maintain a constant OF with regard to their surroundings while cruising and landing. To explain how i ld b h i hi When insects are flying forward, the image of the environment sweeps backward across their viewfield and forms an “optic flow” (OF) that depends on both the groundspeed and the distance to the ground (or to the lateral obstacles). Several studies have shown that insects are able to maintain a constant OF with regard to their surroundings while cruising and landing. To explain how insects could behave in this way, we introduced the concept of an optic flow regulator, that is, a feedback control system that adjusts a flight force so as to maintain the OF at a fixed set-point [1]. The variable that needs to be measured is neither groundspeed nor range but the ratio groundspeed:range - in other words, the optic flow - which the insect can access directly via motion detecting neurons. The OF regulator concept accounts for a number of seemingly disparate insect behaviours that were reported over the last decades [2]. Most reports are qualitative, but quantitative findings made on honeybees’ landing can also be explained on the basis of this simple control system, including the constant descent angle observed in the bee’s final approach [3]. In a similar vein, a honeybee trained to fly in a corridor [4] may rely on a dual OF regulator that adjusts both its forward and side thrusts - resulting in a forward groundspeed and a clearance to the walls, respectively - without any needs to measure groundspeed or range [5]. The OF regulator concept was simulated and physically implemented on board two kinds of aerial robots: a miniature helicopter for ground avoidance [1] (Fig.1) and a miniature hovercraft for lateral obstacle avoidance and cruise control in straight or tapered corridors [5]. The electronic OF sensors aboard [6] were derived from the housefly EMDs previously analyzed using single neuron recording combined with single photoreceptor stimulation [7]. Fig. 1. HAL Id: hal-02195476 https://amu.hal.science/hal-02195476v1 Submitted on 26 Jul 2019 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 From insects to robots and back Nicolas FRANCESCHINI, Franck RUFFIER and Julien SERRES Biorobotics Lab, Institute of Movement Science CNRS & Uni. of the Mediterranean MARSEILLE, France nicolas.franceschini, franck.ruffier, julien.serres @univmed.fr From insects to robots and back The robot OCTAVE equipped with a ventral OF sensor and an OF regulator mimics insect behaviour in the vertical plane (takeoff, cruising, terrain following, landing, wind reaction) p Fig. 1. The robot OCTAVE equipped with a ventral OF sensor and an OF regulator mimics insect behaviour in the vertical plane (takeoff, cruising, terrain following, landing, wind reaction) Fig. 1. The robot OCTAVE equipped with a ventral OF sensor and an OF regulator mimics insect behaviour in the vertical plane (takeoff, cruising, terrain following, landing, wind reaction) Fig. 1. The robot OCTAVE equipped with a ventral OF sensor and an OF regulator mimics insect behaviour in the vertical plane (takeoff, cruising, terrain following, landing, wind reaction)
https://openalex.org/W3089517540	http://www.odermatol.com/odermatology/2020e/E272.Atopic-ChiriacA.pdf	English	null	Atopic dermatitis in the view of pediatrics	Nasza Dermatologia Online	2,020	cc-by	741	Sir, In 2014 we performed a study on 1000 kindergarten children and we concluded a point prevalence of atopic dermatitis extremely low: 0,22% [1]. In 2014 we performed a study on 1000 kindergarten children and we concluded a point prevalence of atopic dermatitis extremely low: 0,22% [1]. • Seborrheic dermatitis was highly prevalent, but divided into infantile and non-infantile type; • Scalp seborrhea was considered a specific diagnosis; • diaper dermatitis was reported in almost the same proportion as atopic dermatitis; In 2020, in the same geographic area, we performed a retrospective study, between 2013 and 2018, revising all medical records of the children (aged 0-16) hospitalized in the Saint Mary Children Hospital Iasi. We selected only skin diseases (main or second diagnosis). Each diagnosis was performed by pediatrics without any dermatological examination (Table 1). • Total number of skin disorders did not vary too much for the entire period (2013-2018); p • Prevalence of atopic dermatitis was reported to vary from 2.74% to 4.06% of all skin diseases for hospitalized childr en. Atopic dermatitis is a very important diagnosis for pediatrics, more than psoriasis for example, but one can doubt the validity of diagnostic criteria, as well as the identification of other skin diseases. Another Evaluating the medical records some observations were made: • Skin diseases were categorized differently from dermatological point of view; © Our Dermatol Online e.2020 e180.1 How to cite this article: Chiriac AE, Chiriac A, Duceac L, Azoicai D. Atopic dermatitis in the view of pediatrics. Our Dermatol Online. 2020;11(e):e180.1-e180.2. Letter to the Editor Letter to the Editor Letter to the Editor How to cite this article: Chiriac AE, Chiriac A, Duceac L, Azoicai D. Atopic dermatitis in the view of pediatrics. Our Dermatol Online. 2020;11(e):e180.1-e180.2. Submission: 01.04.2020; Acceptance: 08.06.2020 DOI: 10.7241/ourd.2020e.180 Anca E Chiriac1, Anca Chiriac2, Letitia Duceac3, Doina Azoicai4 1University of Medicine and Pharmacy Grigore T. Popa, Faculty of Medicine- PhD Student, Iasi, Romania, 2Apollonia University, Nicolina Medical Center, Dermatology Department, P. Poni Institute of Macromolecular Chemistry, Iasi, Romania, 3Apollonia University, Saint Mary Childrens Hospital, Epidemiology Department, Iasi, Romania, 4University of Medicine and Pharmacy Grigore T. Popa, Faculty of Medicine, Department of Epidemiology, Iasi, Romania Corresponding author: Prof. Anca Chiriac, E-mail: ancachiriac@yahoo.com • Psoriasis, vitiligo, alopecia areata and other were summarized as other dermatitis; • Psoriasis, vitiligo, alopecia areata and other were summarized as other dermatitis; Sir, Submission: 01.04.2020; Acceptance: 08.06.2020 DOI: 10.7241/ourd.2020e.180 Diagnosis 2018 2017 2016 2015 2014 2013 Piodermitis 45 37 24 51 47 43 Pemphigus 1 1 0 1 0 0 Epidermolysis bullosa 1 1 1 0 0 1 Dermatitis herpetiformis 1 3 0 1 1 0 Prurigo 2 4 5 8 9 20 Atopic dermatitis 192 (2,74%) 208 (3,58%) 234 (3,88%) 250 (3,58%) 229 (4,06%) 217 (3,70%) Scalp seborrhea 9 3 5 8 5 8 Infantile seborrheic dermatitis 18 4 4 11 14 13 Seborrheic dermatitis 43 52 40 60 42 47 Diaper dermatitis 167 200 213 225 138 125 Allergic contact dermatitis 51 47 51 66 78 119 Irritant contact dermatitis 22 26 39 23 6 7 Contact dermatitis 2 8 6 13 14 15 Food allergy 1 5 6 16 6 8 Oral contact allergy 1 0 1 0 0 0 Burns 1 - - - - - Other dermatitis 25 34 23 38 25 23 Total 527 580 602 698 564 585 www.odermatol.com explanation could rely on the attention focused on the atopic dermatitis, ignoring other skin diseases. will be made to conceal their identity, but anonymity cannot be guaranteed. © Our Dermatol Online e.2020 Consent 1. Chiriac A, Foia L, Gorduza VE, Chiriac AE, Uliliuc T, Kezic S, et al. The puzzled low prevalence of atopic dermatitis in kindergarten children in Romania. Pediatr Allergy Immunol. 2014;25:96-7. The examination of the patient was conducted according to the Declaration of Helsinki principles. The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts Copyright by Anca E Chiriac, 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. e180.2 © Our Dermatol Online e.2020
https://openalex.org/W4391530652	https://www.researchsquare.com/article/rs-3921845/latest.pdf	English	null	Is it bad apples or bad barrels? Undergraduate Medical Students Attitude towards Plagiarism; Transcultural Study	Research Square (Research Square)	2,024	cc-by	9,523	Is it bad apples or bad barrels? Undergraduate Medical Students Attitude towards Plagiarism; Transcultural Study Asmaa Abdelnasser (  asmaa_mohamed@med. Suez Canal University Enjy Abouzeid Suez Canal University Enas M A Mostafa Suez Canal University Manal Ibrahim Hanafi Mahmoud Ibn Sina National College for Medical Studies Nourhan F. Wasfy Suez Canal University Shaimaa A Shehata Suez Canal University Results A total of 387 medical students completed the questionnaires, 182 participants from Egyptian Medical School and 205 from Saudi Medical School. A majority (73.9%) had not received previous plagiarism training. The category-wise distribution of the study participants in the two medical schools in all components of the ATP scale showed that a vast majority fell in the moderate category. The overall mean (SD) scores were for Egyptian and Saudi Medical Students’ positive attitude (35, 33), negative attitude (23, 22), and subjective norms (31, 30). The thematic analysis of the focus group discussions categorized the student responses under three themes: Understanding Plagiarism, Drivers of Plagiarism, and Proactive Preventive Measures for Plagiarism. Methods This two-phase observational mixed-method study utilized a probability-stratified random sample technique to include medical students from two schools, in Egypt and Saudi Arabia. The attitude of the students was investigated using The Attitudes Towards Plagiarism (ATP) questionnaire, a validated online self-administered questionnaire. It was distributed electronically to 387 medical students from both schools. Additionally, two focus group sessions were conducted following an inductive approach and underwent thematic analysis. Aim to explore the attitude of medical students towards plagiarism and identify the underlying factors that may influence plagiarism using a mixed quantitative and qualitative transcultural approach. Research Article Posted Date: February 5th, 2024 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 Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Page 1/24 Page 1/24 Background Plagiarism is a significant violation of academic integrity since it diminishes the value of original and honest academic work. Plagiarism is becoming increasingly common in medical colleges around the world, posing significant obstacles for educators, faculty, and policymakers in addressing such ethical dilemmas and limiting potential risks and liabilities as well. Introduction Plagiarism, fabrication, falsification, and fraud are all examples of academic misconduct practices [1]. Student plagiarism has grown to be a serious issue in education [2]. According to the findings of one Australian study, 30–50% of university students endeavored various types of plagiarism while studying [3]. Several study reports indicated various plagiarism rates of 19–81% in undergraduate study [4]. It is considered a significant violation of academic integrity since it diminishes the value of original and honest academic work. It has always been a problem in academic contexts, and it appears to become worse. In today's environment, it is regarded as the most widespread challenge in the academic world [4– 6]. Barnhart (1988) described the origin of the word 'plagiarism'; it was derived from the Latin word 'plagarius' meaning the “kidnapper, seducer, plunderer, or literary thief” [7]. It is hard to sum up "plagiarism" in just a few words. Nonetheless, utilizing portions of the text without citing, presenting someone else's ideas as one's own, and quoting direct text or sections of the text without acknowledgment" are all examples of plagiarism [8]. Barnhart (1988) described the origin of the word 'plagiarism'; it was derived from the Latin word 'plagarius' meaning the “kidnapper, seducer, plunderer, or literary thief” [7]. It is hard to sum up "plagiarism" in just a few words. Nonetheless, utilizing portions of the text without citing, presenting someone else's ideas as one's own, and quoting direct text or sections of the text without acknowledgment" are all examples of plagiarism [8]. In medical education, such considerations are more significant. Unethical conduct by physicians might risk patient safety, jeopardize professional relationships, and generate chaos [9]. Plagiarism is becoming increasingly common in medical colleges and schools around the world, posing significant obstacles for educators, faculty, and policymakers in addressing such ethical dilemmas and limiting potential risks and liabilities as well [5, 9]. This type of academic misconduct has ramifications for not only students and faculty but also the institution's reputation and award integrity [10]. Husain et al. (2017) denoted five main factors that contributed to the commission of plagiarism. These are institutional, academic, personal, technological, and external factors [11]. Previous research has found that the cultural context has a substantial impact on the development of moral behavior and attitudes and that such cultural standards in a given community have a considerable impact on students' attitudes [12]. Conclusion This study provides an insightful analysis of students' understanding of plagiarism, particularly in the context of academic writing. Key factors identified as contributing to plagiarism include language barriers, poor academic writing skills, the challenging nature of assignments, strict deadlines, and an Page 2/24 assessment focus on scores. The study also notes the dual role of technology in this context and underscores the significant impact of cultural influences on students' perceptions of plagiarism. Introduction Furthermore, the technological breakthrough enabling students to readily plagiarize others' ideas and words utilizing a 'copy and paste' methodology, is one of the key causes for the growth of this misconduct [13]. The recent advancement in machine learning and Artificial Intelligence (AI) technology is transforming education, learning, and research globally. An AI-based chatbot, ChatGPT is considered the most popular, easily accessible language processing technique used worldwide [14]. Despite its popularity, ChatGPT has created new challenges and threats to education [2]. Educators have sounded alarm bells about the increased risk of plagiarism and cheating if students use ChatGPT to prepare or write essay-type questions in exams [14]. Recently, several universities have banned ChatGPT over fears of student plagiarism that may hinder academic integrity [15]. Page 3/24 Page 3/24 In medical education, particularly following the adaptations in assessment methods post-COVID-19, students frequently face the challenge of managing various assignments and research projects. This situation is made difficult by the ease of accessing information online and a general shortage of formal training on academic writing and proper citation practices. Consequently, understanding the concept of plagiarism becomes crucial in the context of medical training and education. While existing literature has pointed out the influence of cultural factors on student attitudes, there is a lack of previously published studies assessing the attitude towards plagiarism among different cultures and the factors contributing to such attitudes through a mixed-method approach. Accordingly, in this study, we aimed to explore the attitude of medical students towards plagiarism and identify the underlying factors that may influence plagiarism, among different cultures, using a mixed quantitative and qualitative approach. ● Sampling: A proportionate stratified random sampling was used to allocate a sample for students in each year (years 2 to 6). Furthermore, a simple random sampling was employed to select students from each year during the academic year 2021/2022. As students enter university directly from high school, they are unfamiliar with terminologies such as plagiarism. Therefore, Year 1 excluded students from the sample. The total number of students in the Egyptian Medical School was 1109 and 1139 from Saudi Medical School. A sample size of 329 undergraduate students from both medical schools was calculated. Study design and setting: The study is a two-phase observational mixed-method study that included a sample of medical students from two different cultural contexts: Egyptian and Saudi Medical schools. The Attitudes Towards Plagiarism Questionnaire (ATP) ATP is a validated questionnaire developed by Mavrinac et al. (2010). ATP questionnaire is divided into three sections, the first of which measures acceptance of plagiarism (a positive attitude), the second, rejection of plagiarism (a negative attitude), and the third, social and normative factors that may influence acceptance of the issue (subjective norms) [16]. According to Farooq and Sultana 2022, the scale exhibited strong internal consistency, composite reliability, and construct validity. All three attitudinal factors were convergent. Because of this, this tool is useful for determining the plagiarist's intentions as a function of attitudes (both positive and negative), and subjective norms [17]. Page 4/24 Page 4/24 Page 4/24 The Arabic translation was used based on a validated Arabic version of the ATP questionnaire [18]. It consisted of 29 statements on a 5-point Likert scale, graded (1 - strongly disagree and 5 - strongly agree) for each statement. The face and content validity of the questionnaire were examined by 10 medical education experts’ opinions. Reliability was calculated using Cronbach alpha. The questionnaire was presented in bilingual statements (Arabic and English), uploaded on an electronic platform (Microsoft Form), and disseminated through official platforms and study groups. The total number of students who consented and filled out the questionnaires in the two schools was 387 (182 from Egyptian Medical school and 205 from Saudi Medical school). ● Scoring According to that scale, points were designated for each answer and scores were calculated by summing. Minimal and maximal possible scores were calculated for each factor and ranges were divided into three equal parts representing low, moderate, and high score scales. The scoring system is summarized in Table 1. Table 1 The Scoring System of the Attitudes Towards Plagiarism Questionnaire (ATP) Subthemes Low score range Moderate Score range High score range Positive Attitude (12 statements) 12–28 29–45 46–60 Negative Attitude (7 statements) 7–16 17–26 27–26 Subjective Norm (10 statements) 10–23 24–37 38–50 Mean Score/statement 1.0-2.3 2.4–3.7 3.8-5.0 ●Statistical analysis Table 1 ● Statistical analysis Data were entered in SPSS V.25 for statistical analysis. Numerical variables were measured as mean and standard deviations while categorical variables were expressed as frequencies and percentages. The level of significance was less than 0.05. Means were compared using an independent sample T-test. 2- Phase two: Qualitative Phase-Focus groups (FGs) ● Sampling: ● Sampling: A purposive sample of students from 2–6 years in both medical schools was invited to two focus groups. Any student in the two medical schools, who completed/did not complete the questionnaire, had the same chance to be included in the focus group. Each focus group was conducted in a two-hour session Page 5/24 using a virtual platform such as Zoom video conference. The authors ensured heterogeneity of the groups' participants in terms of gender and academic years. The sessions were recorded after getting permission (informed consent) verbally from all participants. ● Data collection tool: A deductive qualitative grounded theory approach was adopted in the conduction of the focus groups. The focus groups aimed to explore the students' perspectives on the factors that influence plagiarism in medical education. The discussion was guided by specific questions to explore in depth the students’ attitudes toward plagiarism. The authors prepared a focus group guide as shown in Fig. 1 Statistical analysis All focus groups were audio-recorded and transcribed anonymously by the authors. The transcripts were checked for accuracy and unclear data identified were excluded. The focus groups were conducted in spoken Arabic and translation of the recordings into English was done by two authors EA and NFW. All the authors agreed on the translation before proceeding with the analysis. Thematic analysis was applied to identify common themes following the constructivism research philosophy. The Qualitative Data were coded as follows: S Student, EM Egyptian Medical School, SM Saudi Medical School, and the number of students. For example, student number 1 in the Egyptian Medical school group was coded as SEM1, and student number 1 in the Saudi Medical school group was coded as SSM1. Ethical considerations: Administrative approval was obtained from the Deans of the medical schools included in the study before initiating data collection. Ethical approval was secured from the Research Ethics Committee of the Faculty of Medicine-Suez Canal University, Egypt (Research 4610#), and the Institutional Research Review Board at Ibn Sina National College, Saudi Arabia (IRRB-21-09062021) before the commencement of data collection. Section 1: Quantitative data analysis Two medical schools were included in the study. The number of students who consented and filled out the questionnaires was N = 387; 182 participants from Egyptian Medical School and 205 from Saudi Medical School were included in the final analysis. The demographic characteristics of all participants are summarized as follows: approximately 216 (55.8%) are female students and 171 (44.2%) are male students. There was a good representation of students from all medical study years. A vast majority of them 286 (73.9%) have yet to gain previous training in plagiarism. However, about one-third of the total students in Saudi Medical School (32.2%) significantly got training on plagiarism compared to only 19.2% of Egyptian Medical School (p = 0.004). Page 6/24 Page 6/24 Attitude towards plagiarism (N = 387): The study employed the validated ATP tool to collect data on the attitude of students from two medical schools and different levels of study. The reliability study of the collected data revealed high internal consistency (Cronbach alpha = 0.83 Egyptian Medical School and 0.88 Saudi Medical School), with an average of 0.85 that supports the findings of the study. Table 2 summarizes the category-wise distribution of the study participants in the two medical schools in all components of the ATP scale and it was observed that a vast majority fell in the moderate category (Egyptian and Saudi Medical Schools). The overall mean (SD) scores were for Egyptian and Saudi Medical Students’ positive attitude (35, 33), negative attitude (23, 22), and subjective norms (31, 30). ble 2: Attitude towards plagiarism among the study groups (N=387) Parameters Egyptian Medical School Saudi Medical School Count Column Valid N % Count Column Valid N % Positive Attitude Low 45 24.7% 57 27.8% Moderate 114 62.6% 129 62.9% High 23 12.6% 19 9.3% Negative Attitude Low 14 7.7% 24 11.7% Moderate 139 76.4% 154 75.1% High 29 15.9% 27 13.2% Subjective Norms Low 18 9.9% 34 16.6% Moderate 138 75.8% 146 71.2% High 26 14.3% 25 12.2% Table 2: Attitude towards plagiarism among the study groups (N=387) le 2: Attitude towards plagiarism among the study groups (N=387) Table 3 shows the mean scores of the participants' positive attitudes towards plagiarism. The statements, ‘It is justified to use previous descriptions of a method’, ‘Self-plagiarism is not punishable’, ‘Young researchers should receive milder punishment’, ‘A scientific paper couldn't be written without plagiarizing’, ‘Short deadlines give the right to plagiarize a bit, ‘Sometimes one cannot avoid using other people’s words’ and ‘Self-plagiarism should not be punishable in the same way as plagiarism is’ showed the highest mean in Egyptian Medical School. The same statements had the highest mean score among Saudi Medical School students except ‘Self-plagiarism is not punishable’ and ‘Sometimes one cannot avoid using other people’s words. Most of these statements showed statistically significant differences Table 3 shows the mean scores of the participants' positive attitudes towards plagiarism. Attitude towards plagiarism (N = 387): The statements, ‘It is justified to use previous descriptions of a method’, ‘Self-plagiarism is not punishable’, ‘Young researchers should receive milder punishment’, ‘A scientific paper couldn't be written without plagiarizing’, ‘Short deadlines give the right to plagiarize a bit, ‘Sometimes one cannot avoid using other people’s words’ and ‘Self-plagiarism should not be punishable in the same way as plagiarism is’ showed the highest mean in Egyptian Medical School. The same statements had the highest mean score among Saudi Medical School students except ‘Self-plagiarism is not punishable’ and ‘Sometimes one cannot avoid using other people’s words. Most of these statements showed statistically significant differences Page 7/24 Page 7/24 between the two medical schools. In addition, the statement ‘If one cannot write well, it is justified to copy parts of a similar paper’ showed statistically significant differences between the two medical schools. Table 3 The mean scores of the participants' positive attitudes towards Plagiarism (N = 387) Positive Attitudes Items Egyptian Medical School Mean (Sd) Saudi Medical School. (Mean (Sd) t-test (P- value) It is justified to use previous descriptions of a method 3.2 (1.149) 3.05 (1.15) 1.231 (0.219) Self-plagiarism is not punishable 3.06 (1.359) 2.76 (1.406) 2.124 (0.034) Plagiarized parts of a paper may be ignored 2.72 (1.306) 2.49 (1.353) 1.711 (0.088) Young researchers should receive milder punishment 3.48 (1.303) 3.11 (1.290) 2.124 (0.034) If one cannot write well, it is justified to copy parts of a similar paper 2.59 (1.262) 2.45 (1.222) 2.849 (0.005) A scientific paper couldn't be written without plagiarizing 3.42 (1.293) 3.20 (1.332) 1.062 (0.289) Short deadlines give the right to plagiarize a bit 3.16 (1.297) 2.78 (1.297) 2.868 (0.004) When students do not know what to write, they translate a part of a paper from a foreign language without citing the source 2.34 (1.125) 2.57 (1.205) -1.934 (0.054) It is justified to use one’s own previously published work without providing a citation 2.55 (1.228) 2.73 (1.262) -1.355 (0.176) If a colleague allows a student to copy from her/his paper, he/she is NOT doing anything bad, because he/she has his/her permission 2.82 (1.222) 2.73 (1.265) 0.729 (0.466) Sometimes one cannot avoid using other people’s words 3.08 (1.210) 2.75 (1.246) 2.641 (0.009) Self-plagiarism should not be punishable in the same way as plagiarism is. Attitude towards plagiarism (N = 387): 3.16 (1.297) 2.78 (1.297) 2.868 (0.004) P- value is significant at ≤ 0.05 level Table 4 shows the mean scores of the participants' negative attitudes towards plagiarism The Page 8/24 Table 4 shows the mean scores of the participants' negative attitudes towards plagiarism. The statements ‘The names of the authors who plagiarize should be disclosed to the scientific community’, ‘It is important to discuss issues like plagiarism and self-plagiarism’, ‘Plagiarizing is as bad as stealing an exam’, and ‘Plagiarism impoverishes the investigative spirit’ showed the highest mean in both Egyptian and Saudi Medical Schools. All these statements showed statistically significant differences between the two medical schools except ‘Plagiarism impoverishes the investigative spirit’. Table 4 The Mean Scores of the Participants' Negative Attitudes Towards Plagiarism (N = 387) Negative Attitudes Items Egyptian Medical School Mean (Sd) Saudi Medical School. (Mean (Sd) t-test (P- value) Plagiarists do not belong in the scientific community 2.70 (1.208) 2.81 (1.290) -0.835 (0.404) The names of the authors who plagiarize should be disclosed to the scientific community 3.26 (1.140) 3.00 (1.174) 2.195 (0.029) It is important to discuss issues like plagiarism and self-plagiarism 3.80 (1.115) 3.56 (1.173) 2.067 (0.039) Plagiarizing is as bad as stealing an exam 3.73 (1.240) 3.40 (1.255) 2.560 (0.011) Plagiarism impoverishes the investigative spirit 3.64 (1.226) 3.51 (1.211) 1.048 (0.295) A plagiarized paper does no harm to science 2.77 (1.231) 2.68 (1.242) 0.724 (0.470) It should NOT be considered as a serious offense 2.50 (1.291) 2.51 (1.297) -0.056 (0.956) P- value is significant at ≤ 0.05 level Table 5 shows the mean scores of the participants' subjective norms toward plagiarism. The statements ‘Authors say they do NOT plagiarize, when in fact they do’, ‘Those who say they have never plagiarized are lying’, and ‘Sometimes, it is necessary to plagiarize’ showed the highest mean score in both Egyptian and Saudi Medical Schools. In the subjective norms domain, six statements showed statistically significant differences between the two medical schools. Page 9/24 Table 5 The Mean Scores of the Participants' Subjective Norms Towards Plagiarism (N = 387) Subjective Norms items Egyptian Medical School Mean (Sd) Saudi Medical School. Section 2: Qualitative data analysis: Two focus groups were conducted to explore the students’ perspectives towards plagiarism and the factors that influence plagiarism in depth. The first focus group included ten students from Saudi Medical School, while the second one included eight students from Egyptian Medical School. The thematic analysis of the qualitative data is demonstrated in Fig. 2. Attitude towards plagiarism (N = 387): (Mean (Sd) t-test (P- value) Authors say they do NOT plagiarize, when in fact they do 3.60 (1.165) 3.30 (1.206) 2.492 (0.013) Those who say they have never plagiarized are lying 3.41 (1.117) 3.32 (1.210) 0.721 (0.477) Sometimes plagiarism is tempted because everyone is doing it 3.07 (1.226) 2.93 (1.221) 1.161 (0.246) Plagiarism is continued because it is not detected 3.21 (1.208) 2.94 (1.251) 2.132 (0.034) My institution has a plagiarism-free environment 2.16 (1.090) 2.51 (1.136) -3.060 (0.002) Plagiarism is not a big deal 2.71 (1.169) 2.73 (1.65) -0.101 (0.920) Sometimes a sentence or two may be copied just to inspire further writing 3.23 (1.098) 2.83 (1.245) 3.305 (0.001) Someone should not feel guilty when copying verbatim a sentence or two from his/her previous work 3.16 (1.213) 3.01 (1.274) 1.141 (0.255) Plagiarism is justified if I currently have more important obligations or tasks to do 3.08 (1.252) 2.77 (1.176) 2.524 (0.012) Sometimes, it is necessary to plagiarize 3.64 (1.112) 3.22 (1.312) 3.402 (0.001) P- value is significant at ≤ 0.05 level Section 2: Qualitative data analysis: Table 5 1.2 Consent and Collaboration However, some students put forth the argument that if the use of another's work is done with the consent and approval of the original author, it should not be considered plagiarism. One student, SEM5, emphasized the necessity of consent and stated, "Consent and approval of the work's author are mandatory." The proponents of this perspective contended that collaborative efforts and knowledge sharing among students are crucial for enhancing learning and collaboration, as well as mitigating feelings of isolation in the educational process. Likewise, students SEM2, SEM3, and SEM8 disagreed regarding the act of copying and submitting another scholar's or student's research or paper as one's own as a form of plagiarism. They argued that such instances should be viewed as a source of guidance, with SEM2 stating, "Others' work can be considered a guide for us." 1.4 Plagiarism as Theft However, an opposing viewpoint was presented by student SEM1, who posited that "plagiarism is theft, whatever the drive or the reasons behind it." This perspective was not endorsed by the rest of the group, who contended that certain behaviors might persist in student societies without appropriate penalties, eventually becoming accepted, despite their potential to be considered attempts at plagiarism. 1.1 Proper citation According to the input of the two groups, Plagiarism is a concept widely known and understood by students, who acknowledge that it involves utilizing someone else's work without proper citation. A Page 10/24 Page 10/24 student SSM5 stated, “Plagiarism is getting an idea from a resource without mentioning the original resource”. The majority of students within the study groups agreed that the absence of proper citations when incorporating someone else's work is considered an act of plagiarism. 1.3 Confusion between Guide and Plagiarism The students expressed confusion regarding the distinction between guidance others and attempting plagiarism. They highlighted that unintentional plagiarism may occur due to a lack of awareness of the university rules or task guidelines. However, they also mentioned instances where students attempted to help others and share information, but unknowingly engaged in unintentional plagiarism. In this regard, SEM4 suggested gradually implementing a strict system within the institution for detecting plagiarism to ensure that all students have a clear understanding of the concept. 2.1 Student maturity and experience Furthermore, the rate of plagiarism was found to be influenced by factors such as the academic year. One student SSM2 highlighted the relationship between student maturity and the prevalence of plagiarism, suggesting that it is more common in the early years of academic study due to student immaturity. “Poor knowledge of plagiarism in the early academic years can cause someone to plagiarize”, student SSM3 added. 1.5 Impact on Students Another student raised the concern that repeated plagiarism attempts could have a detrimental impact on students' attitudes. It was argued that students would become dependent and lack self-regulated learning skills, perpetuating a habit of seeking easy ways to obtain marks and succeed without genuine learning. As one student SSM3 remarked, "The student that plagiarized is harming himself more than the others. He may pass the task, but he will lose in the long run. The student will be dependent and will not learn from the task." 1.6 Expanding the definition of plagiarism. Page 11/24 Page 11/24 One student, SSM4, contended that plagiarism encompasses the use of others' thoughts and ideas, not just their work. It was suggested that giving appropriate credit to others serves as motivation for individuals to think critically and generate their own ideas. Moreover, recognizing the contributions of others represents a fair acknowledgment of their time and effort. Similarly, SEM5 shared the sentiment, stating, "Copying others' ideas as ours is plagiarism." In terms of self-plagiarism, most students agreed that utilizing their own previous work in subsequent tasks is not considered misconduct. They believe that building upon previous work can save time and effort. However, they emphasized the importance of referencing their previous work and notifying any authors who were involved in the previous work. Likewise, student SEM7 declared, "Copying my own work and resubmitting it is not plagiarism." However, one student disagreed with this notion and argued that they should strive to produce special work for each new task, viewing it as an opportunity for personal development. Another student acknowledged that the content and data from previous work could guide them in deciding whether to reuse it or not but agreed that it could be used if applicable. Additionally, it was mentioned that it is generally accepted for someone to seek paid assistance if they are unable to complete a task. However, a student disagreed with this opinion, asserting that individuals should prioritize self-learning and self-improvement before resorting to paid help. Furthermore, the distinction between cheating and plagiarism appeared to be a source of confusion among students. They agreed that cheating primarily pertains to exams, whereas plagiarism is associated with research or academic work. However, they recognized that both cheating and plagiarism are forms of misconduct. The students believed that if these transgressions carried serious consequences, their behavior might change accordingly. 2- Drivers of Plagiarism rate Students unanimously agreed that plagiarism is highly prevalent in the academic context. Student SSM7 stated, “It is very common in many universities and high schools”. However, they acknowledged that the specific type or form of plagiarism may vary depending on the situation. 2.3 Student academic writing skills and language barriers Students' skills played a role, with one student, SSM5, emphasizing the significance of students' confidence and proficiency in their writing skills as factors influencing their likelihood of engaging in plagiarism. SSM10 stated, “Plagiarism could happen because of having difficulties paraphrasing the original text”. Language fluency was also mentioned, with SSM3 stating, "Language barriers, such as English not being the mother language, can contribute to plagiarism if a student lacks confidence in their language skills." 2.4 Culture and Educational Practices Some students defended such misconduct based on mutual moral justifications, which directly contributed to the prevalence of plagiarism. The influence of culture and habitual experiences regarding plagiarism were identified as factors that have shaped students' mindsets and acceptance of this behavior. SEM4 compared Western and Eastern institutions, ‘The culture within these institutions emphasizes the importance and gravity of plagiarism, which helps to minimize its occurrence. Additionally, Western institutions tend to be stringent in dealing with misconduct and copyright violations. On the other hand, Eastern countries exhibit more flexibility and misconceptions, creating a more lenient culture surrounding plagiarism’. Furthermore, while some students may view plagiarism attempts as a stigma and feel shame when accused of such misconduct, others do not consider it shameful due to the absence of appropriate consequences or because of misconceptions about this behaviour. 2.2 Assessment and Workload The students identified several factors underlying plagiarism in medical schools. They agreed that tight deadlines and a high volume of assignments can lead to unintentional plagiarism. The student SSM3 added “We do not have enough time to meet the deadline to submit the assigned tasks” Moreover, one student, SSM2, noted that the nature and type of the task can either facilitate or deter plagiarism. Tasks Page 12/24 Page 12/24 Page 12/24 that primarily require factual recall of knowledge tend to have a higher rate of plagiarism, particularly when students are working from similar resources. Similarly, SEM1 stated, “Non-creative tasks could be a reason behind plagiarism” Conversely, tasks that encourage reflection and innovation tend to foster idea generation and reduce the rate of plagiarism. SEM2 also mentioned that difficult assignments that exceed students' capabilities may drive them to engage in plagiarism. Regarding this point, student SSM2 stated “When a student is given a work, and he doesn’t understand it, then he will end up plagiarizing or taking other people’s work.” Assessment of the task was identified as another influential factor. Tasks that are primarily focused on scores and outcomes may promote plagiarism and encourage students to prioritize quantity and completion over the quality of their work. Additionally, high workloads and strict deadlines were recognized as potential contributors to plagiarism. SEM7 noted, "Time constraints with the academic workload could be reasons for using others' work." 2.5 Technology's Dual Role Technology was identified as a dual factor, facilitating both plagiarism and its detection. The availability of online materials has made research easier but has also created opportunities for copying and stealing Page 13/24 Page 13/24 others' work. One student, SEM4, reflected on the COVID-19 pandemic, noting that ‘the sudden shift to online learning without adequate preparation had facilitated plagiarism’. 2.6 Institutional Role Both student groups unanimously agreed that the institution plays a pivotal role in the prevalence of plagiarism. They emphasized that institutions that consistently and transparently address the issue of plagiarism tend to have lower rates of it. Furthermore, the attitudes and beliefs of the staff members are indirectly and directly transmitted to the students as part of the hidden curriculum. Finally, all the students agreed that firm and publicly announced consequences are crucial in addressing the prevalence of plagiarism within an institution. SSM8 stated, "If the institution declares that students can use others' work without it being considered plagiarism, then it is not plagiarism." Another student SEM8 pointed out that ‘the institution's guidelines and scoring rubrics, which emphasize referencing as an important assessment criterion, have contributed to a decrease in the prevalence of plagiarism’. 3- Proactive preventive measures for addressing Plagiarism Finally, students suggested some proactive steps as shown in Table 6 Page 14/24 Table 6 Plagiarism proactive preventive measures suggested by the Egyptian and Saudi Medical Schools’ students. students. Regulation and assessment ● Announce and communicate the evaluation rules, institutional policies, and regulations regarding plagiarism to all students, ensuring they are aware of the consequences and expectations. ● Modify the nature and structure of assignments to encourage creativity and critical thinking rather than simply requiring the recall of factual knowledge. ● Incorporate elements of mutual benefit in tasks, providing additional incentive or rewards beyond just a score. ● Establish clear and explicit criteria for assignments, including specific guidelin for referencing and citation, as well as clearly defined expectations for acceptabl levels of plagiarism, aligning them with the institution's evaluation rules and policies. Student support ● Provide a comprehensive student guide. ● Conduct frequent orientation sessions. ● Offer close guidance and mentorship. ● Provide continuous feedback. ● Showcase success stories and Introduce examples of students who have excelled in their research endeavors without resorting to plagiarism. ● Develop writing and research skills. ● Address language barriers. ● Foster peer teaching and support. Detection ● Provide students with free access to plagiarism checker programs to encourag them to incorporate plagiarism checks as a routine step in their work process. ● Incorporate plagiarism detection software into the assessment platforms used by the institution. ● Offer faculty development programs focused on enhancing their knowledge a skills in detecting different types of plagiarism. Consequences ● Tailor the penalty of plagiarism attempts according to circumstances: ● They should vary in severity according to the maturity, level of expertise, guidance, and instructions. ● Penalties vary from redoing the work to failure of the task or the year. students. Regulation and assessment ● Announce and communicate the evaluation rules, institutional policies, and regulations regarding plagiarism to all students, ensuring they are aware of the consequences and expectations. ● Modify the nature and structure of assignments to encourage creativity and critical thinking rather than simply requiring the recall of factual knowledge. ● Incorporate elements of mutual benefit in tasks, providing additional incentiv or rewards beyond just a score. ● Establish clear and explicit criteria for assignments, including specific guidel for referencing and citation, as well as clearly defined expectations for acceptab levels of plagiarism, aligning them with the institution's evaluation rules and policies. 3- Proactive preventive measures for addressing Plagiarism ● Incorporate elements of mutual benefit in tasks, providing additional incentives or rewards beyond just a score. ● Establish clear and explicit criteria for assignments, including specific guidelines for referencing and citation, as well as clearly defined expectations for acceptable levels of plagiarism, aligning them with the institution's evaluation rules and policies. Culture ● Foster a shift in students' mindset from an exam/score-oriented attitude to one that focuses on future benefits and learning. ● Cultivate a culture within the institution that promotes and respects copyrights, both among students and faculty members. ● Emphasize rewards and recognition for academic achievements and integrity instead of solely relying on punitive measures. ● Integrate teachings on ethics and moral philosophy into the curriculum. Regulation and assessment ● Announce and communicate the evaluation rules, institutional policies, and regulations regarding plagiarism to all students, ensuring they are aware of the consequences and expectations. ● Modify the nature and structure of assignments to encourage creativity and critical thinking rather than simply requiring the recall of factual knowledge. ● Incorporate elements of mutual benefit in tasks, providing additional incentives or rewards beyond just a score. ● Establish clear and explicit criteria for assignments, including specific guidelines for referencing and citation, as well as clearly defined expectations for acceptable levels of plagiarism, aligning them with the institution's evaluation rules and policies. Culture ● Foster a shift in students' mindset from an exam/score-oriented attitude to one that focuses on future benefits and learning. ● Cultivate a culture within the institution that promotes and respects copyrights, both among students and faculty members. ● Emphasize rewards and recognition for academic achievements and integrity instead of solely relying on punitive measures. ● Integrate teachings on ethics and moral philosophy into the curriculum. ● Integrate teachings on ethics and moral philosophy into the curriculum. 3- Proactive preventive measures for addressing Plagiarism Student support ● Provide a comprehensive student guide. ● Conduct frequent orientation sessions. ● Offer close guidance and mentorship. ● Provide continuous feedback. ● Showcase success stories and Introduce examples of students who have excelled in their research endeavors without resorting to plagiarism. ● Develop writing and research skills. ● Address language barriers. ● Foster peer teaching and support. Detection ● Provide students with free access to plagiarism checker programs to encoura them to incorporate plagiarism checks as a routine step in their work process. ● Incorporate plagiarism detection software into the assessment platforms use by the institution. ● Offer faculty development programs focused on enhancing their knowledge skills in detecting different types of plagiarism. Consequences ● Tailor the penalty of plagiarism attempts according to circumstances: ● They should vary in severity according to the maturity, level of expertise, guidance, and instructions. ● Penalties vary from redoing the work to failure of the task or the year. e e s o p ag a s , a g g t e t t e st tut o s e a uat o u es a d policies. Student support ● Provide a comprehensive student guide. ● Conduct frequent orientation sessions. ● Offer close guidance and mentorship. ● Provide continuous feedback. ● Showcase success stories and Introduce examples of students who have excelled in their research endeavors without resorting to plagiarism. ● Develop writing and research skills. ● Address language barriers. ● Foster peer teaching and support. Detection ● Provide students with free access to plagiarism checker programs to encourage them to incorporate plagiarism checks as a routine step in their work process. ● Incorporate plagiarism detection software into the assessment platforms used by the institution. ● Offer faculty development programs focused on enhancing their knowledge and skills in detecting different types of plagiarism. Consequences ● Tailor the penalty of plagiarism attempts according to circumstances: ● They should vary in severity according to the maturity, level of expertise, guidance, and instructions. ● Penalties vary from redoing the work to failure of the task or the year. Page 15/24 Page 15/24 Regulation and assessment ● Announce and communicate the evaluation rules, institutional policies, and regulations regarding plagiarism to all students, ensuring they are aware of the consequences and expectations. ● Modify the nature and structure of assignments to encourage creativity and critical thinking rather than simply requiring the recall of factual knowledge. Discussion Among these factors are the language barriers and poor academic writing skills as shown by the viewpoints expressed by the results. This has been confirmed by numerous previous studies in which students justified plagiarism from papers written in a foreign language [20, 24, 29, 31, 32]. Moreover, Babelghaith et al. 2022 mentioned that students considered that since they are still learners, they have some freedom to plagiarize to some extent [24]. In addition, students who are not native English speakers may thus carefully follow the original sentence to avoid expressing the topic inaccurately by having poor paraphrasing skills [33]. On the other hand, Smith, Ghazali, and Minhad 2007 showed that there was no concrete evidence to support the idea that a lack of proficiency in the English language was a factor contributing to plagiarist action [34]. Furthermore, the quality and quantity of the given assignments, their strict due dates, and the assessment focusing primarily on scores and outcomes are yet another potential contributor to plagiarism. These factors may result in encouraging students to prioritize quantity and completion over the quality of their work [35], even going so far as to seek financial aid if they are unable to finish an assignment. A common cultural framework that prioritizes social impressions over academic integrity may have an impact on plagiarism among university students. When this happens, students may copy work from others because they are afraid of failing or are hesitant to submit their work at risk [9]. Moreover, the COVID-19 pandemic and the abrupt switch to online learning without prior preparation, which made plagiarism easier. Such expanded internet use during the COVID-19 lockdown has made it much simpler for students to plagiarize whatever they come across. Accordingly, the attitudes about plagiarism have been made worse by such an unexpected surge in online education [31]. Technology was another crucial factor contributing to plagiarism as noted by the students in the FGs. Technology has led to a surge in plagiarism in multiple sectors, including institutions of higher education. It may be somewhat held accountable for the propensity for plagiarism by keeping two factors in mind: Copying convenience, as well as the easy accessibility to materials [8, 13, 32]. Sadly, higher education might move too slowly to introduce new policies and adopt new practices to keep up with these technological breakthroughs [36]. Discussion In this two-phase observational mixed-method study, students in FGs unanimously agreed that plagiarism is highly prevalent in the academic context. This was in line with the ATP questionnaire results in which participating students in both medical schools had moderate mean scores for subjective Norms. This outcome is in line with what was found in previous studies [19, 20]. This is a serious indicator as the theory of planned behavior claims that a person's perception of his/her own behavioral control and subjective standards might influence their intention to engage in action [13]. Accordingly, Students who think others are plagiarizing may be more prone to engage in plagiarism themselves. Students may view plagiarism as the usual and a less serious transgression if they believe that its prevalence is reasonably high [9, 17]. It is noteworthy that students participating in the FGs demonstrated a clear understanding of the term plagiarism. However, concerning the ATP questionnaire components, most surveyed students fell into the moderate category, consistent with prior research [21–26]. This moderate stance may suggest a lack of seriousness or awareness regarding plagiarism. It implies that, even if students acknowledge plagiarism as unethical, they may overlook it under certain circumstances or in an academic setting [27]. On the other hand, the results highlight an argument suggesting that if the use of another's work is done with the consent and approval of the original author, it should not be deemed plagiarism. This perspective is rooted in the concept of 'peer responsibilities' reflecting the cultural norm of Arab students assisting friends and peers [28]. Addressing this, it becomes crucial to guide students in distinguishing between false and genuine definitions of collaborative work. Page 16/24 Page 16/24 Surprisingly, the ATP survey in this study revealed inconsistent and unclear attitudes towards plagiarism. Despite moderate mean scores in both medical schools on the ATP questionnaire, which characterized plagiarism as equally reprehensible as exam stealing and detrimental to the spirit of investigation, there were also moderate mean scores for statements justifying plagiarism. This inconsistency aligns with previous studies [20, 29], suggesting that participants may not have received adequate education about plagiarism, potentially making them more prone to accepting it when deemed necessary. Given the concerning findings of this study and the complex nature of plagiarism's root causes, further exploration into contributing factors was warranted [30]. Interestingly, this study identified a range of factors underlying plagiarism. Discussion It is crucial for higher education institutions to consistently respond to student plagiarism to foster academic integrity, as the absence of negative repercussions may create an environment conducive to plagiarism [10, 20, 35]. Moreover, FG participants believed that repeated successful plagiarism attempts could negatively impact students' attitudes, normalizing plagiarism as a social norm in scientific writing. The absence of consequences may lead students to perceive plagiarism as widely accepted, contributing to its persistence, especially when students receive scores for plagiarized work [20, 32, 41, 44]. Additionally, the likelihood of the incident going unnoticed. This highlighted the need not only to establish strict institutional anti-plagiarism regulations but also to cultivate a negative attitude against plagiarism to discourage it, especially in situations when it is encouraged by everybody around. Establishing a culture in academic institutions that clearly values ethical behavior will reduce the likelihood of plagiarism among students [9]. Discussion Accordingly, because of concerns about student plagiarism, many colleges have already outlawed ChatGPT, and other nations have restricted it. Concerns have been raised that ChatGPT may render the present technologies for detecting plagiarism ineffective. This has already spurred the creation of further tools that can determine if writing contains AI [14]. This should act as an Page 17/24 Page 17/24 alarm to the university officials to carefully consider strategies to guarantee that academic dishonesty is clearly stated to students and reduced, regardless of what occurs on the technological side [37]. alarm to the university officials to carefully consider strategies to guarantee that academic dishonesty is clearly stated to students and reduced, regardless of what occurs on the technological side [37]. However, students also recognized technology as a double-edged weapon. Despite its role in facilitating plagiarism, it can aid in its detection. However, ignorance of the use of such plagiarism-detection software in the academic setting may also hinder it from being a deterrent to plagiarism [38]. Accordingly, universities should make a conscious decision to secure licenses for this software and establish guidance for instructors to use it properly [9]. Moreover, all students should be made aware that their work will be checked for plagiarism, and they need to be well-trained on how to use it before submission [39]. These anti-plagiarism strategies have been also noted by the students in the FGs. It's noteworthy to consider that a variety of complex, culturally specific aspects may have an impact on people's attitudes and propensity to plagiarize, given that different cultures have different learning techniques and norms [20, 40]. Accordingly, this study demonstrated how cultural variations have influenced how people see plagiarism. Students in the FGs concluded that culture significantly influences students' understanding of plagiarism as well as maturity. Interestingly, the participants from both medical schools agreed that young researchers should receive milder punishment, in line with previous studies [24, 41, 42]. The fact that they are still learning might serve as justification for this perspective. However, no one should be exempted from the consequences of plagiarism. The ethical and legal issues with plagiarism cannot be justified by either inexperience or ignorance [43]. Furthermore, the students highlighted that robust institutional regulations against plagiarism and staff members' negative attitudes toward it could serve as significant deterrents. Declarations Ethics approval and consent to participate: Ethical approval was secured from the Research Ethics Committee of the Faculty of Medicine-Suez Canal University, Egypt (Research 4610#), and the Institutional Research Review Board at Ibn Sina National College, Saudi Arabia (IRRB-21-09062021) before the commencement of data collection. Consent for publication: All authors approve to publish the work. Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors report there are no competing interests to declare Competing interests: The authors report there are no competing interests to declare Funding: This work was self-funded by authors. Authors Contributions: Conceptualization, AA and SA; Data curation: AA, EA, SA, EM, and NW; Formal analysis, MI, EA, NW, and AA; Methods: SA, NW, EA, AA and EM; Project administration, AA, EA, SA, EM and NW; Writing – original draft, AA, EA, SA, EM, and NW; Writing – review & editing, AA, EA, SA, EM, MI and NW. All authors have read and approved the manuscript. Conclusion The conclusion of this work demonstrated a comprehensive understand It suggested that the use of another person's work, when done with the c original author, should not be considered plagiarism. The study found th plagiarism, under the belief that building upon previous work can save ti highlighted several factors contributing to plagiarism, such as language writing skills, the high demands and volume of assignments, tight deadl overly fixated on scores and outcomes. Additionally, the study recognize in this context. To tackle this problem effectively, it is crucial to establish various elements. This includes increasing student awareness, refining e regularly updating anti-plagiarism technology. By adopting this compreh discourage plagiarism and promote a culture of academic integrity. The conclusion of this work demonstrated a comprehensive understanding of the concept of 'plagiarism.' It suggested that the use of another person's work, when done with the consent and approval of the original author, should not be considered plagiarism. The study found that students often tolerate self- plagiarism, under the belief that building upon previous work can save time and effort. The research highlighted several factors contributing to plagiarism, such as language barriers, insufficient academic writing skills, the high demands and volume of assignments, tight deadlines, and an assessment system overly fixated on scores and outcomes. Additionally, the study recognized the dual nature of technology in this context. To tackle this problem effectively, it is crucial to establish a positive cycle that integrates various elements. This includes increasing student awareness, refining ethics course content, and regularly updating anti-plagiarism technology. By adopting this comprehensive approach, the goal is to discourage plagiarism and promote a culture of academic integrity. Strengths and limitations The study's strengths lie in its transcultural, mixed-method approach and the thorough exploration of various student characteristics. However, limitations exist. First, the representation of only one medical school from each of the two Middle Eastern countries may limit generalizability to other regional medical schools. Future studies should include a broader sample of medical schools in the region. Furthermore, Page 18/24 the cross-sectional nature of the quantitative phase prevents establishing causal inferences. Finally, selection bias in the qualitative phase due to purposive sampling may affect generalizability. Acknowledgments: We would like to extend our sincere gratitude and appreciation to the students who actively participated in the study from both Egyptian and Saudi medical schools. Our special thanks go to Dr. Rashad Hassan Page 19/24 Al Kashgari, the Dean of Ibn Sina National College for Medical Studies, and Dr. Ranya Mostafa, Vice Dean of the Education sector at FOM-SCU Egypt, for their unwavering assistance and limitless support. Authors' information: 1Medical Education Department, Faculty of Medicine, Suez Canal University, Egypt 2Health Professions Education Center, Ibn Sina National College for Medical Studies, Saudi Arabia 3Faculty of Medicine, Ulster University, UK 4Forensic Medicine and Clinical Toxicology Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt 5Family and Community Medicine Department, Ibn Sina National College for Medical Studies, Saudi Arabia 6Community Medicine Department, Faculty of Medicine, Alexandria University, Egypt Asmaa Abdelnasser: https://orcid.org/0000-0002-1276-5014 Enjy Abouzeid: https://orcid/.org/0000-0002-9431-6019 Enas M A Mostafa: https://orcid.org/0000-0003-3843-2899 Manal Ibrahim Hanafi Mahmoud: https://orcid.org/0000-0001-5451-6924 Nourhan F. Wasfy: https://orcid.org/0000-0002-2896-9142 Shaimaa A Shehata: https://orcid.org/0000-0002-2810-3613 Nourhan F. Wasfy: https://orcid.org/0000-0002-2896-9142 Nourhan F. 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Factors Influencing Plagiarism Amongst Undergraduate Students at an Institution of Higher Learning: Kwazulu-Natal. In: Makua M, Akinlolu M, Sithole M, Gumede P, Nyondo C, editors. Proceedings of The Focus Conference (TFC 2022). Paris: Atlantis Press SARL; 2023. p. 60–71. 33. De Lima JÁ, Sousa Á, Medeiros A, Misturada B, Novo C. Understanding Undergraduate Plagiarism in the Context of Students’ Academic Experience. J Acad Ethics. 2022;20:147–68. 34. Smith M, Ghazali N, Minhad SFN. Attitudes towards plagiarism among undergraduate accounting students: Malaysian evidence. Asian Rev Acc. 2007;15:122–46. 35. Hussein MG. The awareness of plagiarism among postgraduate students at Taif University and its relationship to certain variables. Cogent Soc Sci. 2022;8:2142357. 36. Lee J, Soylu MY. ChatGPT and Assessment in Higher Education. 2023. 36. Lee J, Soylu MY. ChatGPT and Assessment in Higher Education. 2023. 37. Cotton D, Cotton P, Shipway JR. Chatting and Cheating. Ensuring academic integrity in the era of ChatGPT. Innov Educ Teach Int. 2023. https://doi.org/10.1080/14703297.2023.2190148. 38. Tayan BM. Academic Misconduct: An Investigation into Male Students’ Perceptions, Experiences & Attitudes towards Cheating and Plagiarism in a Middle Eastern University Context. J Educ Learn. 2017;6:158–66. 39. Nahas MN. Survey and Comparison between Plagiarism Detection Tools. Am J Data Min Knowl Discov. 2017;2:50–3. Page 22/24 40. References Abbas A, Fatima A, Arrona-Palacios A, Haruna H, Hosseini S. Research ethics dilemma in higher education: Impact of internet access, ethical controls, and teaching factors on student plagiarism. Educ Inf Technol. 2021;26:6109–21. 41. Gomez MSS, Nagesh L, Sujatha BK. Assessment of the attitude towards Plagiarism among dental postgraduate students and faculty members in Bapuji Dental College and Hospital, Davangere – A cross sectional survey. IOSR J Dent Med Sci. 2014;13:01–6. 42. 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Figure 1 Questions of the Focus Group Guide Questions of the Focus Group Guide Questions of the Focus Group Guide Page 23/24 Figure 2 Thematic Analysis of the Qualitative Data Thematic Analysis of the Qualitative Data Page 24/24
https://openalex.org/W2041842730	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0077438&type=printable	English	null	Characteristics of Acupuncture Treatment Associated with Outcome: An Individual Patient Meta-Analysis of 17,922 Patients with Chronic Pain in Randomised Controlled Trials	PloS one	2,013	cc-by	7,906	Hugh MacPherson1, Alexandra C. Maschino2, George Lewith3, Nadine E. Foster4, Claudia Witt5, Andrew J. Vickers2, on behalf of the Acupuncture Trialists' Collaboration¶ 1 Department of Health Sciences, University of York, York, United Kingdom, 2 Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America, 3 University of Southampton, Southampton, United Kingdom, 4 Arthritis Research UK Primary Care Centre, Institute of Primary Care and Health Sciences, Keele University, Keele, United Kingdom, 5 Institute for Social Medicine, Epidemiology and Health Economics,Charité Universitätsmedizin, Berlin, Germany Abstract Background: Recent evidence shows that acupuncture is effective for chronic pain. However we do not know whether there are characteristics of acupuncture or acupuncturists that are associated with better or worse outcomes. Methods: An existing dataset, developed by the Acupuncture Trialists’ Collaboration, included 29 trials of acupuncture for chronic pain with individual data involving 17,922 patients. The available data on characteristics of acupuncture included style of acupuncture, point prescription, location of needles, use of electrical stimulation and moxibustion, number, frequency and duration of sessions, number of needles used and acupuncturist experience. We used random-effects meta-regression to test the effect of each characteristic on the main effect estimate of pain. Where sufficient patient-level data were available, we conducted patient-level analyses. Results: When comparing acupuncture to sham controls, there was little evidence that the effects of acupuncture on pain were modified by any of the acupuncture characteristics evaluated, including style of acupuncture, the number or placement of needles, the number, frequency or duration of sessions, patient-practitioner interactions and the experience of the acupuncturist. When comparing acupuncture to non-acupuncture controls, there was little evidence that these characteristics modified the effect of acupuncture, except better pain outcomes were observed when more needles were used (p=0.010) and, from patient level analysis involving a sub-set of five trials, when a higher number of acupuncture treatment sessions were provided (p<0.001). Conclusion: There was little evidence that different characteristics of acupuncture or acupuncturists modified the effect of treatment on pain outcomes. Increased number of needles and more sessions appear to be associated with better outcomes when comparing acupuncture to non-acupuncture controls, suggesting that dose is important. Potential confounders include differences in control group and sample size between trials. Trials to evaluate potentially small differences in outcome associated with different acupuncture characteristics are likely to require large sample sizes. Citation: MacPherson H, Maschino AC, Lewith G, Foster NE, Witt C, et al. (2013) Characteristics of Acupuncture Treatment Associated with Outcome: An Individual Patient Meta-Analysis of 17,922 Patients with Chronic Pain in Randomised Controlled Trials . PLoS ONE 8(10): e77438. doi:10.1371/ journal.pone.0077438 Editor: Sam Eldabe, The James Cook University Hospital, United Kingdom Received March 15, 2013; Accepted September 2, 2013; Published October 11, 2013 Received March 15, 2013; Accepted September 2, 2013; Published October 11, 2013 Copyright: © 2013 MacPherson et al. Citation: MacPherson H, Maschino AC, Lewith G, Foster NE, Witt C, et al. (2013) Characteristics of Acupuncture Treatment Associated with Outcome: An Individual Patient Meta-Analysis of 17,922 Patients with Chronic Pain in Randomised Controlled Trials . PLoS ONE 8(10): e77438. doi:10.1371/ journal.pone.0077438 Characteristics of Acupuncture Treatment Associated with Outcome: An Individual Patient Meta-Analysis of 17,922 Patients with Chronic Pain in Randomised Controlled Trials Hugh MacPherson1, Alexandra C. Maschino2, George Lewith3, Nadine E. Foster4, Claudia Witt5, Andrew J. Vickers2, on behalf of the Acupuncture Trialists' Collaboration¶ 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. Funding: The Acupuncture Trialists’ Collaboration is funded by an R21 (AT004189I from the National Center for Complementary and Alternative Medicine (NCCAM) at the National Institutes of Health (NIH) to AV) and by a grant from the Samueli Institute. HM’s work has been supported in part by the United Kingdom National Institute for Health Research (NIHR) under its Programme Grants for Applied Research scheme (RP-PG-0707-10186). CW’s work has been supported by the Carstens Foundation within the grant for the Chair for Complementary Medicine Research. NF is supported by a Research Professorship from the National Institute of Health Research. The views expressed in this publication are those of the author(s) and not necessarily those of the NCCAM, NHS, NIHR or the Department of Health in England. No sponsor had any role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript. 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. ¶ Members of the Acupuncture Trialists’ Collaboration are listed in the acknowledgements. October 2013 \| Volume 8 \| Issue 10 \| e77438 PLOS ONE \| www.plosone.org 1 Characteristics of Acupuncture and Outcome Acupuncture Characteristics Responding authors of the trials were asked to select from these three options. Trials were categorized as flexible needle formula if trialists indicated that acupuncture was semi-standardized, flexible with fixed points, or both fixed and flexible formulas. The analyses compare flexible and individualized formula to the reference group, fixed needle formula. Outcome The primary outcome used for this analysis was pain as defined as primary outcome by the responding author of each study. Where multiple criteria were considered in the primary outcome (e.g. a response defined as either a 33% reduction in pain or a 50% reduction in pain medication) or if the primary outcome was inherently categorical, we used a continuous measure of pain measured at the same time point as the original primary outcome. To make the various outcome measurements comparable between different trials, the primary endpoint of each was standardized by dividing by pooled standard deviation. Data Acquisition Individual patient data were obtained from 29 trials. Data on the trial-level characteristics of the acupuncture intervention were obtained directly from responding trialists by use of a questionnaire and are presented in Appendix S1. Acupuncture is not a single standardized intervention. In routine clinical practice, the same patient may receive acupuncture with different characteristics from different practitioners. These differences include specific characteristics of treatment - such as the number and frequency of sessions or the additional use of electrical stimulation - as well as the overall “style” of acupuncture. A distinction is often made between traditional Chinese acupuncture, in which diagnosis and treatment are based on a theoretical framework involving patterns of symptoms and concepts such as yin, yang and the strength of qi, and Westernized approaches, involving a neuro- anatomical basis for diagnosis and treatment[2]. The estimates of effect sizes such as those reported by the Acupuncture Trialists' Collaboration are averages across different acupuncturists and styles of acupuncture[1]. It is natural to ask, therefore, whether there are characteristics of acupuncture style or acupuncturists that are associated with patient outcome, that is, whether there are modifiers of acupuncture effect. Introduction described[1]. The search included trials of acupuncture for chronic pain published prior to November 2008 and included only trials where allocation concealment was determined unambiguously to be adequate. Eligible pain types were non- specific back or neck pain, shoulder pain, chronic headache or osteoarthritis - with the additional criterion that the current episode of pain must be of at least four weeks duration. This search resulted in the identification of 31 trials. A recent individual patient data meta-analysis conducted by the Acupuncture Trialists' Collaboration provided clear evidence that acupuncture is of benefit for chronic pain[1]. The study included close to 18,000 patients with one of four chronic pain conditions: osteoarthritis, headache, back and neck pain, shoulder pain. Comparisons were made between acupuncture and sham acupuncture, as well as between acupuncture and a range of non-acupuncture controls, such as wait list and usual care. Benefits in terms of effect sizes (standardised mean differences) were in the order of 0.15 to 0.23 for the comparison between acupuncture and sham and 0.5 for the comparison with non-acupuncture controls[1]. Acupuncture Characteristics The characteristics of acupuncture we investigated included the style of acupuncture, the point prescription, the location of needles, the use of electrical stimulation and moxibustion, whether acupuncture-specific patient practitioner interactions were allowed, acupuncturist experience, the number, frequency and duration of sessions, and the number of needles used. Some trials recorded patient-level data on certain acupuncture characteristics, such as the number of sessions, the duration of sessions and the number of needles used. In these cases a summary statistic was calculated and entered into analysis. If no such data were available, the values reported by trialists on the data abstraction sheets were used. Assessing effect modification in clinical trials is associated with considerable power concerns [3,4]. Trials are usually designed with only sufficient power to test the primary hypothesis in which case they will be inherently underpowered to determine whether individual characteristics of treatment might be associated with affect outcome. The sample size needed to detect an interaction has to be many times greater than that needed to detect a primary effect[5]. Combining summary data from a number of trials in a meta-analysis can overcome the problem of limited power to some extent. An individual patient data meta-analysis is more powerful as it allows patient-level analysis[6]. Style of acupuncture. Trialists defined the style of acupuncture as based on traditional Chinese theory, or contemporary Western acupuncture or a mixture of both approaches. For our main analysis, we compared trials that used at least some traditional approaches to those where the acupuncture was purely Western; as a sensitivity analysis, we compared trials using purely a Chinese traditional approach to those that used at least some Western techniques. The large individual patient dataset developed by the Acupuncture Trialists Collaboration[1] incorporates raw data from high quality randomised controlled trials of acupuncture for chronic pain. In this study, we analyse this dataset to determine whether there are characteristics of acupuncture or acupuncturists that act as effect modifiers for acupuncture treatment outcome. Point Prescription. The point prescription was defined as fixed, flexible or individualized. Responding authors of the trials were asked to select from these three options. Trials were categorized as flexible needle formula if trialists indicated that acupuncture was semi-standardized, flexible with fixed points, or both fixed and flexible formulas. The analyses compare flexible and individualized formula to the reference group, fixed needle formula. Point Prescription. The point prescription was defined as fixed, flexible or individualized. Statistical Methods Data on effect modifiers were either at the individual or trial level. Using number of treatment sessions as an example, this would constitute individual level data if the number of acupuncture sessions received by each patient had been recorded on the study database, but constitute trial level data if either all patients received the same number of sessions or data were not recorded at the patient level and an estimate of the average number of sessions was provided by the trialists. Minimum years of practice required. Trialists were asked to report the minimum number of years of practice as an acupuncturist required to participate in their trial. If trialists did not indicate a requirement, the trial was included in the analysis as having zero years required, even if trialists noted that all acupuncturists were experienced. Otherwise, the minimum number of years required was entered into the analyses as a continuous variable. For each trial, we identified the primary outcome defined by the study authors in terms of both the scale and time point. We kept endpoints on the continuous scale. If multiple criteria are considered in the primary outcome, or if the primary outcome is inherently categorical, we will use a continuous measure of pain measured at the same time point as the original primary endpoint. For analyses that include trials with different primary endpoints, we will create a standardized primary endpoint by dividing by standard deviation. Maximum Number of sessions allowed. The maximum number of acupuncture treatment sessions allowed during the trial period was reported by each trialist. If participants were allowed extra sessions pending partially successful treatment, these were not counted towards a maximum since it was frequently the case that extra sessions were only offered to partial responders. If patient-level data were available an average was taken. Data were analyzed per 5 session increments. For trial-level analyses, we used random-effects meta- regression to test the effect of each characteristic on the main effect estimate using the Stata command metareg. We first calculated the effect size and standard error for each trial as described in our main paper[1]. For each treatment characteristic under study, we then entered the effect size and standard error for each trial into a meta-regression along with the trial level average for that characteristic. patient Acupuncture-specific patient practitioner interactions. Acupuncture-specific interactions between the patient and the acupuncturist can occur through explanations of treatment, advice, support, and suggestions about helpful lifestyle changes, which are driven by acupuncture theory and intended to influence outcome. These non-needling aspects of treatment can be considered integral to and characteristic of acupuncture. Trialists were asked if their trial allowed or encouraged these interactions and associated patient self-help actions. If the trialist replied that these kinds of interactions were not prohibited at the trial level it was assumed that they might have taken place since they are often part of standard practice. Location of Needles. The placement of acupuncture needles was categorized as local (at or near the location of pain) or distal to the location of pain), or both. Included Trials Trials included in these analyses were identified through a systematic literature review that has been previously October 2013 \| Volume 8 \| Issue 10 \| e77438 PLOS ONE \| www.plosone.org 2 Characteristics of Acupuncture and Outcome Characteristics of Acupuncture and Outcome Stimulation. Trialists were asked to report if their trial allowed electrical stimulation to be added to the needles during acupuncture sessions. Stimulation. Trialists were asked to report if their trial allowed electrical stimulation to be added to the needles during acupuncture sessions. trial, the author specified that 9 needles were used in each affected leg. The paper published from this trial reported that 25% of patients in the acupuncture group had both knees affected so therefore this trial was included in the analysis has having an average of 11 needles. Trials were excluded from this analysis if the number of needles was unknown. If patient- level data were available an average was included. The average number of needles used was analyzed as a continuous variable, with the coefficient reported in 5 needle increments. Moxibustion. Trialists were asked to report if acupuncturists were allowed to prescribe moxibustion. Moxibustion. Trialists were asked to report if acupuncturists were allowed to prescribe moxibustion. De Qi. Trial level information on de qi needle sensation was collected. If acupuncturists attempted to elicit de qi, whether felt by acupuncturist or patient, then the trial was considered to have attempted de qi. De Qi. Trial level information on de qi needle sensation was collected. If acupuncturists attempted to elicit de qi, whether felt by acupuncturist or patient, then the trial was considered to have attempted de qi. October 2013 \| Volume 8 \| Issue 10 \| e77438 Statistical Methods The coefficients obtained from these analyses are estimates, in standard deviations, of the effect of each acupuncture characteristic on the main treatment effect. Using session time to illustrate, this analysis addresses questions about effect modification in the form of: “Do trials where the practitioners provided patients with longer session times on average show larger or smaller effects of acupuncture than trials where the average session time was shorter?” Frequency of sessions. The frequency of sessions was recorded and analyzed continuously, as a weekly average (ie. typical number of sessions per week). If a range was given, or if the frequency varied over the study period, the mid-point was taken. Duration of sessions. The duration of sessions was reported as the average length of a session in minutes among the patients receiving acupuncture. The mid-point was used if a range was given. We used average needle retention time for two trials that reported this characteristic but not overall treatment session time. Patient-level data were used where available by taking the mean duration of patients’ sessions. Trials were not included in this analysis if treatment was individualized and no individual level data were available. Duration of sessions was included as a continuous variable in the analyses and results reported per 5 minute increments. Patient-level analyses included the number of sessions, the number of needles, and the age and gender of the acupuncturist. For each trial, we created a linear regression as for the main analysis of effect size, but included the characteristic and an interaction term between the characteristic and treatment allocation. The coefficient and standard error for the interaction term represents the change in the outcome score in standard deviations associated with the acupuncture characteristic in the acupuncture treatment group. This was then entered into a meta-analysis, using the Stata Average Number of needles used. Trialists were asked to report the average number of needles used per treatment session. If a range was given, the mid-point was taken. For one PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77438 3 Characteristics of Acupuncture and Outcome acupuncture controlled trials included only 5 trials and 8 292 Table 1. Trial-level acupuncture characteristics. Frequency (%). N=29. Statistical Methods Style of acupuncture Traditional Chinese techniques 17 (59%) ’Western’ 4 (14%) Combination of traditional Chinese and Western 8 (28%) Point Prescription Fixed needle formula 4 (14%) Flexible formula 16 (55%) Individualized 9 (31%) Location of needles Local Points Only 0 (0%) Distal Points Only 1 (3%) Both Local and Distal Points 28 (97%) Electrical stimulation allowed 7 (24%) Moxibustion allowed 4 (14%) De Qi attempted (n=25) 25 (100%) Acupuncture-specific patient practitioner interactions 12 (41%) Minimum years of experience required No requirement specified (0 years) 12 (41%) 6 months to 2 years 5 (17%) 3 years 9 (31%) 5 years 2 (7%) 10 years 1 (3%) Maximum number of sessions 3-5 3 (10%) 6-10 14 (48%) 11-15 7 (24%) 16-20 3 (10%) 21-30 2 (7%) Frequency of sessions (mean number of sessions per week) 0.88 1 (3%) 1 14 (48%) 1.5 7 (24%) 1.67 1 (3%) 2 6 (21%) Mean duration of sessions, rounded to whole numbers (n=26) 15-19 minutes 1 (4%) 20-24 minutes 4 (16%) 25-29 minutes 6 (24%) 30+ minutes 14 (56%) Mean number of needles used, rounded to whole numbers (n=25) 1-4 1 (4%) 5-9 6 (25%) 10-14 9 (38%) 15-20 8 (33%) doi: 10.1371/journal.pone.0077438.t001 command metan. The interpretation of the results is similar as to the results from the trial level analysis. In both analyses, random effects estimates were used. Table 1. Trial-level acupuncture characteristics. Frequency (%). N=29. A pre-planned sensitivity analysis excluded a set of outlying trials, all by the same team, which had very much larger effect sizes than other trials. This sensitivity analysis has previously been conducted in the primary analysis of effect size[1]. Analyses were conducted separately for sham and non- acupuncture controls. All analyses were conducted using Stata 12.0 (Stata Corp., College Station, TX). Results Characteristics of each individual trial are presented in Appendix S1. Table 1 provides a summary of the trial-level characteristics, while Table 2 provides a summary of patient- level characteristics. In a majority of trials the acupuncture was based on traditional Chinese acupuncture (59%) and had a flexible point prescription (55%). In all 29 trials manual needle stimulation was used in the acupuncture group, while only about quarter of trials allowed the addition of electrical stimulation (n=7) and 14% allowed moxibustion (n=4). Attempts to elicit de qi in the acupuncture group were made in all 25 trials that provided this information. The maximum number of sessions varied widely, from 3 to 30, as did the mean number of needles used (range 1-18) and the mean session duration (range 15 to 32 minutes). The mean session frequency ranged from one session every eight days to two sessions a week. In the trial-level data we did not find evidence that any of the acupuncture characteristics evaluated, including style of acupuncture, the number or placement of needles, the number, frequency or duration of sessions, patient-practitioner interactions or the experience of the acupuncturist, significantly changed the effect of acupuncture on pain (all p>0.05) in sham controlled trials (Table 3). We also found little evidence that these characteristics modify the effect of acupuncture when acupuncture was compared to non-acupuncture controls. The exception was that acupuncture effects increased in comparison to non-acupuncture controls when more needles were used (increase in effect size per 5 needles of 0.33; 95% CI 0.08, 0.58; p=0.010). The results of the sensitivity analysis excluding three outlying trials, which were all sham controlled, all by the same team, and with very much larger effect sizes than other trials, are presented in Table 4. This analysis showed that trials allowing electrical stimulation had a significantly stronger effect of acupuncture compared to sham controls (β= 0.27; 95% CI 0.03, 0.51; p=0.027) and those with a longer average treatment session duration had a smaller effect compared to sham controls (β=-0.14 per 5 minutes; 95% CI -0.22, -0.06; p=0.001). Patient-level analyses mirrored trial level analyses; the direction of the effect of the acupuncture characteristics was unchanged (Table 5). Importantly, the confidence intervals were much tighter around the patient-level estimates despite the fact that fewer trials could be included in each analysis. Results For each analysis, there were no more than five trials with patient- level data available for the specific predictor being analyzed. The patient-level analysis for number of sessions in non- The results of the sensitivity analysis excluding three outlying trials, which were all sham controlled, all by the same team, and with very much larger effect sizes than other trials, are presented in Table 4. This analysis showed that trials allowing electrical stimulation had a significantly stronger effect of acupuncture compared to sham controls (β= 0.27; 95% CI 0.03, 0.51; p=0.027) and those with a longer average treatment session duration had a smaller effect compared to sham controls (β=-0.14 per 5 minutes; 95% CI -0.22, -0.06; p=0.001). doi: 10.1371/journal.pone.0077438.t001 acupuncture controlled trials included only 5 trials and 8,292 patients compared to trial-level analysis which included 18 trials with a total of 14,597 patients. The patient-level analysis suggested that a higher number of acupuncture treatment sessions improves the effect of acupuncture (β=0.11; 95% CI 0.01, 0.21; p=0.0007). We considered the possibility of interactions either between for number of sessions and duration of sessions or between the duration of sessions and Patient-level analyses mirrored trial level analyses; the direction of the effect of the acupuncture characteristics was unchanged (Table 5). Importantly, the confidence intervals were much tighter around the patient-level estimates despite the fact that fewer trials could be included in each analysis. For each analysis, there were no more than five trials with patient- level data available for the specific predictor being analyzed. The patient-level analysis for number of sessions in non- PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77438 4 Characteristics of Acupuncture and Outcome Table 2. Patient-level acupuncture characteristics. Frequency (%). N=18,434. Results Number of Sessions 0 383 (2%) 1-5 402 (2%) 6-10 7161 (39%) 11-15 1998 (11%) 16-20 45 (<1%) 21-30 16 (<1%) Missing 1806 (10%) Not reported 6623 (36%) Average Session Duration 2-15 166 (1%) 15-30 2552 (14%) 31-45 406 (2%) 46-60 60 (<1%) 60+ 3 (<1%) Missing 1257 (7%) Not reported 13990 (76%) Average Number of Needles 2-5 20 (<1%) 6-10 610 (3%) 11-15 717 (4%) 16-20 627 (3%) 21-25 177 (1%) 26+ 27 (<1%) Missing 2529 (14%) Not reported 13727 (74%) Age of Physician/Acupuncturist 30-35 298 (2%) 36-40 2119 (11%) 41-45 2630 (14%) 46-50 2407 (13%) 51-55 1701 (9%) 56-60 872 (5%) 60+ 303 (2%) Missing 368 (2%) Not reported 7736 (42%) Male Physician/Acupuncturist Male 7002 (66%) Female 3626 (20%) Missing 0 (0%) Not reported 7806 (42%) doi: 10.1371/journal.pone.0077438.t002 evidence of a modifying effect on pain outcomes, including many characteristics that might have been commonly expected to modify outcomes, such as style of acupuncture, use of electrical stimulation, addition of moxibustion, experience of the Table 3. Results of univariate meta-regression analyses. ACUPUNCTURE VS. SHAM n=20 ACUPUNCTURE VS. NON- ACUPUNCTURE n=18 β* 95% CI P value β* 95% CI P value Style of acupuncture Some TCM v Western only 0.05 -0.52, 0.63 0.9 0.13 -0.51, 0.77 0.7 TCM only v Some Western 0.20 -0.20, 0.61 0.3 -0.10 -0.38, 0.19 0.5 Point Prescription Fixed needle formula ref ref Flexible formula -0.08 -0.58, 0.43 0.8 0.02 -0.64, 0.68 >0.9 Individualized -0.15 -1.16, 0.86 0.8 -0.08 -0.74, 0.59 0.8 Electrical stimulation allowed 0.34 -0.13, 0.80 0.15 -0.19 -0.56, 0.17 0.3 Manual stimulation allowed all allowed all allowed Moxibustion allowed all did not allow -0.28 -0.63, 0.06 0.11 De Qi attempted all allowed all allowed Acupuncture- specific patient practitioner interactions allowed -0.22 -0.70, 0.26 0.4 0.06 -0.23, 0.35 0.7 Minimum experience required (years) 0.01 -0.08, 0.10 0.8 0.05 -0.05, 0.16 0.3 Maximum number of sessions (per 5 sessions) -0.14 -0.37, 0.08 0.2 0.02 -0.07, 0.12 0.6 Frequency of sessions (per week) -0.19 -0.66, 0.27 0.4 0.09 -0.31, 0.49 0.7 Duration of sessions (per 5 minutes) a -0.10 -0.30, 0.11 0.4 -0.01 -0.26, 0.24 0.9 Number of needles used (per 5 needles) b -0.17 -0.37, 0.03 0.095 0.33 0.08, 0.58 0.01 . Results β is an estimate of the change in the effect of acupuncture in terms of standardized difference compared to controls for each characteristic; a positive β indicates a larger effect of acupuncture compared to controls for trials with the given the characteristic versus the referent level of the characteristic. a. 4 trials excluded missing data b. 5 trials excluded missing data doi: 10.1371/journal.pone.0077438.t003 Table 3. Results of univariate meta-regression analyses. doi: 10.1371/journal.pone.0077438.t002 number of needles, and we found that neither one of these interactions was significantly associated with the effect of acupuncture. Table 5. Results of patient-level analysis of acupuncture characteristics. Table 5. Results of patient-level analysis of acupuncture characteristics. SHAM NON-ACUPUNCTURE N β 95% CI P value N β 95% CI P value Number of sessions (per 5 sessions) 3 (646/648) -0.76 -1.75, 0.22 0.13 5 (8292/9321) 0.11 0.01, 0.21 0.0007 Duration of sessions (per 5 minutes) 5 (2444/2482) -0.03 -0.08, 0.03 0.3 less than 3 trials Number of needles used (per 5 needles) 5 (1769/2484) -0.11 -0.35, 0.14 0.4 less than 3 trials Age of acupuncturist (per 5 years) less than 3 trials 6 (9127/9446) -0.01 -0.04, 0.02 0.5 Male acupuncturist less than 3 trials 6 (9384/9446) -0.07 -0.16, 0.02 0.084 N = Number of trials included in analysis (number of patients included in analysis /total number of patients in included trials) doi: 10.1371/journal.pone.0077438.t005 physiotherapy-led course of advice and supervised exercise to all participants. This evidence-based package of care[10] provided to all trial participants may have led to a ceiling effect making it difficult to identify any additional effect of acupuncture. We also found in a patient level analysis involving a limited dataset from a sub-set of five trials that more treatment sessions was associated with better pain outcomes in acupuncture treatment groups compared to non-acupuncture controls (p<0.001). This suggests that dose of acupuncture could have an impact on treatment outcomes. Table 4. Results of meta-regression for acupuncture trials with sham controls, excluding outlying trials. Table 4. Results of meta-regression for acupuncture trials with sham controls, excluding outlying trials. ACUPUNCTURE vs. SHAM n=17 N Β 95% CI P value Style of acupuncture 17 ’Western’ only ref Traditional Chinese -0.14 -0.46, 0.17 0.4 Point Prescription 17 Fixed needle formula ref Flexible formula -0.25 -0.50, 0.00 0.054 Individualized -0.11 -0.58, 0.36 0.6 Electrical stimulation allowed 17 0.27 0.03, 0.51 0.027 Manual stimulation allowed 17 all allowed Moxibustion allowed 17 all did not allow De Qi elicited (n=26) 17 all allowed Acupuncture-specific patient practitioner interactions allowed 17 -0.04 -0.28, 0.20 0.8 Minimum experience required (years) 17 0.00 -0.05, 0.05 >0.9 Maximum number of sessions (per 5 sessions) 17 -0.05 -0.18, 0.08 0.4 Frequency of sessions (per week) 17 -0.04 -0.29, 0.21 0.8 Duration of sessions (per 5 minutes) 17 -0.14 -0.22, -0.06 0.001 Number of needles used (per 5 needles) 17 -0.08 -0.22, 0.05 0.2 *. Table 5. Results of patient-level analysis of acupuncture characteristics. β is an estimate of the change in the effect of acupuncture compared to controls for each characteristic; a positive β indicates a larger effect of acupuncture compared to controls for trials with the given the characteristic versus the referent level of the characteristic. doi: 10.1371/journal.pone.0077438.t004 When interpreting these analyses, we carefully considered the results in the context of testing multiple hypotheses. Testing a large number of hypotheses increases the risk of falsely rejecting at least one null hypothesis. Given the largely null results, we did not feel that formal statistical correction to account for multiple testing was justified. Principal findings evidence of a modifying effect on pain outcomes, including many characteristics that might have been commonly expected to modify outcomes, such as style of acupuncture, use of electrical stimulation, addition of moxibustion, experience of the Our results provide the best information available on treatment effect modifiers in acupuncture trials for chronic pain. For most treatment-related characteristics, we found no PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77438 5 Characteristics of Acupuncture and Outcome Table 5. Results of patient-level analysis of acupuncture characteristics. Table 5. Results of patient-level analysis of acupuncture characteristics. October 2013 \| Volume 8 \| Issue 10 \| e77438 Strengths and Limitations When exploring practitioner-related effects on outcome, the few characteristics of acupuncturists that were reported sufficiently consistently by trialists, namely age, gender and minimum experience as an acupuncture practitioner, are likely to be too simple. It is possible that there is variation associated with individual practitioners, that some have better results than others. A similar situation has arisen for other therapist-led interventions[16,17]. To better understand the reasons for any heterogeneity between acupuncturists, more sophisticated measures are likely to be needed. For example, empathy has been shown to correlate with enablement, which in turn correlates with outcome[18], and further research into other measures such as the therapeutic alliance or success of patient-practitioner interactions may be useful lines of enquiry. Our findings are consistent with a widely accepted principle underlying traditional Chinese medicine that it is not the techniques and methods used but the cultivation of the practitioner that is the key to effective practice[19]. Further qualitative work might usefully explore the ways that acupuncturists perceive that they are effective, including the ways that they cultivate themselves as practitioners. The benefits of an individual patient data meta-analysis as opposed to a meta-analysis of summary data include increased power and the facility to conduct further types of analyses, including the patient level analyses of treatment effect modifiers that we report here. Indeed, it is highly doubtful that questions about effect modification could be addressed outside the context of an meta-analysis. The main limitation, as for any meta-analysis, was data availability. The total number of trials was relatively modest, and analyses with individual patient data included no more than five trials. As a result, many of our analyses had relatively low power, with wide confidence intervals around central estimates. Furthermore, heterogeneity of treatment characteristics was relatively limited. For example, nearly 75% of trials involved between 6 and 15 treatments, and in no trial was acupuncture administered more than twice a week. It is not unusual in China for acupuncture to be given four or five times a week. We were unable to test whether such a level of acupuncture frequency has additional benefit as none of the included primary trials delivered acupuncture this frequently. We also did not have data on syndrome differentiation, a feature that characterizes acupuncture when practiced according to the principles of traditional Chinese medicine, and could therefore draw no conclusions regarding their impact on outcome. Comparison with other studies We did not observe large variations in outcome associated with the style of acupuncture practice. This contrasts with findings from qualitative studies that purported to identify the importance of the theoretical affiliation and institutional context[11,12]. Hughes et al describe how theoretical affiliation can impact on “almost all aspects of treatment”, with “demonstrable implications for the practice and research of acupuncture”.[11] For example, the authors argued that the outcomes from trials of acupuncture “fail to reflect [acupuncture’s] effectiveness in clinical practice”, and hypothesize that therapeutic benefits are likely to be reduced due to an absence of a traditional acupuncture approach that incorporates the notion of “energy work”.[11] Similarly, Paterson and Britten argue that without a theoretical approach to acupuncture that is “holistic”, with an emphasis that includes the process of care, there is likely to be “reduced or absent treatment effects”.[12] These theoretical positions are not supported by our empirical data in patients with chronic pain, although we accept that it is theoretically possible that effect modifiers of acupuncture may be condition specific. acupuncturist, and the frequency and duration of sessions. We found that the effect of acupuncture increased in comparison to non-acupuncture controls when more needles were used. This association should be considered in the context of the large number of our hypotheses tested and the rather implausible estimate of an increase in effect size per 5 needles of 0.33, larger than the mean difference between true and sham acupuncture [1]. Additionally, this result is driven by the two trials with the largest positive effect size (Brinkhaus et. al for LBP [7] and Witt et. al 2005 [8] for OA) also used the most needles. These two trials have true waiting list controls while the trial by Foster et al [9], which had the lowest effect size estimate and the lowest number of needles, provided a Our results are in concordance with a pooled analysis of four German-based trials of acupuncture for chronic pain [13], all of which are included in our study. We also found that neither the duration of training or experience as an acupuncturist are associated with an effect of acupuncture. Conclusions We found little evidence of important effects on pain outcomes associated with different characteristics of acupuncture or acupuncturists, including style of acupuncture, the frequency or duration of sessions, patient-practitioner interactions or the years of experience of the acupuncturists. Increased number of needles and more sessions appear to be associated with better outcomes when comparing acupuncture to non-acupuncture controls. This suggests that the dose of acupuncture is important. Trials designed to evaluate the potentially small differences in outcome associated with different acupuncture or acupuncturist characteristics are likely to require large sample sizes. There is room for a diversity of practice in acupuncture, and no strong evidence that such diversity leads to some patients receiving sub-optimal outcomes. Strengths and Limitations And finally, we conducted a large number of tests, and our results must be considered in this context. Comparison with other studies In that previous study, the only physician characteristic that had a significant October 2013 \| Volume 8 \| Issue 10 \| e77438 PLOS ONE \| www.plosone.org 6 Characteristics of Acupuncture and Outcome influence on outcome in the German-based trials was that internists performed slightly better than an average physician and orthopedists slightly worse. As physician specialty was only recorded in these German studies, this analysis was not repeated here. duration of sessions, needle prescriptions, and training and experience of acupuncturists - are appropriate. This is because the variations in outcome associated with these factors are likely to be small. We also know that in order to design a trial with sufficient power to detect the small differences in outcome that might be associated with different acupuncture characteristics, very large sample sizes will be needed. This study would suggest that the most useful characteristics to test out would be the number of needles and number of treatment sessions. By way of an example, if we hypothesize that the difference in outcome between two individual acupuncture characteristics of treatment is associated with an effect size of 0.05, then a trial comparing these would require approximately 12,500 patients. Given the results of the primary research [1] showing small differences between real and sham acupuncture, it is not surprising that the current analysis showed little evidence of substantial differences between alternative approaches to acupuncture. Each individual component of acupuncture cannot be expected to make more than a small contribution to any overall effect. Implications for research Compared to meta-regression of trial level data, we have shown how individual patient data meta-analysis can dramatically increase the power to explore treatment effect modifiers in patients with chronic pain. We recommend that future trials should record patient level data and make these data available to other researchers. In this meta-analysis, we only included studies published prior to 2008, however the Acupuncture Trialists Collaboration is to incorporate subsequent trials in the individual patient database which will lead to an update of the results. There is no reason to believe that contemporary acupuncture trials systematically underestimate treatment effects. Though acupuncturists have long been concerned about what constitutes “correct” practice [14,15], it can be argued that the consensus methods that are often used to determine acupuncture characteristics - number of treatment sessions, Supporting Information Appendix S1. Trial and patient level information. Acknowledgements Jonas, MD, Samueli Institute, Washington; Kai Kronfeld, PhD, Interdisciplinary Centre for Clinical Trials (IZKS Mainz), University Medical Centre Mainz, Mainz, Germany; Lixing Lao, PhD, University of Maryland and Center for Integrative Medicine, College Park; (DOCX) Hugh MacPherson, PhD, Complementary Medicine Research Group, University of York, York, England; Eric Manheimer, MS, Center for Integrative Medicine, University of Maryland School of Medicine, College Park. Maryland; Hugh MacPherson, PhD, Complementary Medicine Research Group, University of York, York, England; Author Contributions George Lewith, MD, FRCP, Complementary and Integrated Medicine Research Unit, Southampton Medical School, Southampton, England; Conceived and designed the experiments: HM AM GL CW AV. Analyzed the data: AM AV. Wrote the manuscript: HM AM GL NF CW AV. Klaus Linde, MD, Institute of General Practice, Technische Klaus Linde, MD, Institute of General Practice, Technische Universität München Munich; Universität München, Munich; Acknowledgements Vickers (collaboration chair), DPhil, Memorial Sloan- Remy Coeytaux, MD, PhD, Department of Community and Family Medicine, Duke University, Durham, North Carolina; Remy Coeytaux, MD, PhD, Department of Community and Family Medicine, Duke University, Durham, North Carolina; Andrew J. Vickers (collaboration chair), DPhil, Memorial Sloan- Kettering Cancer Center; Kettering Cancer Center; Norbert Victor, PhD (deceased), Institute of Medical Biometrics and Informatics, University of Heidelberg, Heidelberg, Germany; Angel M. Cronin, MS, Dana-Farber Cancer Institute, Boston, Massachusetts; Hans-Christoph Diener, MD, PhD, Department of Neurology, University of Duisburg-Essen, Germany; Hans-Christoph Diener, MD, PhD, Department of Neurology, University of Duisburg-Essen, Germany; Peter White, PhD, School of Health Sciences, University of Southampton; Heinz G. Endres, MD, Ruhr University Bochum, Bochum, Germany; Heinz G. Endres, MD, Ruhr University Bochum, Bochum, Germany; Lyn Williamson, MD, MA (Oxon), MRCGP, FRCP, Great Western Hospital, Swindon, and Oxford University, Oxford, England; Nadine Foster, DPhil, BSc(Hons), Arthritis Research UK Primary Care Centre, Keele University, Newcastle-under-Lyme, Staffordshire, England; Stefan N. Willich, MD, MPH, MBA, Institute for Social Medicine, Epidemiology, and Health Economics, Charité University Medical Center, Berlin; Stefan N. Willich, MD, MPH, MBA, Institute for Social Medicine, Epidemiology, and Health Economics, Charité University Medical Center, Berlin; Staffordshire, England; Juan Antonio Guerra de Hoyos, MD, Andalusian Integral Plan for Pain Management, and Andalusian Health Service Project for Improving Primary Care Research, Sevilla, Spain; Claudia M. Witt, MD, MBA, University Medical Center Charité and Institute for Social Medicine, Epidemiology and Health Economics, Berlin. Michael Haake, MD, PhD, Department of Orthopedics and Traumatology, SLK Hospitals, Heilbronn, Germany; Michael Haake, MD, PhD, Department of Orthopedics and Traumatology, SLK Hospitals, Heilbronn, Germany; Data sharing policy. The Acupuncture Trialists' Collaboration obtained some data that cannot be publically deposited as this was a condition of us receiving the data from third parties. All summary data for the trial level analyses will immediately be made available to investigators on request; requests for individual patient data will be considered on a case-by-case basis depending on the trials involved for the analysis concerned. Such data are fully deidentified. Richard Hammerschlag, PhD, Oregon College of Oriental Medicine, Portland; Dominik Irnich, MD, Interdisciplinary Pain Centre, University of Munich Munich Germany; p y y Munich, Munich, Germany; Munich, Munich, Germany; Wayne B. Jonas, MD, Samueli Institute, Washington; Wayne B. Acknowledgements Eric Manheimer, MS, Center for Integrative Medicine, University of Maryland School of Medicine, College Park. Maryland; The study has been supported by the Acupuncture Trialists' Collaboration, which includes physicians, clinical trialists, biostatisticians, practicing acupuncturists and others. The collaborators within the Acupuncture Trialists’ Collaboration are: Alexandra Maschino, BS, Memorial Sloan-Kettering Cancer Center, New York, New York; Center, New York, New York; Dieter Melchart, MD, PhD, Centre for Complementary Medicine Research (Znf) Technische Universität München Munich; Dieter Melchart, MD, PhD, Centre for Complementary Medicine Research (Znf), Technische Universität München, Munich; Albrecht Molsberger, MD, PhD, German Acupuncture R h G D ld f G Albrecht Molsberger, MD, PhD, German Acupuncture Research Group, Duesseldorf, Germany; Claire Allen, BS, Cochrane Collaboration Secretariat, Oxford, England; Claire Allen, BS, Cochrane Collaboration Secretariat, Oxford, England; Karen J. Sherman, PhD, MPH, Group Health Research Institute, Seattle, Washington; Mac Beckner, MIS, Information Technology and Data Management Center, Samueli Institute, Alexandria, Virginia; Mac Beckner, MIS, Information Technology and Data Management Center, Samueli Institute, Alexandria, Virginia; Management Center, Samueli Institute, Alexandria, Virginia; Brian Berman, MD, University of Maryland School of Medicine and Center for Integrative Medicine, College Park; Maryland; Benno Brinkhaus, MD, Institute for Social Medicine, Epidemiology and Health Economics, Charité University Medical Center, Berlin, Germany; Hans Trampisch, PhD, Department of Medical Statistics and Epidemiology, Ruhr University Bochum; Brian Berman, MD, University of Maryland School of Medicine and Center for Integrative Medicine, College Park; Maryland; Brian Berman, MD, University of Maryland School of Medicine and Center for Integrative Medicine, College Park; Maryland; Brian Berman, MD, University of Maryland School of Medicine and Center for Integrative Medicine, College Park; Maryland; Benno Brinkhaus, MD, Institute for Social Medicine, Epidemiology and Health Economics, Charité University Medical Center, Berlin, Germany; Jorge Vas, MD, PhD, Pain Treatment Unit, Dos Hermanas Primary Care Health Center (Andalusia Public Health System), Dos Hermanas, Spain; Benno Brinkhaus, MD, Institute for Social Medicine, Epidemiology and Health Economics, Charité University Medical Center, Berlin, Germany; Andrew J. Vickers (collaboration chair), DPhil, Memorial Sloan- Kettering Cancer Center; Norbert Victor, PhD (deceased), Institute of Medical Biometrics and Informatics, University of Heidelberg, Heidelberg, Germany; Andrew J. Vickers (collaboration chair), DPhil, Memorial Sloan- Kettering Cancer Center; Norbert Victor, PhD (deceased), Institute of Medical Biometrics Andrew J. 3. Klaber Moffett JA, Carr J, Howarth E (2004) High fear-avoiders of physical activity benefit from an exercise program for patients with back pain. Spine. 29(11): 1167-1172. doi: 10.1097/00007632-200406010-00002. PubMed: 15167652. 4. Underwood MR, Morton V, Farrin A (2007) Do baseline characteristics predict response to treatment for low back pain? Secondary analysis of the UK BEAM dataset [ISRCTN32683578]. Rheumatology (Oxf). 46(8): 1297-1302. doi:10.1093/rheumatology/kem113. Appendix S1. Trial and patient level information. October 2013 \| Volume 8 \| Issue 10 \| e77438 PLOS ONE \| www.plosone.org 7 Characteristics of Acupuncture and Outcome 4. Underwood MR, Morton V, Farrin A (2007) Do baseline characteristics predict response to treatment for low back pain? Secondary analysis of the UK BEAM dataset [ISRCTN32683578]. Rheumatology (Oxf). 46(8): 1297-1302. doi:10.1093/rheumatology/kem113. 3. Klaber Moffett JA, Carr J, Howarth E (2004) High fear-avoiders of physical activity benefit from an exercise program for patients with back pain. Spine. 29(11): 1167-1172. doi: 10.1097/00007632-200406010-00002. PubMed: 15167652. References 3. Klaber Moffett JA, Carr J, Howarth E (2004) High fear-avoiders of physical activity benefit from an exercise program for patients with back pain. Spine. 29(11): 1167-1172. doi: 10.1097/00007632-200406010-00002. PubMed: 15167652. 1. Vickers AJ, Cronin AM, Maschino AC, Lewith G, MacPherson H et al. (2012) Acupuncture for Chronic Pain: Individual Patient Data Meta- analysis. Arch Intern Med 172(19): 1444-1453. doi:10.1001/ archinternmed.2012.3654. PubMed: 22965186. 2. Hopton AK, Curnoe S, Kanaan M, MacPherson H (2012) Acupuncture in practice: mapping the providers, the patients and the settings in a national cross-sectional survey. BMJ Open. 2: e000456. doi:10.1136/ bmjopen-2011-000456. PubMed: 22240649. 2. Hopton AK, Curnoe S, Kanaan M, MacPherson H (2012) Acupuncture in practice: mapping the providers, the patients and the settings in a national cross-sectional survey. BMJ Open. 2: e000456. doi:10.1136/ bmjopen-2011-000456. PubMed: 22240649. PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77438 8 Characteristics of Acupuncture and Outcome 5. Brookes ST, Whitely E, Egger M, Smith GD, Mulheran PA, Peters TJ (2004) Subgroup analyses in randomized trials: risks of subgroup- specific analyses; power and sample size for the interaction test. J Clin Epidemiol57(3): 229-236. doi:10.1016/j.jclinepi.2003.08.009. PubMed: 15066682. 13. Witt CM, Lüdtke R, Wegscheider K, Willich SN (2010) Physician characteristics and variation in treatment outcomes: are better qualified and experienced physicians more successful in treating patients with chronic pain with acupuncture? J Pain. 11(5): 431-435. doi:10.1016/ j.jpain.2009.08.010. PubMed: 20439056. j jp 14. Birch S (2007) Reflections on the German Acupuncture Studies. Journal of Chinese Medicine.83: 12-17. 6. Stewart LA, Tierney JF (2002) To IPD or not to IPD? Advantages and disadvantages of systematic reviews using individual patient data. Eval Health Prof 25(1): 76-97. doi:10.1177/0163278702025001006. PubMed: 11868447. 15. Hanfileti L (2013) Medical acupuncture and traditional Chinese medicine: What licensed acupuncturists need to know about the training and qualifications of physicians providing medical acupuncture. Retrieved onpublished at whilst December year 1111 from http:// www.insights-for-acupuncturists.com/medical-acupuncture.html. Accessed 16th September 2013. 7. Brinkhaus B, Witt CM, Jena S, Linde K, Streng A et al. (2006) Acupuncture in patients with chronic low back pain: a randomized controlled trial. Arch Intern Med 166: 450-457. doi:10.1001/archinte. 166.4.450. PubMed: 16505266. 8. Witt C, Brinkhaus B, Jena S, Linde K, Streng A et al. (2005) Acupuncture in patients with osteoarthritis of the knee: a randomised trial. Lancet 366: 136-143. doi:10.1016/S0140-6736(05)66871-7. PubMed: 16005336. 16. Characteristics of Acupuncture and Outcome References Wampold BE, Brown GS (2005) Estimating variability in outcomes attributable to therapists: a naturalistic study of outcomes in managed care. J Consult Clin Psychol 73(5): 914-923. doi:10.1037/0022-006X. 73.5.914. PubMed: 16287391. 9. Foster NE, Thomas E, Barlas P, Hill JC, Young J et al. (2007) Acupuncture as an adjunct to exercise based physiotherapy for osteoarthritis of the knee: randomised controlled trial. BMJ. 335(7617): 436. doi:10.1136/bmj.39280.509803.BE. PubMed: 17699546. 17. Lewis M, Morley S, van der Windt DA, Hay E, Jellema P et al. (2010) Measuring practitioner/therapist effects in randomised trials of low back pain and neck pain interventions in primary care settings. Eur J Pain. 14(10): 1033-1039. doi:10.1016/j.ejpain.2010.04.002. PubMed: 20444631. 436. doi:10.1136/bmj.39280.509803.BE. PubMed: 17699546 10. National Institute for Health and Clinical Excellence (2009) Low back pain: Early management of persistent non-specific low back pain National Institute for Health and Clinical Excellence. Available: http:// www.nice.org.uk/cg88. Accessed 16th September 2013. 18. Price S, Mercer SW, MacPherson H (2006) Practitioner empathy, patient enablement and health outcomes: a prospective study of acupuncture patients. Patient Educ Couns 63(1-2): 239-245. doi: 10.1016/j.pec.2005.11.006. PubMed: 16455221. g g 11. Hughes JG, Goldbart J, Fairhurst E, Knowles K (2007) Exploring acupuncturists' perceptions of treating patients with rheumatoid arthritis. Complement Ther Med. 15(2): 101-108. doi:10.1016/j.ctim. 2006.09.008. PubMed: 17544860. j p 19. Scheid V (2012) Defining best practice or cultivating the best practitioner. In: V ScheidH MacPherson. Integrating East Asian Medicine into Contemporary Healthcare.Edinburgh: Churchill Livingstone. pp. 13-38. 12. Paterson C, Britten N (2004) Acupuncture as a complex intervention: a holistic model. J Altern Complement Med 10(5): 791-801. doi: 10.1089/1075553042476669. PubMed: 15650468. PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77438 9
https://openalex.org/W2921439506	https://www.nature.com/articles/s41598-019-40900-3.pdf	English	null	Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations	Scientific reports	2,019	cc-by	15,973	Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations Received: 5 October 2017 Accepted: 21 January 2019 Published: xx xx xxxx Yann Becker1, Renée-Claude Loignon2, Anne-Sophie Julien3, Geneviève Marcoux1, Isabelle Allaeys1, Tania Lévesque1, Emmanuelle Rollet-Labelle1, Hadrien Benk-Fortin1, Nathalie Cloutier1, Imène Melki1, Lihi Eder4, Éric Wagner5, Martin Pelletier1,6, Hassan El Hajj7, Marie-Ève Tremblay 7, Clémence Belleannée8, Marie-Josée Hébert9, Mélanie Dieudé9, Joyce Rauch10, Paul R. Fortin2,6 & Eric Boilard1,6 Mitochondria are organelles that govern energy supply and control cell death. Mitochondria also express bacterial features, such as the presence of inner membrane cardiolipin and a circular genome rich in hypomethylated CpG motifs. While mitochondrial extrusion by damaged organs or activated cells is thought to trigger innate immunity, it is unclear whether extracellular mitochondria also stimulate an adaptive immune response. We describe the development of novel assays to detect autoantibodies specific to two distinct components of the mitochondrion: the mitochondrial outer membrane and mitochondrial DNA. Antibodies to these two mitochondrial constituents were increased in both human and murine systemic lupus erythematosus (SLE), compared to controls, and were present at higher levels than in patients with antiphospholipid syndrome or primary biliary cirrhosis. In both bi- and multi-variate regression models, antibodies to mitochondrial DNA, but not whole mitochondria, were associated with increased anti-dsDNA antibodies and lupus nephritis. This study describes new and optimized methods for the assessment of anti-mitochondrial antibodies, and demonstrates their presence in both human and murine SLE. These findings suggest that different mitochondrial components are immunogenic in SLE, and support the concept that extracellular mitochondria may provide an important source of circulating autoantigens in SLE. The roles of mitochondria in bioenergetics and the control of cell proliferation or death are well-documented1,2. Furthermore, mitochondria share several similarities with bacteria3,4. Like bacteria, mitochondria are formed of an outer and an inner membrane (inner contains cardiolipin)4,5, express formylated peptides6,7, and contain a circular genome with hypomethylated DNA CpG motifs, referred to as mitochondrial DNA (mtDNA)8,9. g y y Various cellular lineages are capable of extruding their mitochondria. Activated mast cells10, T-cells11, eosin- ophils12, hepatocytes13, neutrophils14–16 and platelets17,18, in addition to damaged organs or tissues7,13,19,20, release 1Centre de Recherche du CHU de Québec – Université Laval, Département de microbiologie et immunologie, Faculté de Médecine de l′Université Laval, Québec, Qc, Canada. 2Division de Rhumatologie, Département de Médecine, CHU de Québec – Université Laval, Québec, Qc, Canada. 3Département de mathématiques et statistique, Université Laval, Québec, Qc, Canada. 4Women’s College Hospital and University of Toronto, Toronto, ON, Canada. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 5 October 2017 Accepted: 21 January 2019 Published: xx xx xxxx Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations aELISA performed on sub-mitochondrial particles (sonicated “crude” mitochondria). bELISA performed on purified antigen. cThe exact antigen recognized by M7 antibodies is yet to fully characterize. APS, anti-phospholipid syndrome; CFT, complement fixation test; ELISA, enzyme-linked immunosorbent assay; IIF, indirect immunofluorescence on rodent and/or human tissues; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; PBC, primary biliary cirrhosis; PLE, pseudolupus erythematosus; SLE, systemic lupus erythematosus; SS: Sjögren syndrome. extracellular mitochondria. Mitochondria and their components (e.g. N-formylated peptides and mtDNA) are recognized as damage-associated molecular patterns (DAMPs), which activate the innate immune system and elicit an inflammatory response6,21–23. Moreover, ATP and reactive oxygen species (ROS), produced by mito- chondria are triggers of the nuclear oligomerization domain (NOD)-like receptors and contribute to inflammas- ome activation21,22,24. Extracellular mitochondria have been described in various clinical conditions, including trauma25,26, burn injury27, cancer28, rheumatoid arthritis17,29, systemic lupus erythematosus (SLE)15,16 and trans- fusion adverse reactions17,18,30,31. Their pro-inflammatory potential is generally thought to occur through activa- tion of Toll-like receptors (TLR), formyl peptide receptors, and cytosolic pathogen recognition receptors, all key components of the innate immune system6,21–24.hh p y The adaptive immune system can also recognize mitochondria. This concept is important, as the immune response initiated by mitochondrial DAMPs may be different if the adaptive immune system is also impli- cated32,33. Different sets of anti-mitochondrial antibodies (AMA), namely AMA-M1 to -M9, have been character- ized34 (recapitulated in Table 1). The AMA-M2, -M4, -M8, and -M9 classes are well-described in primary biliary cirrhosis (PBC)35–37, an autoimmune disease characterized by a progressive destruction of the bile ducts due to the infiltration of autoreactive T-cells38. These antibodies target distinct mitochondrial proteins notably implicated in oxidative phosphorylation, and their differential induction depends on disease severity or stage. Conversely, AMA-M6 autoantibodies have been described in iatrogenic hepatitis induced by iproniazid39, while the AMA-M7 class of antibodies targets mitochondrial epitopes, identified as sarcosine dehydrogenase and enzymes associated with flavin adenine dinucleotide, in patients with cardiomyopathy and myocarditis40. l p y p y y SLE is an autoimmune disease characterized by the presence of circulating antigen-autoantibody immune complexes and inflammation in multiple organs and tissues. In SLE, neutrophils were shown to release mtDNA through the generation of ROS, a process leading to activation of the DNA sensor stimulator of interferon genes (STING) and type-I IFN production15,16. Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations 5Immunology and Histocompatibility Laboratory, Department of Medical Biology CHU de Québec - Université Laval; Department of Microbiology, Infectious Diseases and Immunology, Université Laval, Québec, Qc, Canada. 6Axe maladies infectieuses et inflammatoires, Centre de recherche du CHU de Québec – Université Laval, Québec, Qc, Canada. 7Axe Neurosciences, Centre de Recherche du CHU de Québec – Université Laval, Québec, Qc, Canada. 8Axe Reproduction, Santé de la mère et de l’enfant, Centre de recherche du CHU de Québec – Université Laval, Québec, Qc, Canada. 9Centre de recherche du CHU de Montréal, Montréal Québec, Québec, Qc, Canada. 10Division of Rheumatology, Department of Medicine, Research Institute of the McGill University Health Centre, Montreal, Qc, H4A 3J1, Canada. Correspondence and requests for materials should be addressed to P.R.F. (email: paul.fortin@crchudequebec.ulaval.ca) or E.B. (email: eric.boilard@crchudequebec.ulaval.ca) 1 Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 www.nature.com/scientificreports/ Type of anti- mitochondrial antibody: Molecular target(s): Localization: Associated disease(s): Method(s) of detection: References: Anti-M1 Cardiolipin IMM APS, SLE, secondary syphilis IIF, ELISAa, CFT 87 Anti-M2 2-oxoacid dehydrogenase complex IMM PBC IIF, ELISAa, CFT 58 Anti-M3 Unknown OMM Venocuran-induced PLE IIF, CFT 55 Anti-M4 Sulfite oxidase OMM PBC ELISAb, CFT 88 Anti-M5 Unknown OMM, IMM APS, SLE, SS, haemolytic anemia IIF, CFT 56 Anti-M6 Monoamine oxydase B OMM Iproniazid-induced hepatitis IIF, ELISAb, CFT 39,89 Anti-M7 Sarcosine dehydrogenase IMM Cardiomyopathies ELISAa–c 90 Anti-M8 Unknown OMM PBC CFT 89 Anti-M9 Glycogen phosphorylase OMM PBC ELISA 91,92 Table 1. Various types of anti-mitochondrial antibodies implicated in human diseases. aELISA performed on sub-mitochondrial particles (sonicated “crude” mitochondria). bELISA performed on purified antigen. cThe exact antigen recognized by M7 antibodies is yet to fully characterize. APS, anti-phospholipid syndrome; CFT, complement fixation test; ELISA, enzyme-linked immunosorbent assay; IIF, indirect immunofluorescence on rodent and/or human tissues; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; PBC, primary biliary cirrhosis; PLE, pseudolupus erythematosus; SLE, systemic lupus erythematosus; SS: Sjögren syndrome. Table 1. Various types of anti-mitochondrial antibodies implicated in human diseases. aELISA performed on sub-mitochondrial particles (sonicated “crude” mitochondria). bELISA performed on purified antigen. cThe exact antigen recognized by M7 antibodies is yet to fully characterize. APS, anti-phospholipid syndrome; CFT, complement fixation test; ELISA, enzyme-linked immunosorbent assay; IIF, indirect immunofluorescence on rodent and/or human tissues; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; PBC, primary biliary cirrhosis; PLE, pseudolupus erythematosus; SLE, systemic lupus erythematosus; SS: Sjögren syndrome. Table 1. Various types of anti-mitochondrial antibodies implicated in human diseases. Results Diff Different methodologies exist for the isolation of mitochondria, and several improvements have been introduced in recent years to enhance their purity and quality. We used a combination of previously published protocols to obtain highly purified mitochondria61,62. Mitochondria were isolated from mouse liver or a human hepatocyte cell line (Hep-G2) using a combination of previously published protocols61,62 (Fig. 1a). The Percoll gradient included in the purification protocol eliminated contaminants from the endoplasmic reticulum and the proteasome (Supplementary Fig. 1a)62. The purity of the mitochondrial preparations was high, based on the extremely low content of cytosolic and nuclear proteins, and the enrichment of three mitochondrial proteins [voltage-dependent anion channel (VDAC), cytochrome C, and translocase of the outer membrane 22 (TOMM22)] (Fig. 1b). The iso- lated mitochondria maintained their cytochrome C (Fig. 1b) and respiratory functions, suggesting that integrity was conserved during the isolation process (Fig. 1c). Mitochondria were homogeneous in size, with a main peak detected at 820 nm by dynamic light scatter- ing (Fig. 1d, left panel), and retained their canonical morphology when observed in electron microscopy (910 ± 210 nm) (Fig. 1d, right panel). High sensitivity flow cytometry, which permits the identification of submi- cron particles, was used as a quantitative approach. We estimated that 6.6 ± 1.9 × 106 mitochondria (Mitotracker+ TOMM22+) (or 4.33 ± 1.17 µg mitochondrial proteins) could be isolated per mg of mouse liver (n = 6), which was sufficient to prepare approximately 400 wells in half-area 96-well microplates using one mouse liver (Fig. 1e). The yield obtained with Hep-G2 cells was lower, as 3.5 ± 0.18 µg of mitochondrial protein were isolated per 106 Hep-G2 cells (7.0 × ± 2.1 × 108 Hep-G2 cells were harvested for each 175 cm2 flask at confluence), which can be used to prepare approximately two half-area 96-well microplates.hi p p pp y p The quantity of purified mitochondrial protein (12.5 µg mitochondrial protein/well) required for coating half-area 96-well microplates ELISA plates was optimized using increasing concentrations of mitochondria (Supplementary Fig. 1b). Wells were saturated with phosphate buffered saline (PBS) containing fetal calf serum (FCS) and gelatin, which proved optimal for blocking nonspecific binding of antibodies in comparison to non-fat dry milk or bovine serum albumin (data not shown). We used an inducible murine model of SLE to determine whether AwMA could be detected in serum63. www.nature.com/scientificreports/ www.nature.com/scientificreports/ AMA-M3 were described in patients using a drug called venocuran who developed a drug-induced syndrome (“pseudolupus”) with clinical manifestations of arthralgia, fever, serositis, and lymphopenia. The immunolog- ical profile of pseudolupus is characterized by the absence of antinuclear antibody (ANA), but the presence of AMA-M355. The antigenic target of AMA-M3 is different than that of the other AMA classes described in PBC55. It is resistant to trypsin and it is extracted by solvent, pointing to its lipid nature. AMA-M3 are no longer encoun- tered clinically since the withdrawal of the drug venocuran. y g AMA-M5 comprise another class of anti-mitochondrial antibodies identified in patients with SLE, as well in APS, Sjögren syndrome, recurrent fetal loss, and hemolytic anemia56. The precise antigenic target(s) of AMA-M5 is undefined, but lack of competition by cardiolipin-containing liposomes suggests that it is distinct from the target of AMA-M157. Indirect immunofluorescence on human or rodent tissues is used to identify anti-M5 anti- bodies58. While immunofluorescence and complement fixation test revealed that only 2% of SLE patients were positive for AMA-M5, approximately 40% were positive by enzyme-linked immunosorbent assay (ELISA)59. However, in the latter approach, mitochondria were only partially purified and were sonicated, thus revealing antigenic epitopes that might not be recognized in tissues60. g p p g g Emerging evidence supports the liberation of mitochondria by activated cells and their potential contribution to inflammation in SLE15,16. Identification of mitochondrial antigens recognized by autoantibodies in SLE may provide information on the roles of extracellular mitochondria in autoimmunity and systemic inflammation. Herein, we developed new methods to detect the presence of two distinct types of circulating AMA: anti-whole mitochondria antibodies (AwMA) and AmtDNA. We then determined their usefulness in a murine model of SLE, as well as in a cohort of SLE patients. We evaluated these AMA in parallel with antibodies to the mito- chondrial chaperonin HSP60 (a known mitochondrial target in SLE), and determined the associations of these autoantibodies with disease characteristics. To our knowledge, this is the first study that examines the association of these AMA with disease manifestations in SLE. Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations Studies showed that anti-mtDNA antibodies (AmtDNA) are induced in a subset of SLE patients, suggesting that extruded mtDNA could be a relevant source of antigen for the anti-double stranded DNA (anti-dsDNA) antibodies that prevail in SLE16,41,42. Clinically, anti-DNA antibodies are screened initially by indirect immunofluorescence using human epithelial type 2 cells (HEp-2) and enzyme immunoassay using double-stranded DNA43. Presence of anti-DNA antibodies can also be assessed by indirect immunofluorescence using Crithidia luciliae, an hemoflagellate parasite of blow-flies that possesses a uniquely large mitochondrion that contains a high concentration of DNA, the kinetoplast44–46. Another method commonly used is Farr radioimmunoassay, which involves the precipitation of antibody-bound radiolabeled DNA and its detection with a scintillation counter47.h Antibodies targeting mitochondrial components other than mtDNA are also found in SLE. They include anti-cardiolipin [AMA-M1, also known as anti-cardiolipin antibodies (ACA)], anti-60kDa heat shock protein (anti-HSP60), as well as AMA-M3 and AMA-M5 antibodies. AMA-M1 antibodies recognize cardiolipin and were originally identified in syphilis-infected patients. Anti-cardiolipin antibodies are also found in patients with SLE and antiphospholipid syndrome (APS), resulting in false positive results in earlier syphilis detection assays34,48. Interestingly, cardiolipin is a phospholipid that is found uniquely in the inner mitochondrial membrane in eukaryotic cells. However, damaged mitochondria may externalize their cardiolipin49, that may become accessi- ble and induce the development of antibodies to cardiolipin. Clinically, the presence of ACA is associated with a greater risk of thrombotic events and thrombocytopenia50,51. Another mitochondrial antigen, HSP60, is a mitochondrial chaperonin implicated in mitochondrial pro- tein import52. Patients with SLE have antibodies against HSP6053, and their presence (when concomitant with anti-phospholid antibodies) is associated with vascular events54. Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 2 Results Diff (b) Cytoplasmic (C), nuclear (N) and mitochondrial (M) markers were assessed by western blotting in murine (left) and human (right) mitochondrial preparations (25 µg protein per lane). Results are representative of three distinct preparations. Blots separated by dashed lines are non- contiguous but from same membrane. Blots separated by full lines were performed on distinct membranes; (c) Functionality of murine mitochondria was determined by measurement of the oxygen consumption rate (OCR) of 10 µg mitochondria treated successively with 2 µM rotenone, 10 mM succinate, 40 µM antimycin A and 100 µM N,N,N’,N’-Tetramethyl-p-phenylenediamine (TMPD) along with 10 mM ascorbate (Asc). (d) The hydrodynamic size of the murine mitochondria was determined using zetasizer nano ZS (n = 3, left panel) and their morphology was visualized by electron microscopy (right panel). Inner membrane (black arrowhead), outer membrane (white arrowhead), cristae (black arrow), mitochondrial matrix (white arrow) are presented (scale bar account for 500 nm); (e) Size representation of purified murine mitochondria using high sensitivity- fl t t D bl iti it h d i (Mit t k + TOMM22+) d f tifi ti Figure 1. Assessment of the mitochondrial preparations. (a) Mitochondria were isolated from either mouse liver or human Hep-G2 cell line by differential centrifugations and further purified by ultracentrifugation against Percoll gradient. Alkaline lysis was performed to retrieve mtDNA. Pure mitochondria or mtDNA were used as coating antigens in direct ELISAs. (b) Cytoplasmic (C), nuclear (N) and mitochondrial (M) markers were assessed by western blotting in murine (left) and human (right) mitochondrial preparations (25 µg protein per lane). Results are representative of three distinct preparations. Blots separated by dashed lines are non- contiguous but from same membrane. Blots separated by full lines were performed on distinct membranes; (c) Functionality of murine mitochondria was determined by measurement of the oxygen consumption rate (OCR) of 10 µg mitochondria treated successively with 2 µM rotenone, 10 mM succinate, 40 µM antimycin A and 100 µM N,N,N’,N’-Tetramethyl-p-phenylenediamine (TMPD) along with 10 mM ascorbate (Asc). (d) The hydrodynamic size of the murine mitochondria was determined using zetasizer nano ZS (n = 3, left panel) and their morphology was visualized by electron microscopy (right panel). Inner membrane (black arrowhead), outer membrane (white arrowhead), cristae (black arrow), mitochondrial matrix (white arrow) are presented (scale bar account for 500 nm); (e) Size representation of purified murine mitochondria using high sensitivity- flow cytometry. Results Diff This model is known to produce autoantibodies to nuclear and cellular antigens63, but the presence of AwMA has never been explored. We found high serum levels of AwMA in this induced murine lupus model, compared to healthy control mice (Fig. 2a).l Reactive oxygen species are generated under inflammatory conditions, and were reported during the release of mitochondria15,16. Thus, we assessed whether oxidation of mitochondria could impact mitochondrial recognition by AwMA. Isolated mitochondria were treated with increasing concentrations of the oxidant tert-butyl hydrop- eroxide (TBHP), and the oxidized protein and lipid contents were confirmed using commercial assays (Fig. 2b,c and Supplementary Fig. 2). We found that oxidation had no or very little impact on recognition of mitochondria by SLE antibodies (Fig. 2d) (Fold increase: 1.2 ± 0.2). The data suggest that mitochondria are immunogenic in SLE regardless of the oxidation status of their antigens. g g We next used our quantitative AwMA ELISA to screen human sera. We included 175 SLE patients and 43 healthy controls (76% female, mean age 42 ± 12) (Table 2). We also evaluated sera from APS patients (n = 12), given the high levels of anti-cardiolipin antibodies (AMA-M1) in APS, as well as sera from PBC patients (n = 12) confirmed positive for AMA by indirect immunofluorescence on mouse stomach/kidney slides (MSK). il Given the higher yield and purity of the mitochondria isolated from mouse liver, and the fact that mice are readily accessible in most research laboratories, we used intact murine mitochondria as coating antigens in our assay for the detection of autoantibodies targeting the outer mitochondrial membrane in humans. We found that AwMA were present in all healthy controls, but at much lower levels than those encountered in a large proportion of the SLE patients. SLE patients were more frequently positive and at higher levels for AwMA than healthy controls. APS and PBC patients also presented a significant increase in AwMA compared to healthy Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Assessment of the mitochondrial preparations. (a) Mitochondria were isolated from either mouse liver or human Hep-G2 cell line by differential centrifugations and further purified by ultracentrifugation against Percoll gradient. Alkaline lysis was performed to retrieve mtDNA. Pure mitochondria or mtDNA were used as coating antigens in direct ELISAs. Results Diff sonicated mitochondria) were used as coating antigens, suggesting that certain antigens relevant to PBC may be exposed in these conditions (Supplementary Fig. 3). Human and murine mitochondria were also compared in our assay using sera from a subset of SLE patients. Both sources of mitochondria (human Hep-G2 cells and mouse liver) were similarly recognized by human AwMA (Fig. 3b), suggesting that antigenic epitopes may be conserved across these species. Hence, mitochondria from murine, bovine and porcine tissues are routinely used to assess AwMA in humans, suggesting that interspecies differences play a negligible role, if any, in mitochondrial antigenicity. g y Membrane-bound vesicles in the extracellular milieu, known as extracellular vesicles or microparticles, are proposed contributors to the antigenic load in SLE64–66. To determine whether AwMA could also recog- nize microparticles derived from membranes from cells or particles other than mitochondria, we utilized red blood cell microparticles (RBCMP) as blood-borne microparticles devoid of mitochondria as a competitor in our AwMA-ELISA, and compared it to extracellular mitochondria in solution. Whereas increased concentrations of competing mitochondria decreased AwMA binding by up to 49.84 ± 15.01%, RBCMP showed no inhibition of binding of the SLE antibodies in our assay (Fig. 3c). While these results suggest that the antibodies detected in the AwMA-ELISA might have a preferred substrate originating in mitochondrial membrane, we cannot exclude the possibility that other membrane bound microparticles or even cells may also be recognized by AwMA, given the probable occurrence of numerous protein and non-protein antigens in mitochondria. To determine whether mtDNA also represents an antigenic target of SLE autoantibodies, we isolated mtDNA from crude mitochondria preparations, using standard DNA extraction by alkaline lysis. Digestion of the mtDNA by Hae II, a restriction enzyme with a single restriction site on the murine mitochondrial genome (Supplementary Fig. 4a) yielded a single fragment of 16,569 base pairs, indicating the isolation of mtDNA with its circular confor- mation. As expected, digestion by Pst I generated two fragments of 12,751 and 3818 base pairs, further confirming the expected size of the isolated mtDNA. Moreover, we confirmed enrichment of mtDNA relative to genomic DNA (Supplementary Fig. 4b). Up to 1.55 ± 0.35 µg mtDNA was obtained for each mg of mitochondrial protein used. Results Diff (Control: N = 8, SLE: N = 12, Student’s t-test); (b) Lipid peroxidation following in-vitro oxidation of the mitochondria by 500 µM tert-buthyl hydroperoxide (TBHP) was quantified by thiobarbituric reactive substances (TBARS) assay (N = 3, Wilcoxon test); (c) Protein oxidation was determined by carbonyl assay (n = 6, Wilcoxon test); (d) The effect of oxidation of mitochondrial epitopes on their recognition by serum AwMA (1:20) was assessed by direct ELISA, using either native (grey symbols) or oxidized mitochondria (black symbols) as coating antigens (N = 13, two-way ANOVA with multiple comparisons; Sidak’s correction). All experiment presented in the figure were performed using mouse mitochondria. Data are mean ± SD. p < 0.05. p < 0.01. p < 0.001. *p < 0.0001. donors but signals detected for these patients were lower than those measured in SLE patients (Fig. 3a). As the ELISA is performed on intact mitochondria, these results suggest that AwMA are induced in SLE, and recog- nize autoantigens on the outer mitochondrial membrane that are distinct from the epitopes in APS (cardiolipin) and PBC (pyruvate dehydrogenase complex E2-component, PDC-E2), both located in the mitochondrial inner membrane. Consistent with this, we identified PBC patients positive for AMA when submitochondrial particles (i.e. sonicated mitochondria) were used as coating antigens, suggesting that certain antigens relevant to PBC may be exposed in these conditions (Supplementary Fig. 3). Human and murine mitochondria were also compared in our assay using sera from a subset of SLE patients. Both sources of mitochondria (human Hep-G2 cells and mouse liver) were similarly recognized by human AwMA (Fig. 3b), suggesting that antigenic epitopes may be conserved across these species. Hence, mitochondria from murine, bovine and porcine tissues are routinely used to assess AwMA in humans, suggesting that interspecies differences play a negligible role, if any, in mitochondrial antigenicity. donors but signals detected for these patients were lower than those measured in SLE patients (Fig. 3a). As the ELISA is performed on intact mitochondria, these results suggest that AwMA are induced in SLE, and recog- nize autoantigens on the outer mitochondrial membrane that are distinct from the epitopes in APS (cardiolipin) and PBC (pyruvate dehydrogenase complex E2-component, PDC-E2), both located in the mitochondrial inner membrane. Consistent with this, we identified PBC patients positive for AMA when submitochondrial particles (i.e. Results Diff Double-positive mitochondria (Mitotracker+ TOMM22+) were used for quantification. Silica beads were used to determine 100–1000 nm size scale. Data are mean ± SD. Anti-A: antimycin A; CytC: cytochtome C; FSC: forward scatter; Mito: mitochondria PCNA: proliferating cell nuclear antigen; SSC: side scatter; Total: total starting material; TOMM22: translocase of the outer mitochondrial membrane; VDAC: voltage-dependent anion channel. Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 www.nature.com/scientificreports/ Figure 2. Antibodies targeting mitochondrial antigens are produced in a murine model of SLE. (a) Elevated levels of anti-whole mitochondria antibodies (AwMA) were detected by direct ELISA in sera (1:150) from an inducible murine model of systemic lupus erythematosus (SLE) compared to control mice. An isotype-matched monoclonal mouse anti-translocase of the outer mitochondrial membrane 22 (TOMM22) antibody (clone IC9- 2. 4 µg/mL) was included as a positive assay control (dotted line). (Control: N = 8, SLE: N = 12, Student’s t-test); (b) Lipid peroxidation following in-vitro oxidation of the mitochondria by 500 µM tert-buthyl hydroperoxide (TBHP) was quantified by thiobarbituric reactive substances (TBARS) assay (N = 3, Wilcoxon test); (c) Protein oxidation was determined by carbonyl assay (n = 6, Wilcoxon test); (d) The effect of oxidation of mitochondrial epitopes on their recognition by serum AwMA (1:20) was assessed by direct ELISA, using either native (grey symbols) or oxidized mitochondria (black symbols) as coating antigens (N = 13, two-way ANOVA with multiple comparisons; Sidak’s correction). All experiment presented in the figure were performed using mouse mitochondria. Data are mean ± SD. p < 0.05. p < 0.01. p < 0.001. **p < 0.0001. Figure 2. Antibodies targeting mitochondrial antigens are produced in a murine model of SLE. (a) Elevated l l f h l h d b d d d b d f Figure 2. Antibodies targeting mitochondrial antigens are produced in a murine model of SLE. (a) Elevated levels of anti-whole mitochondria antibodies (AwMA) were detected by direct ELISA in sera (1:150) from an inducible murine model of systemic lupus erythematosus (SLE) compared to control mice. An isotype-matched monoclonal mouse anti-translocase of the outer mitochondrial membrane 22 (TOMM22) antibody (clone IC9- 2. 4 µg/mL) was included as a positive assay control (dotted line). www.nature.com/scientificreports/ Characteristics SLE patients Age Range, years 20–78 Mean ± S.D, years 47 ± 15 Disease duration Range, years 0–57 Mean ± S.D, years 18 ± 12 Gender, female, n (%) 175 (100) Thrombotic events, n (%) 35 (20) SLEDAI-2K ≥ 4, n (%) 57 (33) SDI ≥ 1, n (%) 124 (71) Increased anti-dsDNA, n (%) 59 (34) Lupus nephritis, n (%) (n = 172) 67 (39) Currently Prescribed Medication, n (%) Anticoagulation or anti-platelet (n = 174) 40 (23) Antimalarial 127 (73) Prednisone 81 (46) Lipid lowering 26 (15) Diabetes medication 6 (3) Malar rash 127 (72.6) Discoid rash 24 (13.7) Photosensitivity 113 (64.6) Oral ulcers 108 (61.7) Arthritis (≥2 peripheral joints) 151 (86.3) Serositis 67 (38.3) Neurologic disorder (seizure or psychosis) 24 (12.0) Renal disorderA 100 (57.1) eGFR (n = 160) Range, mL/min/1.73 m2 17–121 Mean ± S.D, mL/min/1.73 m2 84.38 ± 24.70 <60 mL/min/1.73 m2, n (%) 26 (16.3) Hematologic disorderB 155 (88.6) Immunologic disorderC 159 (90.9) Anti-nuclear antibodies (ANA) 170 (97.1) American College of Rheumatology criteria (ACR) score Range 03-Nov Mean ± S.D 6.83 ± 1.62 Table 2. Demographics and clinical characteristics (ACR criteria) for SLE patients included in the study (n = 175). A>0.5 g per day of protein in urine or cellular cast or end-stage renal disease. BHemolytic anemia (low red blood cell count) or leukopenia (White blood cells < 4000/µl), lymphopenia (<100 000/µl) in the absence of offending drug. CPositive anti-Smith, anti-dsDNA, antiphospholipid antibody and/or false positive serological test for syphilis. eGFR: estimated glomerular filtration test. Table 2. Demographics and clinical characteristics (ACR criteria) for SLE patients included in the study (n = 175). A>0.5 g per day of protein in urine or cellular cast or end-stage renal disease. BHemolytic anemia (low red blood cell count) or leukopenia (White blood cells < 4000/µl), lymphopenia (<100 000/µl) in the absence of offending drug. CPositive anti-Smith, anti-dsDNA, antiphospholipid antibody and/or false positive serological test for syphilis. eGFR: estimated glomerular filtration test. Little is known about the association of AwMA and AmtDNA with the clinical characteristics of SLE. We assessed whether AwMA and AmtDNA were associated with disease manifestations in 175 SLE patients for whom detailed clinical information was available. AwMA levels correlated with AmtDNA levels in SLE patients (rs = 0.23, p = 0.003), but not in healthy controls (rs = 0.15, p = 0.33). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Characteristics SLE patients Age Range, years 20–78 Mean ± S.D, years 47 ± 15 Disease duration Range, years 0–57 Mean ± S.D, years 18 ± 12 Gender, female, n (%) 175 (100) Thrombotic events, n (%) 35 (20) SLEDAI-2K ≥ 4, n (%) 57 (33) SDI ≥ 1, n (%) 124 (71) Increased anti-dsDNA, n (%) 59 (34) Lupus nephritis, n (%) (n = 172) 67 (39) Currently Prescribed Medication, n (%) Anticoagulation or anti-platelet (n = 174) 40 (23) Antimalarial 127 (73) Prednisone 81 (46) Lipid lowering 26 (15) Diabetes medication 6 (3) Malar rash 127 (72.6) Discoid rash 24 (13.7) Photosensitivity 113 (64.6) Oral ulcers 108 (61.7) Arthritis (≥2 peripheral joints) 151 (86.3) Serositis 67 (38.3) Neurologic disorder (seizure or psychosis) 24 (12.0) Renal disorderA 100 (57.1) eGFR (n = 160) Range, mL/min/1.73 m2 17–121 Mean ± S.D, mL/min/1.73 m2 84.38 ± 24.70 <60 mL/min/1.73 m2, n (%) 26 (16.3) Hematologic disorderB 155 (88.6) Immunologic disorderC 159 (90.9) Anti-nuclear antibodies (ANA) 170 (97.1) American College of Rheumatology criteria (ACR) score Range 03-Nov Mean ± S.D 6.83 ± 1.62 Table 2. Demographics and clinical characteristics (ACR criteria) for SLE patients included in the study (n = 175). A>0.5 g per day of protein in urine or cellular cast or end-stage renal disease. BHemolytic anemia (low red blood cell count) or leukopenia (White blood cells < 4000/µl), lymphopenia (<100 000/µl) in the absence of offending drug. CPositive anti-Smith, anti-dsDNA, antiphospholipid antibody and/or false positive serological test for syphilis. eGFR: estimated glomerular filtration test. Results Diff Plate adhesion of different concentrations of mtDNA was enhanced by using plates pre-treated with pro- tamine sulfate, and binding specificity was increased by blocking the plates with a PBS solution containing FCS and gelatin (Supplementary Fig. 4c). Of interest, sera from mice with induced SLE were positive for AmtDNA, compared to control mice (Fig. 4a). Moreover, AmtDNA was significantly increased in SLE patients, but not in patients with APS or PBC, relative to healthy controls (Fig. 4b). Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 5 www.nature.com/scientificreports/ In contrast, AwMA did not correlate with antibodies against other mitochondrial antigens (i.e. HSP60 and cardiolipin) and was not found to be associated with clinical outcomes (Tables 3 and 4). Interestingly, AmtDNA was associated with both increased anti-dsDNA antibodies (p = 0.02) and with a history of lupus nephritis (p = 0.007), but not with any of the other clinical out- comes (Table 4). When the duration of the disease, the age of the patients, their BMI, the use of prednisone and/ or antimalarial drugs, and circulating cholesterol LDL were taken into account, the associations of AmtDNA with increased anti-dsDNA antibodies and lupus nephritis remained significant in a multivariate logistic regression (p = 0.01 for both), indicating an association between AmtDNA, and these two clinical parameters. However, AmtDNA did not correlate with anti-dsDNA as measured by Farr assay in the cohort, suggesting that the results measured by our AmtDNA-ELISA and those obtained by Farr assay may not be redundant. Cut-off values were identified for AwMA and AmtDNA (Figs 3, 4 and Table 5). Our two ELISAs displayed high specificities (AwMA: 0.88; AmtDNA: 0.74) and allowed us to efficiently discriminate SLE patients from healthy donors (p < 0.001 for both AmtDNA and AwMA). Using these values, we determined that 110 patients were positive for AmtDNA (62.86%) and 101 patients (57.71%) were positive for AwMA. Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 6 www.nature.com/scientificreports/ Figure 3. Detection of anti-mitochondrial antibodies in SLE patients and specificity of the assay. (a) Increased amounts of anti-whole mitochondria antibodies (AwMA) were detected by direct ELISA in sera (1:150) from systemic lupus erythematosus (SLE), anti-phospholipid syndrome (APS) and primary biliary cirrhosis (PBC) patients. Healthy: N = 43. SLE: N = 175. APS: N = 12, PBC: N = 12. The dotted line corresponds to the cutoff value as determined by Youden’s Index (see Table 5). Kruskal-Wallis test with multiple comparisons to healthy donors; Dunn’s correction. (b) No significant differences were detected by direct ELISA when either murine (Mm, gray symbols) or human mitochondria (Hs, black symbols) were used as coating antigens to detect AwMAs in control and SLE patient sera (1:100). Two-way ANOVA. (c) AwMA binding to coating mitochondria is inhibited in presence of mitochondria (filled circles) but not by red blood cells microparticles (filled squares). Two-way repeated measures ANOVA with multiple comparison (Dunnett’s correction) to signals detected without competitors (i.e. 100%). Discussion Mitochondrial DAMPs are known to stimulate the innate immune response, but it is less clear whether mitochon- drial antigens stimulate an antibody response and whether these antibodies impact the inflammatory reaction. Interestingly, in PBC, a paradigm of true organ-specific autoimmunity, evidence points to involvement of both innate and adaptive immunity with a specific antibody response to mitochondrial antigens67. i Although the prevalence of AMA in SLE was reported several decades ago59, this observation was not pursued. Our study confirms the presence of AwMA (directed against the mitochondrial surface and not redundant with that of PBC) and AmtDNA (directed against mtDNA) in patients with SLE. However, it remains to be estab- lished whether AwMA are pathogenic initiators of the auto-immune process or whether they are consequences of an a priori cell activation or injury with subsequent release of mitochondria in the extracellular milieu. These “free mitochondria” could subsequently become antigenic in predisposed individuals and be a marker of cell or tissue injury. They could also be a cause of further immune activation through the formation of circulating or in-situ immune complexes, constituting a secondary trigger of inflammation. The recognition of mitochondria by antibodies could also implicate Fc receptors and thus modulate a distinct immune response. For example, the intravenous injection of mtDNA into mice failed to induce proteinuria and kidney damage68, but the response may differ in a recipient with circulating antibodies to mitochondria.i 69 f Epitope modification, such as oxidation, can impact its antigenicity69. Notably, there are reports suggesting that the oxidation of mtDNA occurs during its extrusion from cells, and that the oxidized form is pathogenic15,16. Our findings using in vitro oxidation of the mitochondria suggest that mitochondrial epitopes, regardless of oxi- dation status, are targeted by autoantibodies in SLE. However, this does not exclude the possibility that oxidized mitochondria may be more antigenic in other pathogenesis, such as APS, or more efficient at promoting responses if recognized by the innate immune system. Moreover, it is not excluded that following oxidation, mitochondrial swelling and potential formation of pores within the mitochondrial membrane might have revealed new antigens detected in our anti-whole mitochondria ELISA using TBHP-treated mitochondria, but this treatment may also have caused a release of existing antigens that could have been lost in this modified assay, thus explaining the apparent absence of impact of oxidation of the antigenicity of the mitochondria70. www.nature.com/scientificreports/ Experiments presented in panels 3 a and 3 c were performed using mouse mitochondria, the experiment presented in panel 3 b was performed in parallel on murine and human mitochondria. Data are mean ± SD. Not significant (ns): p > 0.05. p < 0.05. *p < 0.001. *p < 0.0001. Hs: Homo sapiens; Mm: Mus musculus. Figure 3. Detection of anti-mitochondrial antibodies in SLE patients and specificity of the assay. (a) Increased amounts of anti-whole mitochondria antibodies (AwMA) were detected by direct ELISA in sera (1:150) from systemic lupus erythematosus (SLE), anti-phospholipid syndrome (APS) and primary biliary cirrhosis (PBC) patients. Healthy: N = 43. SLE: N = 175. APS: N = 12, PBC: N = 12. The dotted line corresponds to the cutoff value as determined by Youden’s Index (see Table 5). Kruskal-Wallis test with multiple comparisons to healthy donors; Dunn’s correction. (b) No significant differences were detected by direct ELISA when either murine (Mm, gray symbols) or human mitochondria (Hs, black symbols) were used as coating antigens to detect AwMAs in control and SLE patient sera (1:100). Two-way ANOVA. (c) AwMA binding to coating mitochondria is inhibited in presence of mitochondria (filled circles) but not by red blood cells microparticles (filled squares). Two-way repeated measures ANOVA with multiple comparison (Dunnett’s correction) to signals detected without competitors (i.e. 100%). Experiments presented in panels 3 a and 3 c were performed using mouse mitochondria, the experiment presented in panel 3 b was performed in parallel on murine and human mitochondria. Data are mean ± SD. Not significant (ns): p > 0.05. p < 0.05. *p < 0.001. p < 0.0001. Hs: Homo sapiens; Mm: Mus musculus. Thus, although more work is needed to explore the clinical associations of AmtDNA and AwMA in a larger lupus population and over time, different subsets of mitochondrial epitopes (i.e. mtDNA vs. outer membrane antigens) appear to measure different immune responses in SLE patients and may be associated with distinct disease characteristics. Thus, although more work is needed to explore the clinical associations of AmtDNA and AwMA in a larger lupus population and over time, different subsets of mitochondrial epitopes (i.e. mtDNA vs. outer membrane antigens) appear to measure different immune responses in SLE patients and may be associated with distinct disease characteristics. Discussion Future investigations will be needed to determine the relative targeting of native versus oxidized forms of organelle components by immune cells. We could not verify the role of oxidation on the recognition of mtDNA by autoantibodies due to spontane- ous oxidation of isolated mtDNA, an observation also made by other groups despite extreme preventive measures Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 7 www.nature.com/scientificreports/ Figure 4. Antibodies targeting mitochondrial DNA in SLE. (a) Anti-mitochondrial DNA antibodies (AmtDNA) are measured by direct ELISA in sera (1:50) from a mouse model of systemic lupus erythematosus (SLE) and control mice (Control: N = 8, SLE: N = 12, Student’s t-test). An isotype-matched monoclonal mouse anti-DNA antibody (clone 35I9 DNA, 10 µg/mL) was included as a positive assay control (dotted line). (b) Elevated levels of AmtDNA are observed in sera (1:150) from SLE but not from anti-phospholipid syndrome (APS) or primary biliary cirrhosis (PBC) patients. Healthy: N = 43. SLE: N = 175. APS: N = 12. PBC: N = 12. The dotted line corresponds to the cutoff value as determined by Youden’s index (see Table 5). Kruskal-Wallis test with multiple comparisons to controls/healthy donors; Dunn’s correction). All experiment presented in the figure were performed using mouse mtDNA. Data are mean ± SD. Not significant (ns): p > 0.05. p < 0.001. PBC: primary biliary cirrhosis. Figure 4. Antibodies targeting mitochondrial DNA in SLE. (a) Anti-mitochondrial DNA antibodies Figure 4. Antibodies targeting mitochondrial DNA in SLE. (a) Anti-mitochondrial DNA antibodies (AmtDNA) are measured by direct ELISA in sera (1:50) from a mouse model of systemic lupus erythematosus (SLE) and control mice (Control: N = 8, SLE: N = 12, Student’s t-test). An isotype-matched monoclonal mouse anti-DNA antibody (clone 35I9 DNA, 10 µg/mL) was included as a positive assay control (dotted line). (b) Elevated levels of AmtDNA are observed in sera (1:150) from SLE but not from anti-phospholipid syndrome (APS) or primary biliary cirrhosis (PBC) patients. Healthy: N = 43. SLE: N = 175. APS: N = 12. PBC: N = 12. The dotted line corresponds to the cutoff value as determined by Youden’s index (see Table 5). Kruskal-Wallis test with multiple comparisons to controls/healthy donors; Dunn’s correction). All experiment presented in the figure were performed using mouse mtDNA. Data are mean ± SD. Not significant (ns): p > 0.05. p < 0.001. PBC: primary biliary cirrhosis. Figure 4. Antibodies targeting mitochondrial DNA in SLE. Discussion (a) Anti-mitochondrial DNA antibodies (AmtDNA) are measured by direct ELISA in sera (1:50) from a mouse model of systemic lupus erythematosus (SLE) and control mice (Control: N = 8, SLE: N = 12, Student’s t-test). An isotype-matched monoclonal mouse anti-DNA antibody (clone 35I9 DNA, 10 µg/mL) was included as a positive assay control (dotted line). (b) Elevated levels of AmtDNA are observed in sera (1:150) from SLE but not from anti-phospholipid syndrome (APS) or primary biliary cirrhosis (PBC) patients. Healthy: N = 43. SLE: N = 175. APS: N = 12. PBC: N = 12. The dotted line corresponds to the cutoff value as determined by Youden’s index (see Table 5). Kruskal-Wallis test with multiple comparisons to controls/healthy donors; Dunn’s correction). All experiment presented in the figure were performed using mouse mtDNA. Data are mean ± SD. Not significant (ns): p > 0.05. p < 0.001. PBC: primary biliary cirrhosis. AmtDNAA AwMAA DNA (Farr)A ACA (+/−)B Anti-HSP60 0.07 p = 0.37 0.10 p = 0.21 0.02 p = 0.81 (+) 0.28 ± 0.52 (−) 0.52 ± 0.53 p = 0.08 AmtDNA — 0.23, p = 0.003 0.05 p = 0.46 (+) 0.33 ± 0.17 (−) 0.37 ± 0.27 p = 0.40 AwMA — — 0.10 p = 0.19 (+) 0.32 ± 0.20 (−) 0.33 ± 0.23 p = 0.51 Table 3. Intercorrelations of anti-mtDNA, anti-whole mitochondria, anti-dsDNA and anti-cardiolipin antibodies in SLE patients (n = 175). AValues are presented as Spearman correlation coefficient and p-value. BValues presented as median ± IQR and Wilcoxon test p-value for patient positives (+) or negatives (−) for ACA. ACA: anti-cardiolipin antibodies; AwMA: anti-whole mitochondria antibodies. AmtDNA: anti- mitochondrial DNA antibodies; DNA Farr: quantification of anti-dsDNA antibodies by Farr assay; HSP60: heat-shock protein 60 KDa. Table 3. Intercorrelations of anti-mtDNA, anti-whole mitochondria, anti-dsDNA and anti-cardiolipin antibodies in SLE patients (n = 175). AValues are presented as Spearman correlation coefficient and p-value. BValues presented as median ± IQR and Wilcoxon test p-value for patient positives (+) or negatives (−) for ACA. ACA: anti-cardiolipin antibodies; AwMA: anti-whole mitochondria antibodies. AmtDNA: anti- mitochondrial DNA antibodies; DNA Farr: quantification of anti-dsDNA antibodies by Farr assay; HSP60: heat-shock protein 60 KDa. taken to maintain the molecules under their reduced form71. Thus, it is likely that AmtDNA and anti-dsDNA antibodies routinely measured in clinical testing both evaluate antibodies to oxidized DNA. Discussion AmtDNA: anti-mitochondrial DNA antibodies. AUC: area under the curve. OD: optical density. PPV: Positive Predictive Value. NPV: Negative Predictive Value. NPP: Negative Predictive Value. Table 5. Performance of cut-off values for AwMA and AmtDNA (Healthy donors: n = 43, SLE: n = 175). AwMA: anti-whole mitochondria antibodies. AmtDNA: anti-mitochondrial DNA antibodies. AUC: area under the curve. OD: optical density. PPV: Positive Predictive Value. NPV: Negative Predictive Value. NPP: Negative Predictive Value. and AmtDNA) likely specific to SLE. Further research is required to identify the mitochondrial antigen(s) and epitope(s) of our AMA assay in SLE. p p y Our analyses of AmtDNA antibodies in human sera showed an association with anti-dsDNA as well as with lupus nephritis, consistent with the documented associations between anti-dsDNA antibodies detected by Farr assay and lupus nephritis74. While dsDNA used in Farr assays is usually isolated from plasmids (i.e. E. coli) and thus may share similarities with mtDNA (e.g. hypomethylated CpG motives, circular tertiary structure)75, the Farr assay has been described for its specificity in the detection of high avidity antibodies of all classes (IgG and IgM, for instance). Our data suggest that the Farr assay probably detects AmtDNA. Consistent with this, nuclear DNA (nDNA) efficiently competed in our AmtDNA ELISA assay (Supplementary Fig. 5a), further point- ing to a certain degree of redundancy between anti-dsDNA and AmtDNA. When nuclear and mtDNA were treated with S1 nuclease, which digests potential single-stranded regions, both could compete in our AmtDNA ELISA assay (Supplementary Fig. 5b), which suggests that most of the antigenicity of mtDNA is provided by its double-stranded conformation. There might exist subsets of antibodies that preferentially recognize mtDNA ver- sus genomic DNA, but there is no evidence of their occurrence at this stage. As extracellular mtDNA is reportedly associated with various pathologies, including trauma25,26, burn injury27, cancer28 and rheumatoid arthritis17,29, it is tempting to postulate that extruded mtDNA, beyond its role as a pro-inflammatory signal for the innate immune system, represents a significant antigenic load available for the formation of immune complexes.i y p gi g p In this study, we focused our attention on immunoglobulin (Ig) G (IgG), but the assays can be modified easily to quantify specific IgG subclasses (e.g. IgG1, IgG2a, IgG2b) or other isotypes (e.g. IgA and IgM), which may reveal distinct functions of these antibodies in disease. Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 Discussion Clinical Outcomes AmtDNA AwMA OR (CI) p OR (CI) p Thrombotic events 0.43 (0.10–1.79) 0.25 0.27 (0.04–1.99) 0.2 [0.35 (0.07–1.67)] [0.19] [0.21 (0.02–1.82)] [0.15] SLEDAI-2K ≥ 4 0.96 (0.37–2.51) 0.93 1.34 (0.49–3.69) 0.57 [1.01 (0.33–3.05)] [0.99] [1.08 (0.37–3.14)] [0.89] SDI ≥ 1 0.91 (0.35–2.42) 0.86 0.85 (0.30–2.40) 0.76 [0.93 (0.32–2.68)] [0.90] [0.69 (0.23–2.09)] [0.52] Increased anti-dsDNAA 3.34 (1.22–9.16) 0.02 1.15 (0.42–3.16) 0.79 [3.94 (1.33–11.69)] [0.01] [1.16 (0.40–3.35)] [0.79] Lupus nephritis 4.45 (1.50–13.20) 0.007 1.06 (0.39–2.90) 0.91 [4.60 (1.41–14.99)] [0.01] [0.97 (0.34–2.73)] [0.95] Table 4. AmtDNA and AwMA associations with clinical manifestations in SLE patients (n = 175). AOccurrences of patients with anti-dsDNA antibodies above the clinical threshold. AwMA: anti-whole mitochondria antibodies. AmtDNA: anti-mitochondrial DNA antibodies. SDI: lupus severity disease index. SLEDAI-2K: systemic lupus erythematosus disease activity index - 2000. OR (CI): Odds ratios (95% Wald Confidence Interval). P from logistic regressions. Bivariate results (N = 175) are followed by multivariate results in square brackets (N = 169). Table 4. AmtDNA and AwMA associations with clinical manifestations in SLE patients (n = 175). AOccurrences of patients with anti-dsDNA antibodies above the clinical threshold. AwMA: anti-whole mitochondria antibodies. AmtDNA: anti-mitochondrial DNA antibodies. SDI: lupus severity disease index. SLEDAI-2K: systemic lupus erythematosus disease activity index - 2000. OR (CI): Odds ratios (95% Wald Confidence Interval). P from logistic regressions. Bivariate results (N = 175) are followed by multivariate results in square brackets (N = 169). Occurrences of patients with anti dsDNA antibodies above the clinical threshold. AwMA: anti whole mitochondria antibodies. AmtDNA: anti-mitochondrial DNA antibodies. SDI: lupus severity disease index. SLEDAI-2K: systemic lupus erythematosus disease activity index - 2000. OR (CI): Odds ratios (95% Wald Confidence Interval). P from logistic regressions. Bivariate results (N = 175) are followed by multivariate results in square brackets (N = 169). Cutpoint (OD405 nm) Sensitivity Specificity PPV NPV AUC AwMA 0.30 (0.17–0.32) 0.58 (0.50–0.65) 0.88 (0.75–0.96) 0.95 (0.89–0.98) 0.34 (0.25–0.43) 0.80 (0.73;0.87) AmtDNA 0.30 (0.25–0.45) 0.63 (0.55–0.70) 0.74 (0.59–0.87) 0.91 (0.84–0.95) 0.33 (0.24–0.43) 0.71 (0.63;0.79) Table 5. Performance of cut-off values for AwMA and AmtDNA (Healthy donors: n = 43, SLE: n = 175). AwMA: anti-whole mitochondria antibodies. AmtDNA: anti-mitochondrial DNA antibodies. AUC: area under the curve. OD: optical density. PPV: Positive Predictive Value. NPV: Negative Predictive Value. NPP: Negative Predictive Value. Table 5. Performance of cut-off values for AwMA and AmtDNA (Healthy donors: n = 43, SLE: n = 175). AwMA: anti-whole mitochondria antibodies. Discussion b d h l l l d h h antibodies routinely measured in clinical testing both evaluate antibodies to oxidized DNA. We observed that AwMA levels were more elevated in SLE than in PBC. PBC patients, however, were positive for AMA when sonicated mitochondria were utilized in our ELISA. These observations are consistent with the fact that pyruvate dehydrogenase complex E2 (PDC-E2), the immunodominant epitope in PBC, is located in the mitochondrial inner membrane. Our AwMA-ELISA measures antibodies to epitopes on intact mitochondria, in which inner membrane epitopes remain unavailable72. This contrasts with the sonicated mitochondria used in previous studies on AMA in SLE59, which contained inverted membranous structures and thus revealed antigens located in the mitochondrial inner membrane60. Furthermore, we found that AwMA levels correlated with the levels of AmtDNA, but not with other antibodies directed at antigens located within the inner membrane of the organelle (e.g. cardiolipin or HSP60), pointing to the existence of mitochondrial antigens that remain to be identified73. Our study provides simple and quantitative assays for assessment of two types of AMA (i.e. AwMA Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 8 www.nature.com/scientificreports/ Clinical Outcomes AmtDNA AwMA OR (CI) p OR (CI) p Thrombotic events 0.43 (0.10–1.79) 0.25 0.27 (0.04–1.99) 0.2 [0.35 (0.07–1.67)] [0.19] [0.21 (0.02–1.82)] [0.15] SLEDAI-2K ≥ 4 0.96 (0.37–2.51) 0.93 1.34 (0.49–3.69) 0.57 [1.01 (0.33–3.05)] [0.99] [1.08 (0.37–3.14)] [0.89] SDI ≥ 1 0.91 (0.35–2.42) 0.86 0.85 (0.30–2.40) 0.76 [0.93 (0.32–2.68)] [0.90] [0.69 (0.23–2.09)] [0.52] Increased anti-dsDNAA 3.34 (1.22–9.16) 0.02 1.15 (0.42–3.16) 0.79 [3.94 (1.33–11.69)] [0.01] [1.16 (0.40–3.35)] [0.79] Lupus nephritis 4.45 (1.50–13.20) 0.007 1.06 (0.39–2.90) 0.91 [4.60 (1.41–14.99)] [0.01] [0.97 (0.34–2.73)] [0.95] Table 4. AmtDNA and AwMA associations with clinical manifestations in SLE patients (n = 175). AOccurrences of patients with anti-dsDNA antibodies above the clinical threshold. AwMA: anti-whole mitochondria antibodies. AmtDNA: anti-mitochondrial DNA antibodies. SDI: lupus severity disease index. SLEDAI-2K: systemic lupus erythematosus disease activity index - 2000. OR (CI): Odds ratios (95% Wald Confidence Interval). P from logistic regressions. Bivariate results (N = 175) are followed by multivariate results in square brackets (N = 169). Material and Methods injections of β2GPI (20 µg) (Crystal Chem, Downers Grove, IL, USA) followed 24 h later by a 100 µL i.v. injection of lipopolysaccharide (LPS from E. coli, O111:B4; 10 µg) (Sigma-Aldrich, Saint-Louis, MO, USA). These injections were repeated after 2 and 4 weeks for a total of three rounds of immunizations and mice were bled 48 h after the third immunization. C57BL/6 J mice injected i.v. with PBS under the same schedule were used as controls. Ethics. Throughout the entire study, blood samples were obtained from patients under informed consent. All the methods presented in this study were performed in accordance with the relevant guidelines and regulations for both human and murine donors. Mitochondria isolation. Mitochondria were isolated from the livers of C57BL/6 J mice following a com- bination of published protocols61,62. Mice were sacrificed by cervical dislocation, the liver was swiftly removed, the gallbladder excised, and the liver was rinsed in ice-cold PBS (137 mM NaCl, 3 mM KCl, 19 mM Na2HPO4, 2 mM KH2PO4). The liver was then minced and transferred to a pre-cooled glass/Teflon tissue grinder contain- ing 12 mL of ice-cold mitochondrial isolation buffer (10 mM Tris, 1 mM EGTA, 200 mM sucrose) per gram of liver then ground until an homogeneous suspension was obtained. Intact cells and nuclei were pelleted twice at 700 g, 4 °C, 10 min. Contaminating membranes, proteins and organelles were eliminated by two centrifugations at 7,000 g, 4 °C, 10 min followed by a centrifugation at 10,000 g, 4 °C, 10 min. The crude mitochondrial suspen- sion was further purified by ultracentrifugation against a Percoll gradient (10 mM Tris, 1 mM EGTA, 30% v:v Percoll) at 95,000 g, 4 °C, 30 min. The band containing mitochondria was collected in PBS. A similar approach was used to isolate human mitochondria with the exception that up to 107 Hep-G2 cells were lysed by repeated passages through a narrow-gauge needle. The commercial kit QIAgen Qproteome (QIAgen, Hilden, Germany) was also used, following the manufacturer’s instructions, to isolate mitochondria from Hep-G2 cells for quality comparison by western blotting with the aforementioned protocol. Isolated mitochondria were dosed by the bicinchoninic acid (BCA) method using BCA Protein Assay Kit (Thermo Fisher scientific, Waltham, MA, USA). Freshly isolated mitochondria were used in all experiments with the exception of the submitochondrial particles preparations for which pelleted mitochondria were kept at −80 °C until needed. Submitochondrial particles (SMP) preparation. Material and Methods Study approval. Patient population. The human sera tested in this study were obtained from a University Health Network research ethics board (REB) approved study of SLE and APS patients in Toronto as well as from healthy controls and PBC patients recruited from CHU de Québec – Université Laval REB approved studies in Quebec City. SLE patients had to meet the 1982 ACR classification criteria for SLE revised in 199781,82 and APS patients met the 1999 Sapporo criteria for APS revised in 200683,84. For SLE, consecutive female patients from the University of Toronto Lupus Clinic (UTLC) were approached and provided consent between August 2010 and October 2011 to be part of a study on the role of fatty acids and cardiovascular disease in lupus. They provided one blood specimen and had their anonymized clinical data linked to their biospecimen. Similarly, APS patients seen at the rheumatology clinic in Toronto were approached following a similar procedure. All remaining bio- specimen could be used for future studies on biomarkers of lupus as per the original subjects’ consent. Healthy control volunteers were recruited as part of study on markers of inflammation if they had no known illnesses and did not have infectious symptoms at the time of the blood draw. Age and sex were collected at that time. PBC patients met the 2009 PBC classification criteria, revised in 201838,85 including the positivity for anti-mitochon- drial antibodies. Data from clinical laboratories. For SLE patients, anti-dsDNA were measured using the Farr assay (labo- ratory cut-off of 30%) and the anti-cardiolipin (IgG and IgM – laboratory cut-offs of 40 GPL or MPL units) were measured by ELISA in a clinical laboratory. Cell culture. Hep-G2 human hepatocarcinoma cells (ATCC, Manassas, VA, USA) were cultured at 37 °C – 5% CO2 in Eagle’s Minimum Essential Medium (EMEM) (Wisent) supplemented with 10% fetal bovine serum (FBS) (Wisent), non-essential amino-acids (Wisent) and penicillin/streptomycin (Wisent). Inducible mouse model of SLE. Recommendations of the Canadian Council on Animal Care were fol- lowed in a protocol approved by the Animal Welfare Committee at Université Laval. C57BL/6 J were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in an Elite-Plus specific-pathogen-free ani- mal facility at CHU de Quebec. SLE autoantibodies were induced in these mice as previously described63. In brief, 6–8-week-old male mice received 100 µL i.v. Discussion Both AwMA and AmtDNA were detected at low levels in healthy individuals, suggesting that these antibodies are further induced during SLE pathogenesis. These antibod- ies might be part of a pool of naturally occurring autoantibodies that are thought to contribute to the continual clearance of membrane vesicles in the circulation76,77. Protective natural autoantibodies have been described pre- dominantly as IgM, but other isotypes such as IgG or IgA have also been reported78–80. It is thus tempting to pos- tulate that antibodies targeting mitochondrial epitopes are present in healthy individuals and might be involved in clearance of extracellular mitochondria. A profound change in the balance of natural IgM and pathogenic IgG against mitochondrial epitopes may in part explain the pathogenesis in SLE.i g p p y p p p g While these observations require confirmation in an independent cohort of patients, they suggest that, in SLE, the adaptive immune system recognizes mitochondrial organelles. Furthermore, our findings suggest that clinical associations may differ according to antibody recognition of inner (e.g., ACA) versus outer mitochondrial (e.g., AwMA) membrane components. This leads to the postulation that pathogenicity of these antibodies may depend on whether extracellular mitochondria are intact or not. Evaluation of antibodies to mitochondrial components in SLE may provide novel information on patients, such as their risk for developing nephritis. If these findings are confirmed in a large prospective cohort of SLE patients, AwMA and AmtDNA may prove useful in predicting Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 9 www.nature.com/scientificreports/ disease activity and disease severity, and in stratifying SLE patients. The quantification of mitochondrial antibod- ies may thus open the way to novel directions in autoimmune disease research and may be useful for achieving a better understanding of disease mechanisms. Material and Methods Proteins of interest were labeled overnight at 4 °C with either mouse anti-actin (Clone AC-15, 1:5,000. Sigma-Aldrich), mouse anti-tubulin (Clone DM1A, 0.2 µg/mL. Abcam, Cambridge, UK), mouse anti-proliferating cell nuclear antigen (PCNA. Clone PC10, 1 µg/mL. Santa Cruz biotechnology, Santa Cruz, CA, USA), rabbit anti-VDAC (Clone D73D12, 1:1,000. Cell Signaling Technology, Danvers, MA, USA), mouse anti-TOMM22 (Clone IC9-2, 2 µg/mL. Abcam) or mouse anti-cytochrome C antibody (Clone 7H8.2C12, 0.5 µg/mL. BD Biosciences, Franklin Lakes, NJ, USA), polyclonal rabbit anti-proteasome 20 S (0.8 µg/mL. Abcam), polyclonal rabbit anti-GRP94 (1:2,000, Abcam) diluted in superblock (BioRad). Following a 1-h incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (1:10,000 in superblock) (Jackson Immunoresearch, West Grove, USA), signals were visualized with Western Lightning Chemiluminescence Reagent (Perkin Elmer, Waltham, MA, USA) on a C-DiGit membrane scanner (LI-COR biotechnology, Lincoln, NE, USA). Total proteins isolated from starting materials (i.e. mouse liver or Hep-G2 cells) were used as controls. Full-length blots are presented in Supplementary Fig. 6a–c. Mitochondrial size measurement by dynamic light scattering (DLS). The size of the isolated mito- chondria was measured by DLS, using a Zetasizer Nano ZS device (Malvern instruments, Malvern, UK) equipped with the standard 4 mW, 633 nm He-Ne laser as a light source, set at a detection angle of 173°. Experiments were replicated three times in 1 cm length disposable UV-Cuvettes (Brand GMBH, Wertheim, Germany) containing 100 µL of isolated murine mitochondria diluted in PBS at a concentration of 10 µg proteins/µL. The following parameters were taken into account upon measuring the size of the mitochondrial: refraction index of the solvent: 1.330, viscosity of the sample: 0.8872 mPas, refraction index of the proteins: 1.45, temperature: 25 °C. Electron microscopy. Mitochondria (108) were fixed in 3.5% acrolein for 15 min at room temperature. Fixed samples were rinsed twice in PBS then embedded in 4% agarose. 50 µm sections were cut using a vibra- tome, post-fixed in 1% osmium tetroxide for 30 minutes and embedded in Durcupan resin. Seventy-nanometer ultrathin sections were visualized at 80 kV using a Tecnai G2 Spirit BioTWIN (FEI, Hillsboro, OR, USA) trans- mission electron microscope. Assessment of the mitochondrial oxygen consumption. Material and Methods SMP were prepared as described elsewhere60. Briefly, frozen mitochondria were thawed at room temperature and diluted to 10 mg of mitochondrial proteins in 10 mM 4-Morpholinepropanesulfonic acid (MOPS). Samples were then sonicated using a Fisher Sonic Dismembrator Model 500 (Thermo Scientific) at 20% maximal output for 20 seconds then kept on a salt-ice water bath for 10 minutes. This cycle was repeated nine times. Samples were then centrifuged at 16,000 g, 4 °C, 10 minutes in order to discard unbroken mitochondria and other unwanted debris. Supernatants were collected in a fresh tube and their volumes adjusted to 2 mL in 10 mM MOPS. Samples were then ultracentrifuged at 150,000 g, 4 °C for 45 minutes. Pelleted SMP were resuspended in SMP buffer (225 mM mannitol, 75 mM sucrose, 10 mM HEPES, 0.1 mM EDTA, pH 7.4) and dosed. SMP preparations were kept at −80 °C until needed. Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 10 www.nature.com/scientificreports/ Red blood cells microparticles (RBCMP) preparation. Red blood cells (RBC) were isolated from the blood of healthy human volunteers by centrifugation at 282 g for 10 minutes at RT. Platelet-rich plasma and buffy coat were discarded, and the RBC fraction kept. RBC were counted (Cellometer Auto M10, Nexcelom Bioscience, Lawrence, MA, USA) and adjusted to a concentration of 5 × 108 cells/mL in Tyrode’s buffer (12 mM NaHCO3, 127 mM NaCl, 5 mM KCl, 0.5 mM NaH2PO4, 1 mM MgCl2, 5 mM Glucose, 10 mM HEPES, pH 7.4). 109 cells were then diluted in 45 mL double-distilled water and incubated for 5 minutes. The tonicity of the buffer was then balanced by addition of 5 ml of PBS 10X (filtered through a 0.22-μm membrane). Remnant RBC were removed by centrifugation at 1,300 g for 5 minutes at RT and supernatant was centrifugated at 18,000 g for 90 minutes at 18 °C. RBC microparticle pellet was then suspended in 500 μl PBS. Protein concentration was measured with BCA assay. Western blotting. After quantification, 25 µg of sample per lane were loaded onto a 12% polyacrylamide gel and underwent migration for 1 h 30 min at 100 V (constant voltage). Gels were then transferred overnight onto polyvinylidene difluoride membranes (PVDF. BioRad, Hercules, CA, USA) at 4 °C and 60 mA (constant current). Non-specific binding sites were blocked in Tris-buffered saline (TBS)−0.1% Tween20 containing 5% non-fat dry milk for 4 h at room temperature. Material and Methods Mitochondria were resuspended in mito- chondrial assay solution (MAS: 70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA and 0.2%(w/v) fatty acid-free BSA, pH 7.4 at 37 °C) and supplemented with 10 mM pyruvate, 2 mM malate and 4 μM FCCP [carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone], pH 7.4. An equivalent of 10 μg proteins was seeded on XF-96 plates (Agilent, Santa Clara, CA, USA). Plates were then centrifuged 2,000 g for 20 min at 4 °C. We visualized the distribution of mitochondria under a bright-field microscope to ensure adher- ence and homogeneous repartition. Plates were maintained at 37 °C without CO2 for approximately 40 minutes prior to loading. Oxygen consumption rates (OCR) were measured in accordance with manufacturer instructions (Agilent/Seahorse Bioscience). Experiments were replicated in three wells and averaged for each experimental condition. A total of 3 measurements of oxygen consumption for each condition were made approximately every 6 minutes under basal conditions and after sequential addition of rotenone (2 μM), succinate (10 mM), antimycin A (40 μM) and TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine)/Ascorbate (100 μM/10 mM). Succinate was used as the electron donor for complex II, rotenone as a complex I inhibitor, antimycin A as a complex III inhib- itor and respiration through complex IV was measured using TMPD/ascorbate. High sensitivity flow cytometry. Due to their small size, mitochondria were detected by high sensitivity flow cytometry, using a “small particle option” consisting of a forward scatter (FSC) coupled to a photomultiplier tube (PMT) mounted on a BD FACS Canto II Special Order Research Product (SORP, BD Biosciences). Mitochondria (0.5 µg) were stained with 1 µg of anti-TOMM22-Atto 488 (clone IC9-2, Sigma-Aldrich) and 1 µM of mitotracker deep red (Invitrogen, Carlsbad, CA, USA) for 30 minutes at 37 °C and diluted with PBS to a final volume of 500 µl before flow cytometry analysis. To quantitatively measure the number of mitochondria, we used 3 µm diameter polystyrene micro- sphere (Polysciences, PA, USA). 80,000 microspheres were added to each sample and 500 microspheres were acquired. Silica particles (Kisker Biotech, Steinfurt, Germany) were used to determine 100–1,000 nm size scale. Mitochondrial DNA extraction by alkaline lysis. Isolated mitochondria were pelleted at 10,000 g, 4 °C, 10 min and resuspended in 0.8 mL mtDNA isolation buffer A [50 mM Tris-HCl, 10 mM EDTA, 100 µg/mL RNAse A (QIAgen), pH 7.5] for every 3 mg of mitochondrial proteins. Material and Methods Mitochondria were then lyzed in one volume (v:v) mtDNA isolation buffer B (extemporaneously-prepared. 0.2 M NaOH, 1% SDS) for 3 min. on ice, under gentle Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 11 www.nature.com/scientificreports/ agitation. mtDNA suspensions were neutralized with one volume (v:v) mtDNA isolation buffer C (1.32 M potas- sium acetate, pH 4.8 adjusted with ice-cold acetic acid) for 5 min. on ice, under gentle agitation. Mitochondrial debris were pelleted by centrifugation for 10 minutes at 14,000 g, RT. Supernatants were transferred to fresh tubes. mtDNA was precipitated overnight at −20 °C with 0.1 volume (v:v) potassium acetate (stock solution: 3 M sodium acetate, pH adjusted to 5.2 with ice-cold acetic acid) and 0.7 volume (v:v) absolute isopropanol. mtDNA was then pelleted at 14,000 g, washed thrice with 1.5 mL 70% ethanol and resuspended in DNA resuspension buffer (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). Mitochondrial DNA concentration was determined by spectro- photometry (Nanodrop 1000, Thermo Fisher Scientific). Nuclei isolation from mouse hepatocytes. Nuclei were isolated from mouse livers using published methods86. Briefly, during mitochondria isolation protocols, while supernatants were used for performing mito- chondria isolations, pellets from the first 700 g centrifugation were resuspended in 10 mL of ice-cold nuclei isola- tion buffer A (5 mM MgCl2, 10 mM Tris–HCl, 250 mM Sucrose. pH 7.4) and ground in a pre-cooled glass/Teflon tissue grinder. The suspension was further disrupted by three passages through a 25 G5/8 gauge needle followed by two passages through a 27 G ½ gauge needle. The suspension was then filtered against a 40 µM nylon cell strainer (Thermo Fisher Scientific) and centrifuged at 600 g, 4 °C, 10 min. Pellets were resuspended in 14 mL buffer A and centrifuged at 600 g, 4 °C, 10 min. Pellets were then resuspended in 9 volumes of ice-cold nuclei isolation buffer B (1 mM MgCl2, 10 mM Tris– HCl, 2.0 M sucrose. pH 7.4) and centrifuged at 16,000 g, 4 °C, 30 min. Pellets were resuspended in 200 µL PBS and stored at −20 °C. Whole-cell and nuclear DNA isolation. DNA from either 25 mg of whole mouse liver or the 200 µL or isolated nuclei were extracted using QIAmp® DNA Mini Kit (QIAgen), following instructions from the manufac- turer. Samples were eluted in DNA resuspension buffer. Mitochondrial DNA and nuclear DNA enrichments by qPCR. Material and Methods Thirty nanograms of mitochondrial DNA, nuclear DNA or the same amount of total DNA extracted from whole mouse liver using the aforementioned protocols, were amplified in a Rotor-Gene Q real time qPCR cycler (QIAgen) according to standard protocols with SsoAdvanced Universal SYBR® Green Supermix (BioRad) in a 10 µL reaction volume. Two distinct primer couples were used: one specific to mitochondrial DNA (5′-GGAACAACCCTAGTCGAATGAA-3′/5′-GCTAGGGCCGCGATAATAAA-3′) and the other to nuclear DNA (5′-CCTGCTGCTTATCGTGGCTG-3′/5′-GCCAGGAGAATGAGGTGGTC-3′). The experimental conditions were: 50 °C - 2 minutes in, 95 °C - 10 min and 40 cycles (95 °C - 15 seconds, 60 °C - 1 minutes). Fold changes were calculated with the 2−ΔCt method setting mean value for total liver DNA extracts as 1. Mitochondrial DNA digestion by restriction enzymes. Purified mouse mtDNA was digested by Hae II or Pst I restriction endonucleases (NEB, Whitby, ON, Canada) according to the manufacturer’s protocol. Digested products (1.5 μg) were then resolved on a 0.5% (w/v) agarose gel. Images were acquired on a Chemidoc MP gel documentation system (Bio-Rad). Full-length gel is presented in Supplementary Fig. 6d. S1 nuclease treatment of mitochondrial DNA and nuclear DNA. 30 µg of mtDNA and nDNA were diluted in 200 µL of 1X S1-buffer and treated with 50 U S1 nuclease (Thermo Fischer Scientific) for 5 minutes at 37 °C. Reaction was stopped by addition of 600 mM EDTA (final concentration) and incubation at 70 °C for 10 minutes. Samples were precipitated by addition of 300 mM Sodium Acetate (final concentration) and 2 vol- umes (v:v) absolute ethanol for 16 hours at −20 °C. Samples were pelleted at 14,000 g, RT, 20 minutes, and washed 1 mL 70% ethanol and resuspended in DNA resuspension buffer. For competition assays, untreated nDNA and mtDNA followed the same treatment without addition of the enzyme. Samples were dosed by qPCR. Mitochondria oxidation in-vitro. Dosed mitochondria were pelleted and resuspended at a concentra- tion of 1.5 mg of mitochondrial proteins/mL in PBS containing 500 µM of the oxidant tertbutyl hydroperoxide (TBHP) (Sigma-Aldrich). Mitochondria were oxidized for 1 h 30 min at 37 °C under gentle agitation then rinsed twice with ice-cold PBS. Oxidized mitochondria were subsequently quantified by BCA. Mitochondrial lipid oxidation. Thiobarbituric acid reactive substances (TBARS) formation was assessed, using the TBARS Parameter Kit (R&D systems, Minneapolis, MN, USA), following the manufacturer’s instruc- tions. Material and Methods Two hundred micrograms of mitochondria were lysed, the proteins were precipitated using trichloroacetic acid (TCA) and pelleted at 12,000 g, room temperature, 4 min. The supernatants were transferred to fresh tubes. Samples (75 µL) were incubated with 37.5 µL of thiobarbituric acid in a 96-well plate for 3 h at 50 °C. Absorbances were read at 532 nm on a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA) and TBARS quantification was performed using a standard curve provided in the kit. Oxidized samples were com- pared to control mitochondria that were incubated under the same conditions in PBS devoid of TBHP. Mitochondrial protein oxidation. The formation of carbonyl moieties following in-vitro mitochondrial oxidation was measured using the Protein Carbonyl Content Assay Kit (Sigma Aldrich) following the manufac- turer’s instructions. Oxidized samples were compared to control mitochondria that were incubated under the same conditions in PBS devoid of TBHP. Detection of antibodies targeting mitochondrial epitopes by ELISA. For the detection of anti-whole mitochondrial antibodies (AwMA), murine mitochondria were diluted (500 µg/mL) in 50 mM car- bonate/bicarbonate buffer, pH 9.6 and 25 µL per well were loaded onto 96-well half-area clear flat bottom polysty- rene high-binding microplates (Corning, New York, USA). Plates were coated for 18 h at 4 °C then blocked for 4 h Scientific Reports \| (2019) 9:4530 \| https://doi.org/10.1038/s41598-019-40900-3 12 www.nature.com/scientificreports/ at 37 °C with PBS containing 10% FBS and 0.5% gelatin. After three washes with PBS, sera diluted 1:150 (unless otherwise specified) in PBS-10% FBS-0.3% gelatin were incubated overnight at 4 °C in duplicate. After three washes with PBS, plates were incubated for 1 h at room temperature with alkaline phosphatase-(AP) conjugated goat anti-mouse or anti-human IgG (Sigma-Aldrich) diluted 1:1,000 in PBS-0.4% bovine serum albumin (BSA). Plates were washed thrice with PBS and developed with p-nitrophenol phosphate (p-NPP) for ~30 min at 37 °C and optical densities (OD) were read at 405 nm on a microplate reader. The same protocol was used for the detec- tion of autoantibodies targeting submitochondrial particles by using 25 µL per well of SMP diluted (50 µg/mL) in 50 mM carbonate/bicarbonate buffer, pH 9.6. A similar approach was used for human mitochondria with the following modification: plates were incubated with a horseradish peroxidase-conjugated anti-human IgG secondary antibody (1:3,000), peroxidase activity was revealed at room temperature for ~5 min with 3,3′,5,5′-tetramethylbenzidine (TMB). Material and Methods The reaction was stopped with 2 N H2SO4 and the ODs read at 450 nm. For the anti-mtDNA ELISA, plates were pre-coated with 1% protamine sulfate in double-distilled water for 1 h at room temperature. Plates were washed three times with PBS and coated overnight at 4 °C with 400 ng mtDNA in PBS. All subsequent steps were identical to those used for AwMA-ELISAs. Blank values (mitochondrial antigens and no sera) were substracted from measured values for each patient. During the development of these assays, the proper coating of the wells was tested by using isotype-matched mouse monoclonal antibodies (mAb). A mono- clonal antibody (Clone IV.3, 4 µg/mL) was used as a negative assay control in each instance while different mon- oclonal antibodies were used, depending on the coating antigens, as positive assay controls: an anti-TOMM22 mAb (Clone IC9-2, 4 µg/mL. Abcam) for intact mitochondria, an anti-DNA mAb (Clone 35I9 DNA, 10 µg/mL. Abcam) or an anti-Cytochrome C mAb (Clone 7H8.2C12, 5 µg/mL. BD Biosciences). Competition assay. AwMA-ELISAs were performed as described in the previous sections with the follow- ing modifications: serum samples were pre-incubated in dilution buffer (1:150) spiked with various concentra- tions (0.25, 1 and 3 mg/mL) of competitors (i.e. mitochondria or red blood cells microparticles) for 3 hrs. at RT and incubated in duplicate overnight at 4 °C. Data are presented as the percentage of signal remaining after each competition, compared to the OD (405 nm) measured in absence of competitors. p p p A similar procedure was used for AmtDNA-ELISA, using increased concentrations (0, 3, 9 and 27 ng/µL) of competing DNA (extracted from nuclei or mitochondria, with or without S1 nuclease treatment). Statistics. Comparisons between groups were made using either Student’s t-test, Wilcoxon test, Friedman or Kruskal-Wallis tests, one-way ANOVA, two-way or repeated measures ANOVA depending on the outcome, as well as the number and type of groups. When multiple comparisons were assessed, appropriate post-hoc correc- tion tests were used such as Dunn’s, Dunnett’s, Sidak’s or Bonferroni’s. Associations between AwMA, AmtDNA and anti-HSP60 were computed with Spearman correlations. Distribution of these antibodies according to ACA results were compared using Wilcoxon test. Associations between AwMA or AmtDNA and clinical outcomes were studied by bivariate and multivariate linear and logistic regressions. Material and Methods Clinical outcomes studied are average intima media thickness, percent change of the flow mediated dilatation (FMD) of the brachial artery, presence of FMD, presence of plaque in the carotid, thrombotic event ever, white blood cells count, platelet count, increased DNA binding by Farr assay above normal range for testing laboratory, presence of damage according to SLICC Damage Index (SDI > 0), high activity according to SLEDAI-2K activity score (SLEDAI ≥ 4), presence of lupus nephritis, biopsy class, as well as chronicity and activity index from the biopsy. The latter were adjusted for dis- ease duration, age, body mass index (BMI), low-density lipoproteins (LDL) cholesterol, antimalarial medica- tion and prednisone. Logistic regressions are presented with odd ratios and their 95% Wald confidence interval. Participants’ results were considered positive for AwMA and AmtDNA when their value was above the cut-off value identified after maximizing Youden’s Index. A 95% confidence interval was obtained for the cut-off using 10,000 bootstrap samples. Performance measures are presented with their 95% exact confidence interval. Software. Western blot images were acquired using Image Studio Digits 5.2 (LI-COR biotechnology). mtDNA migration through agarose gel was imaged using Image Lab 6.0.1 (Bio-Rad). DLS studies were car- ried using the built-in Zetasizer 7.10 software (Malvern Instruments). EM images were acquired with the Image Capture Software 601.384 (Advanced Microscopy Techniques Corp., Woburn, MA, USA). Flow cytometry was performed using the BD FACSDiva™ 6.1.3 (BD Biosciences). Yields from DNA isolations were quantified using the ND-1000 3.8.1 software (Thermo Fisher Scientific) and qPCR were performed with RotorGene 6.1 (Corbett Research/QIAgen). 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https://openalex.org/W4297996802	https://www.frontiersin.org/articles/10.3389/fmars.2022.951471/pdf	English	null	Identification of ventilated and submarine glacial meltwaters in the Amundsen Sea, Antarctica, using noble gases	Frontiers in marine science	2,022	cc-by	9,793	TYPE Original Research PUBLISHED 29 September 2022 DOI 10.3389/fmars.2022.951471 TYPE Original Research PUBLISHED 29 September 2022 DOI 10.3389/fmars.2022.951471 glacial meltwater, noble gases, optimum multiparameter analysis, Amundsen Sea, basal melting, meteoric water OPEN ACCESS EDITED BY Mark James Hopwood, Southern University of Science and Technology, China REVIEWED BY Oliver Huhn, University of Bremen, Germany Brice Loose, University of Rhode Island, United States EDITED BY Mark James Hopwood, Southern University of Science and Technology, China DongYoub Shin 1, Doshik Hahm 1, Tae-Wan Kim 2, Tae Siek Rhee 2, SangHoon Lee 2, Keyhong Park 2, Jisoo Park 2, Young Shin Kwon 2,3, Mi Seon Kim 2,4 and Tongsup Lee 1 DongYoub Shin 1, Doshik Hahm 1, Tae-Wan Kim 2, Tae Siek Rhee 2, SangHoon Lee 2, Keyhong Park 2, Jisoo Park 2, Young Shin Kwon 2,3, Mi Seon Kim 2,4 and Tongsup Lee 1 1Department of Oceanography, Pusan National University, Busan, South Korea, 2Division of Ocean Sciences, Korea Polar Research Institute, Incheon, South Korea, 3Korea Institute of Ocean Science and Technology, Busan, South Korea, 4Department of Ocean Environmental Sciences, Chungnam National University, Daejeon, South Korea To delineate the glacial meltwater distribution, we used ﬁve noble gases for optimum multiparameter analysis (OMPA) of the water masses in the Dotson- Getz Trough (DGT), Amundsen Sea. The increased number of tracers allowed us to deﬁne potential source waters at the surface, which have not been possible with a small set of tracers. The highest submarine meltwater (SMW) fraction (~0.6%) was present at the depth of ~450 m near the Dotson Ice Shelf. The SMW appeared to travel beyond the continental shelf break along an isopycnal layer. Air-equilibrated freshwater (up to 1.5%), presumably ventilated SMW (VMW) and surface melts, was present in the surface layer (<100 m). The distribution of SMW indicates that upwelled SMW, known as an important carrier of iron to the upper layer, amounts for 29% of the SMW in the DGT. The clear separation of VMW from SMW enabled partitioning of meltwater into locally-produced and upstream fractions and estimation of the basal melting of 53 – 94 Gt yr-1 for the adjacent ice shelves, assuming that the SMW fractions represent accumulation since the previous Winter Water formation. The Meteoric Water (MET) fractions, consisting of SMW and VMW, comprised 24% of those derived from oxygen isotopes, indicating that the annual input from basal melting is far less than the inventory of meteoric water, represented by MET. COPYRIGHT © 2022 Shin, Hahm, Kim, Rhee, Lee, Park, Park, Kwon, Kim and Lee. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Identiﬁcation of ventilated and submarine glacial meltwaters in the Amundsen Sea, Antarctica, using noble gases OPEN ACCESS EDITED BY Mark James Hopwood, Southern University of Science and Technology, China REVIEWED BY Oliver Huhn, University of Bremen, Germany Brice Loose, University of Rhode Island, United States *CORRESPONDENCE Doshik Hahm hahm@pusan.ac.kr SPECIALTY SECTION This article was submitted to Marine Biogeochemistry, a section of the journal Frontiers in Marine Science RECEIVED 24 May 2022 ACCEPTED 29 August 2022 PUBLISHED 29 September 2022 CITATION Shin DY, Hahm D, Kim T-W, Rhee TS, Lee SH, Park K, Park J, Kwon YS, Kim MS and Lee T (2022) Identiﬁcation of ventilated and submarine glacial meltwaters in the Amundsen Sea, Antarctica, using noble gases. Front. Mar. Sci. 9:951471. doi: 10.3389/fmars.2022.951471 1 Introduction Owing to the limited number of tracers, both the CT and d18O methods assume three-component mixing and consider only a part of the potential components, ignoring components in the surface layer, such as Antarctic Surface Water (AASW) and VMW. Their inertness, wide range of solubilities, and diffusivities make noble gases an ideal suite of tracers for overcoming previous limitations in tracer number. Loose and Jenkins (2014), for the ﬁrst time, used ﬁve noble gases as tracers of OMPA and demonstrated their use in unambiguously distinguishing SMW. This combination of noble gases and OMPA, hereafter referred to as the NG−OMPA method, has been successfully adopted to trace meltwater from Greenland (Beaird et al., 2015; Beaird et al., 2018). In this study, we adopted NG−OMPA, which uses CTD data and noble gases simultaneously, to delineate the spreading of glacial meltwaters in the Amundsen Sea. This study is an extension of previous studies that have used limited sets of noble gases to identify SMW in the Amundsen Sea (Kim et al., 2016b; Biddle et al., 2019). Here, the addition of noble gas tracers enabled the explicit deﬁnition of potential components in the surface layer (AASW and VMW), along with mCDW, WW, and SMW. One of the most frequently adopted methods to identify SMW distribution in Antarctic shelves involves using a pair of tracers, chosen from temperature, salinity, and dissolved oxygen, which are readily available from CTD observations (Jenkins, 1999; Jenkins and Jacobs, 2008). This method assumes that all the observed water properties near Antarctic ice shelves should be mixtures of the properties of three components: modiﬁed Circumpolar Deep Water (mCDW), Winter Water (WW), and SMW. Using this Composite Tracer (CT) method, Wåhlin et al. (2010) ﬁrst reported that the intermediate layer between WW and mCDW in the central Amundsen Sea (Dotson-Getz Trough, DGT) is occupied by a 100 – 150 m-thick mixture of mCDW and SMW. The CT method has also been used to quantify SMW fractions as high as 1 – 2% at the outﬂow from the Dotson Ice Shelf (DIS; Randall-Goodwin et al., 2015). Retaining the same assumption as three-component mixing, Biddle et al. (2017) adopted Optimum Multiparameter Analysis (OMPA; Tomczak and Large, 1989) to deduce the SMW fraction in the Amundsen Sea. 1 Introduction Another method often adopted is based on the oxygen isotope analysis of water molecules (Östlund and Hut, 1984). This d18O method also assumes three-component mixing but with different components. In Antarctic continental shelves, the components often considered are mCDW, sea ice meltwater (SIM), and meteoric water (MET) (Meredith et al., 2008; Meredith et al., 2013; Randall-Goodwin et al., 2015). The d18O method exploits the fact that MET has a markedly lower d18O level (-20 – -30‰; Jenkins and Jacobs, 2008; Randall-Goodwin et al., 2015) than mCDW (~0‰) and SIM (~2‰). MET may not only include SMW, but also inputs through the surface layer, such as precipitation on sea, surface melt runoff, and ventilated SMW (VMW). Therefore, the MET fraction derived from the d18O method is expected to be larger than the SMW fraction, especially near the surface layer. Antarctic ice shelves lose 2400–2800 Gt of mass every year (Depoorter et al., 2013; Rignot et al., 2013). Ice calving, the falling down of ice chunks from the edge of a glacier, has long been considered an important process responsible for the loss of ice shelves (Jacobs et al., 1992; Depoorter et al., 2013). Along with ice calving, recent studies have suggested that basal melting, i.e., melting at depth by warm seawater, is equally responsible for the loss of ice shelves. Studies have estimated that 1300–1500 Gt of ice shelves disappear every year as a result of basal melting (Depoorter et al., 2013; Rignot et al., 2013). g Basal melting introduces freshwater, known as the submarine meltwater (SMW), which inﬂuences the physical and biological processes taking place in high-latitude seas. Cape et al. (2019), for example, argued that SMW is a driving force that contributes to the pumping of nutrient-rich subsurface waters to surface layers, resulting in an increase in primary production. This phenomenon may be an important mechanism for iron supply and might explain the good correlation between the basal melting rate and primary production found in polynyas around Antarctica (Arrigo et al., 2015). Furthermore, the spread of additional SMW may initiate changes in ocean circulation. Recent modeling studies have argued that more than half of the SMW produced in the Amundsen Sea ﬂows westwards and freshens the Ross Sea (Nakayama et al., 2014; Nakayama et al., 2017). The freshening may impede the formation of Antarctic Bottom Water (AABW), leading to changes in global heat distribution. OPEN ACCESS 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 Marine Science 01 frontiersin.org Shin et al. 10.3389/fmars.2022.951471 10.3389/fmars.2022.951471 1 Introduction Because OMPA is an overdetermined system, it requires at least as many tracers as the assumed components, in addition to the mass conservation equation; the three tracers used were temperature, salinity, and oxygen. This method, as pointed out by Biddle et al. (2017), may produce a false meltwater signature owing to biological production and consumption of oxygen. frontiersin.org 2.1 Sample collection and analysis Hydrographic observations and sample collection for noble gas analysis were conducted onboard IBRV Araon in January 2011 along the DGT and near the Dotson and Getz ice shelves (DIS and GIS) in the Amundsen Sea (Figure 1). The temperature, salinity, and dissolved oxygen were measured using the CTD package (SBE911plus and SBE43). Approximately 40 g of seawater samples were collected using Niskin bottles mounted on the CTD/Rosette system into Cu tubes with an ID of 16 mm for noble gas analysis, and the tubes were crimp-sealed using hydraulic equipment. Noble Frontiers in Marine Science 02 frontiersin.org frontiersin.org Shin et al. 10.3389/fmars.2022.951471 FIGURE 1 Map depicting sampling locations. The Amundsen Sea (AS) is situated between the Ross and Bellingshausen Seas (BS in the inset). Water samples for the noble gas analysis were collected along the Dotson-Getz Trough (DGT), which links modiﬁed circumpolar deep water (mCDW) to the Dotson and Getz Ice Shelves (DIS and GIS). Red circles indicate sampling locations. Black polygons depict the areas considered for calculating the volume of the Dotson-Getz Trough for the melting rate calculation as mentioned in Section 3.5. FIGURE 1 Map depicting sampling locations. The Amundsen Sea (AS) is situated between the Ross and Bellingshausen Seas (BS in the inset). Water samples for the noble gas analysis were collected along the Dotson-Getz Trough (DGT), which links modiﬁed circumpolar deep water (mCDW) to the Dotson and Getz Ice Shelves (DIS and GIS). Red circles indicate sampling locations. Black polygons depict the areas considered for calculating the volume of the Dotson-Getz Trough for the melting rate calculation as mentioned in Section 3.5. where Ceq is the concentration of each noble gas at equilibrium with the atmosphere (Wood and Caputi, 1966; Weiss, 1971; Weiss and Kyser, 1978; Hamme and Emerson, 2004). gases in the Cu tubes were extracted under vacuum and analyzed at the Isotope Geochemistry Facility, Woods Hole Oceanographic Institute (Massachusetts, USA). The precisions for the ﬁve noble gases (He, Ne, Ar, Kr, and Xe) were better than 1%, and the accuracy was approximately 1% (Stanley et al., 2009). Isotopic ratios of oxygen in seawater, d18O, were analyzed at the Leibniz Laboratory (Kiel, Germany) using the CO2-H2O equilibration technique on a Finnigan Gas Bench II system coupled to a Finnigan DeltaPlus XL. The overall precision of d18O measurements was ± 0.03‰ or better. 3.1 Vertical distributions of the tracers The vertical proﬁles of the potential temperature and salinity revealed the major water masses (mCDW, WW, and AASW) in the Amundsen Sea (Figure 2A, B). The warm and saline mCDW (q ≈0.73°C and S ≈34.6 psu) occupied the deep layer (>400 m) in the DGT. WW with a relatively low temperature and salinity (q ≈-1.8°C and S ≈34.1 psu) was observed above the mCDW at depths of <300 m. Higher temperatures and salinities were observed at mid-depths (200 – 400 m) in front of the DIS at Station 10. The increase in temperatures and salinities has been ascribed to active vertical mixing between the mCDW and WW near the DIS (Kim et al., 2016b). AASW, with a higher temperature and lower salinity than the WW, resided in the surface layer. Increased solar radiation and ice melting in the summer contributed to the formation of the AASW. (3) where f is the fraction of the source water and the subscript obs stands for the observed values. As tracers have different value ranges, they were normalized with the mean and standard deviation values of each tracer. The tracers were further weighted to account for the different resolving powers of the source waters and reliabilities, according to Beaird et al. (2015) (Table 1). We set the weight of the unity-sum constraint to 100, which is similar to that of the potential temperature. To assess the uncertainty of the NG-OMPA, we performed a Monte Carlo simulation with 10,000 sets of perturbed tracer properties within the uncertainties of the tracers presented in Table S1. The average and standard deviation of 10,000 solutions were The saturation anomalies of light noble gases (DHe and DNe) were higher at depths of 400 – 600 m along the DGT than in the upper and deep layers (Figure 2D, E). The maximum DNe gradually decreased from 14% in front of the DIS to 9% at Station 7, accompanying an increase in the distance from the DIS. This phenomenon has been attributed to mixing with ambient water as glacial meltwater spreads away from the DIS (Kim et al., 2016b). In the upper layer, the saturation anomalies decreased with an increase in gas exchange with the atmosphere. DHe was higher than DNe at similar depths (e.g., 24% vs. 14% at Station 10). 2.2 Optimum multiparameter analysis Optimum multiparameter analysis assumes that the distribution of tracers is the result of conservative linear mixing of predeﬁned source waters. The analysis requires at least the same number of tracers as that of source waters to ﬁnd optimal solutions for the fractions of source waters with an overdetermined set of simultaneous equations (Tomczak and Large, 1989). The optimal solutions are determined as the fractions that minimize the residual (R in Eq. 3) between observations and the linear combination of source waters with non-negative constraints on the fractions. We used ﬁve noble gases and a He isotope (3He), in addition to potential temperature and salinity, for the tracers of the OMPA. The isotopic ratio of He (d 3He) reported in this study is deﬁned as d 3He( % ) = 3He=4He RA −1 100 (1) (1) where RA is the helium isotopic ratio (3He/4He) in the atmosphere (1.384 x 10-6; Clarke et al., 1976). The saturation anomaly of each noble gases (DC) was calculated from the equation Two potential source waters at surface layer (AASW and VMW) were added to mCDW, WW, and SMW, often assumed source waters in the CT method (See Section 3.2 for the deﬁnitions of the source waters). The system of linear DC( % ) = C Ceq −1 ! 100, (2) (2) Frontiers in Marine Science 03 Frontiers in Marine Science 03 frontiersin.org 03 Frontiers in Marine Science Frontiers in Marine Science 10.3389/fmars.2022.951471 Shin et al. equations of the tracers is represented as equations of the tracers is represented as chosen as the ﬁnal solution and error of the NG-OMPA. The SMW and meteoric fractions (SMW plus VMW) calculated using the NG-OMPA method were compared with those obtained from the conventional CT method and the MET from the d18O method. A summary of the CT and d18O methods is provided in the Supplementary Material. 2.2 Optimum multiparameter analysis TmCDW TWW TAASW TSMW TVMW SmCDW SWW SAASW SSMW SVMW 3HemCDW 3HeWW 3HeAASW 3HeSMW 3HeVMW 4HemCDW 4HeWW 4HeAASW 4HeSMW 4HeVMW NemCDW NeWW NeAASW NeSMW NeVMW ARmCDW ArWW ArAASW ArSMW ArVMW KrmCDW KrWW KrAASW KrSMW KrVMW XemCDW XeWW XeAASW XeSMW XeVMW 1 1 1 1 1 0 B B B B B B B B B B B B B B B B B B B B @ 1 C C C C C C C C C C C C C C C C C C C C A fmCDW fWW fAASW fSMW fVMW 0 B B B B B B B B @ 1 C C C C C C C C A = Tobs Sobs 3Heobs 4Heobs Neobs Arobs Krobs Xeobs 1 0 B B B B B B B B B B B B B B B B B B B B @ 1 C C C C C C C C C C C C C C C C C C C C A + R, (3) The superscripts a, b, and c indicate tracers and source waters used in the NG−OMPA, CT, and d18O methods, respectively. The superscripts a, b, and c indicate tracers and source waters used in the NG−OMPA, CT, and d18O methods, respectively. 3.1 Vertical distributions of the tracers The elevated He concentration accompanied by a positive d3He indicates additional input of mantle He from the TABLE 1 Properties of the source waters for water mass analysis. Gas concentration units are mol kg-1. Sourcewater qa,b (°C) Sa,b,c (psu) 3Hea (10-15) 4Hea (10-9) Nea (10-9) Ara (10-5) Kra (10-9) Xea (10-10) d18O (‰) AASWa -0.996 33.81 2.432 1.790 8.208 1.763 4.391 6.879 VMWa 0 0 2.917 2.186 1.009 2.234 5.566 9.072 SMWa,b -95 0 37.22 26.90 93.33 4.087 5.852 4.466 pWWa,b -1.823 34.12 2.843 2.037 8.966 1.739 4.189 6.378 -0.72 mCDWa,b,c 0.790 34.60 2.968 1.974 8.472 1.653 3.944 5.912 -0.32 METc 0 -25 ± 5 SIMc 7 2.1 Weight 112 24340 7.9 7.9 7.5 2.1 1.0 6.5 The superscripts a, b, and c indicate tracers and source waters used in the NG−OMPA, CT, and d18O methods, respectively. 04 Frontiers in Marine Science frontiersin.org Shin et al. 10.3389/fmars.2022.951471 FIGURE 2 Vertical proﬁles of the tracers used for the NG-OMPA. (A) Potential temperature, (B) salinity, and (C) isotopic ratio of helium deﬁned as d3He = [(3He/4He) sample/RA - 1]× 100,where (3He/4He) sample is the measured ratio of a sample and RA is the isotopic ratio of the atmosphere. (D−H) Saturation anomalies of the noble gases, deﬁned as (Csample/Csat - 1)100, where Csample and Csat are the measured and solubility equilibrium concentrations, respectively. FIGURE 2 Vertical proﬁles of the tracers used for the NG-OMPA. (A) Potential temperature, (B) salinity, and (C) isotopic ratio of helium deﬁned as d3He = [(3He/4He) sample/RA - 1]× 100,where (3He/4He) sample is the measured ratio of a sample and RA is the isotopic ratio of the atmosphere. (D−H) Saturation anomalies of the noble gases, deﬁned as (Csample/Csat - 1)100, where Csample and Csat are the measured and solubility equilibrium concentrations, respectively. submarine hydrothermal system. The Paciﬁc sector of the Southern Ocean is known to have a larger mantle He signal because the deep water accumulates hydrothermal helium during meridional overturning circulation, and the Paciﬁc has higher mid-ocean ridge spreading rates (Jenkins, 2020). The highest d3He values in the mCDW layer were also consistent with the input of hydrothermal helium with high 3He/4He ratios (Figure 2C). Crustal He is another source of excess He. Crustal He accumulates in the bedrocks of glaciers by a-decay of U/Th- series radionuclides and has 3He/4He ratio markedly lower than the atmosphere (Craig and Scarsi, 1997). frontiersin.org 3.2.1 Antarctic surface water The AASW, present during spring and summer in the surface layer, has a wide range of temperatures and salinities owing to different degrees of solar insolation and ice melting. We set the temperature and salinity of the AASW to the average of the observed values in the upper 50 m at stations without sea ice (Stations 8 – 10). The concentrations of the noble gases were set to the solubility equilibrium values at the temperature and salinity of the AASW (Wood and Caputi, 1966; Weiss, 1971; Weiss and Kyser, 1978; Hamme and Emerson, 2004). The surface samples plotted close to the mixing line between pWW and AASW (Figure 3B). As the uncertainties in temperature and salinity propagate into the solubility concentrations of gases, the noble gas concentrations in the AASW were perturbed in the range of concentrations deﬁned by the standard deviations in the temperature and salinity of the AASW observed in the Monte Carlo simulation (Table S1). Because both the CT and d18O methods use a pair of tracers, they are bound to represent only three source waters, lacking the representation of potential source waters in the upper layer. In Figure 3A, some of the observed values lie outside the triangle created by the mixing lines among the three often-presumed source waters: mCDW, WW (pure WW, pWW), and SMW. The outliers are more clearly visible in the He vs. salinity plot (Figure 3B, and S1 for other noble gases). Most of the samples from depths <200 m plotted close to binary mixing between pWW and AASW. This ﬁnding indicates that the addition of AASW as a source water and noble gases as tracers can improve the estimation of SMW near the surface layer. We also considered the VMW, in addition to the usual mCDW, WW, and SMW, as a source water at the surface. VMW may largely represent ventilated meltwater either from upwelled SMW or from laterally transported meltwater at the surface, both of which spend sufﬁcient time (>1 – 2 weeks) to equilibrate with the atmosphere. It is noted that the SIM was not considered as a source water in the NG-OMPA. Although the SIM is considered as a source water in the d18O method, its contribution to the Amundsen Sea is negative, indicating the dominance of sea ice formation rather than of sea ice melting (Randall-Goodwin et al., 2015; Bett et al., 2020). 3.1 Vertical distributions of the tracers Although the crustal He is known to be incorporated into glacial meltwater in the Greenland (Beaird et al., 2015; Huhn et al., 2021), this component should be negligible in the Amundsen Sea given that 3He/4He ratios in the Amundsen Sea are explained by the the addition of SMW, which has “air-like” isotopic ratio, to the CDW (Refer to Figure 4 of Kim et al., 2016b). We note that the He (3He and 4He) and Ne data were the same as those reported by Kim et al. (2016b). Although light noble gases showed supersaturation in the DGT, the heavy noble gases (Ar, Kr, and Xe) were undersaturated at all depths (Figure 2F–H). The undersaturation of heavy noble gases is attributed to rapid cooling during water formation (Hamme and Severinghaus, 2007). Because heavy noble gases are more soluble and temperature-sensitive than the light gases, if newly formed water does not remain on the surface long enough for the gases to re-equilibrate with the atmosphere, the heavy gases will remain undersaturated. The lowest saturation anomalies observed at the depths of the WW layer (200 – 500 m) further indicate that sea ice cover obstructs gas exchange (Rutgers van der Loeff et al., 2014) Frontiers in Marine Science 05 frontiersin.org frontiersin.org 10.3389/fmars.2022.951471 Shin et al. during WW-forming vertical mixing, resulting in intensiﬁed undersaturation of the heavy noble gases. In the surface layer above the WW layer, the saturation anomalies increased owing to the temperature increase induced by solar irradiation and enhanced air−sea gas exchange by sea ice melting in summer. negativity when using the NG-OMPA method does not allow for explicit inclusion of the SIM, the effects of sea ice melting in summer and formation in winter were implicitly incorporated into the properties of pWW and AASW, respectively, in the NG- OMPA. The tracer properties of the ﬁve source waters are deﬁned in the following subsections and summarized in Table 1. 3.2.1 Antarctic surface water Although the constraint of non- 3.2.4 ‘Pure’ winter water WW, which is replenished every winter by heat loss to the atmosphere and deep mixing, is colder and fresher than the mCDW. Because the observed WW in summer often contains a small fraction of other source waters, we deﬁned ‘pure’ WW (pWW) according to Jenkins et al. (2018). Brieﬂy, the temperature and salinity were set to the point where the extension of the mixing line between the mCDW and the observed WW met the freezing line in the T−S diagram (Figure 3A). The properties of pWW should be determined from each year’s measurements to properly reﬂect the inter-annual variability of water masses in the Amundsen Sea (Dutrieux et al., 2014; Jenkins et al., 2018). The difference in temperature and salinity properties between the observed WW and pWW indicates that the observed WW was produced by the addition of 0.6% mCDW to pWW. The noble gas concentrations in the pWW were calculated from those in the mCDW and the observed WW, considering the mixed fractions of the water masses. The noble gas concentrations in the observed WW were set to the average values observed at depths of 200 – 350 m at Stations 7 and 8, where sea ice appeared to stabilize the upper water column. Notable fractions of VMW (0.5 – 0.9%), along with the dominant AASW, existed in the surface layer in the DGT. The penetration of VMW was more pronounced at Station 10, with a VMW fraction > 0.4% as deep as 200 m. This higher VMW fraction may be attributed to the direct input of subaerial meltwater from the adjacent DIS, ventilated SMW, and/or the advection of upstream meltwater laden on the westward-ﬂowing coastal currents (Nakayama et al., 2014; St-Laurent et al., 2017). Previous modeling studies have predicted surface meltwater fractions in the range of 0.4 – 1.5% in the DGT ( (Kimura et al., 2017; St-Laurent et al., 2019) consistent with our total SMW and VMW fractions of approximately 1% (Figures 5, 6). The persistently high VMW fractions north of the DIS (Stations 7 and 8) may be ascribed to the clockwise circulation of meltwater around the edges of the DGT (St-Laurent et al., 2017). Alternatively, VMW may result from the melting of icebergs drifting from the east of the trough, which can be an important source of freshwater at the surface (Mazur et al., 2019; Bett et al., 2020). 3.2.5 Submarine meltwater The temperature of the SMW was set to an effective potential temperature of -95°C, which takes account of the heats required for ice to warm to freezing point, for phase change from ice to water, and for mixing with ambient water (Jenkins, 1999). The salinity of SMW was set to 0 psu. The noble gas concentrations were determined as the concentrations in the air bubbles (0.11 cm3 in glacial ice; Martinerie et al., 1992), assuming complete dissolution of the air under high pressure at the depth of basal melting (Schlosser, 1986). 3.2.3 Modiﬁed circumpolar deep water The temperature and salinity of the mCDW were determined as the intersection between the SMW−mCDW and WW−mCDW mixing lines that enveloped the values observed in 2011 (Jenkins et al., 2018). To account for potential sampling bias, the uncertainties in temperature and salinity were set to include the mCDW properties of previous studies (Randall-Goodwin et al., 2015; Jenkins et al., 2018). The noble gas concentrations were set to the averages of the samples with the highest temperature and salinity found at depths > 800 m at Stations 9–11. The SMW fractions were most prominent at Station 10 in front of the DIS, with the highest values at depths of 400 – 600 m (>0.6%). These depths are close to the underside of the DIS at ~400 m depth (Gourmelen et al., 2017). The SMW was observed to spread out along the approximately 500 m-deep layer between mCDW and WW. On reaching Station 7, near the continental shelf break, the SMW fraction decreased to 0.2%. This result is in good agreement with previous studies that reported the spreading of SMW beyond the continental shelf break (Randall-Goodwin et al., 2015; Kim et al., 2016b). 3.2.2 Ventilated meltwater VMW potentially represents all freshwater in solubility equilibrium with the atmosphere, including the surface melt of icebergs, ice shelves, and direct precipitation to the ocean. These freshwaters may be produced in the DGT and transported from the upstream such as Pine Island Bay in the Amundsen Sea, and the Bellingshausen Sea (Nakayama et al., 2014; St-Laurent et al., FIGURE 3 Bivariate plots of (A) potential temperature−salinity and (B) 4He−salinity. The circles, triangles, and stars represent samples collected in the DGT. Many samples in the shallow layer (≤200 m) plot above the line of binary mixing between mCDW and SMW in (A). These samples plot along the line of binary mixing between pWW and AASW in (B), suggesting that AASW is necessary as an additional source water for NG-OMPA. The orange and red shades represent the variabilities of AASW and mCDW, respectively. FIGURE 3 Bivariate plots of (A) potential temperature−salinity and (B) 4He−salinity. The circles, triangles, and stars represent samples collected in the DGT. Many samples in the shallow layer (≤200 m) plot above the line of binary mixing between mCDW and SMW in (A). These samples plot along the line of binary mixing between pWW and AASW in (B), suggesting that AASW is necessary as an additional source water for NG-OMPA. The orange and red shades represent the variabilities of AASW and mCDW, respectively. 06 Frontiers in Marine Science frontiersin.org Shin et al. 10.3389/fmars.2022.951471 3.2.4 ‘Pure’ winter water The proﬁles of the tracers reconstructed from the NG- OMPA predicted fractions of the source waters were comparable to the observed proﬁles of the tracers (Figures S2 and S3); the proﬁles of temperature and salinity with higher weightings for optimization were virtually identical to the observed proﬁles. 3.3 Distributions of source waters 2019). The VMW also includes ventilated SMW, which has reached the surface after the melting of ice shelves and icebergs at depth and spent some time (>1 – 2 weeks) equilibrating with the atmosphere. We set the temperature and salinity of the VMW to 0°C and 0 psu, respectively. The noble gas concentrations were set to the solubility equilibrium values at the given temperature and salinity. The deep layer (>500 m) along the DGT appeared to be occupied by >80% mCDW (Figure 4). This is consistent with previous observations indicating persistent inﬂow of mCDW along the DGT (Wåhlin et al., 2010; Randall-Goodwin et al., 2015; Kim et al., 2016b). The mid-depth layer above the mCDW was occupied by WW. The partially ice-covered areas (near Station 7 with an ice fraction of approximately ~50%) retained large fractions of WW (>70%), even at the surface. In contrast, the surface layer within the Amundsen Sea Polynya (Stations 9 and 10) was largely occupied by AASW (>50%). 3.4 Improvement on the estimation of submarine glacial meltwater Both the NG-OMPA and CT methods yielded similar SMW fractions in the mid-depth layer between 400 and 600 m Frontiers in Marine Science 07 frontiersin.org frontiersin.org Shin et al. 10.3389/fmars.2022.951471 A B D E F C FIGURE 4 Fractional contributions of different source waters (A–F) for the section along the Dotson-Getz Trough calculated by the NG-OMPA. Fractions at the water sample locations (circles) are superimposed on the interpolated values. Five noble gases, in addition to temperature and salinity, were used as tracers for the analysis. The values shown are the averages of 10,000 results produced by Monte Carlo simulation. C B A B C D E F D F E FIGURE 4 Fractional contributions of different source waters (A–F) for the section along the Dotson-Getz Trough calculated by the NG-OMPA. Fractions at the water sample locations (circles) are superimposed on the interpolated values. Five noble gases, in addition to temperature and salinity, were used as tracers for the analysis. The values shown are the averages of 10,000 results produced by Monte Carlo simulation. (Figure 5). One notable discrepancy is the gradual decrease in SMW fractions from near the ice shelf to the continental shelf break (0.6% to 0.2%) when employing the NG-OMPA method (SMWOMPA). This decrease was consistent with the fact that the saturation anomalies of Ne (DNe), which are proportional to the meltwater fractions, decreased gradually from the ice shelf to the shelf break (Figure 2E). In contrast, the fractions obtained using the CT method did not vary remarkably, with values ranging from 0.8% to 0.6%, resulting in an overestimation of 0.4% at Station 7. The degrees of deviation from the WW −mCDW mixing line to the SMW property, which is proportional to the meltwater fraction, were not signiﬁcantly different for the water samples at Stations 7 and 10 (Figure 3A). However, the samples from Station 10 plotted further above the WW−mCDW mixing line in the He salinity plot than did those from Station 7 (Figure 3B), suggesting higher SMW fractions. In FIGURE 5 Comparison of SMW fractions calculated by the NG-OMPA and CT methods. Red lines and shaded areas show the averages and the uncertainties of NG-OMPA method. The uncertainties are one standard deviation of 10,000 fractions calculated by Monte Carlo simulation with perturbed values. SMW fractions calculated by the CT method with different pairs of tracers are shown as teal lines. 3.4 Improvement on the estimation of submarine glacial meltwater While previous hydrographic observations have indicated that strong inﬂow and outﬂow existed on the eastern and western sides of the DIS front, a similar along-shelf hydrographic variation indicating strong ﬂows was not found in front of the GIS (Randall-Goodwin et al., 2015; Kim et al., 2016b). The deviation of GMWDNe from SMWOMPA became most pronounced in the layer occupied by WW (100 – 400 m), where the fraction of pWW was high (Figure 4). The conservation of excess Ne in the WW layer corroborates the suggestion that gas exchange at the time of WW formation is restricted by sea ice cover (Rutgers van der Loeff et al., 2014). It is likely that the SMW in the deep layer is produced within the DGT because the Bear Ridge, located on the eastern side of the DGT, rises to <300 m below the sea surface, prohibiting the direct input of upstream glacial meltwater to the deep layer (St- Laurent et al., 2019; Bett et al., 2020). However, it is difﬁcult to specify the origin of the SMW in the upper layer at a depth of approximately 200 m (Figure 4) because westward-ﬂowing coastal currents may carry upstream glacial meltwater to the DGT (Nakayama et al., 2014). We speculate that the coastal currents mostly carry VMW rather than SMW, based on the observations showing that the coastal currents were mostly conﬁned to the surface layer <100 m (Kim et al., 2016a). Additional support for this speculation comes from the fact that upstream glacial meltwater, mainly from the Pine Island Ice Shelf, travels for several months with a mean westward velocity of 0.03 m s-1 (Nakayama et al., 2014) to reach the DGT, providing enough time to equilibrate with the atmosphere. To deduce the basal melting rates of the ice shelves in the DGT, we assumed that the SMW fraction represents meltwater accumulated after the formation of the WW because pWW is included as a source water in the NG-OMPA. Owing to the lack of direct observation, it is difﬁcult to specify when pWW forms. We used a time series of climatological sea ice concentrations (Figure S2) to infer probable months in which the properties of the pWW were set. 3.4 Improvement on the estimation of submarine glacial meltwater The mean fractions were multiplied by the volume of the layers and the resultant SMW amounts were 8.9 km3 and 22.3 km3 for the upper and deep layers, respectively (Table 2). the upper layer, the three SMW fractions estimated using CT method with different pairs of tracers deviated from one other because there was no source water deﬁned to account for atmospheric interaction and additional freshwater sources (Jenkins and Jacobs, 2008). The overall similarity between the fractions obtained by the salinity−O2 pair and the SMWOMPA fractions also supports the plausibility of the NG-OMPA method. Jenkins and Jacobs (2008) argued that the counteracting effects of freshening of salinity and air–sea equilibrium of O2, respectively, fortuitously compensate for each other and produce the most reliable SMW estimates among the three tracer pairs in the surface layer. The SMWOMPA fractions appeared to be smaller than those estimated using the He and Ne concentrations alone (Kim et al., 2016b). The authors attributed the He and Ne excess over the concentrations at a background site located 400 km north of the continental shelf break of the Amundsen Sea to glacial melting. Intrinsically, the SMW fractions by Kim et al. (2016b) represent meltwater added within the DGT as well as those added during the transit of the water mass from off the continental shelf to the trough. If the water mass overwintered in the trough, the fractions would also include meltwater from the previous year, incorporated through WW-forming vertical mixing. In this respect, a slightly higher meltwater fraction estimated from excess Ne over the background concentrations (GMWDNe) than SMWOMPA (Figure 5) is not surprising because the latter represents meltwater addition after the most recent WW formation. The largest discrepancy between GMWDNe and SMWOMPA (0.7% vs. approximately 0% at 500 m at Station 11) was found in front of the GIS (Figure 5). This ﬁnding indicates that the majority of GMWDNe was added to the layer via vertical mixing in winter, while meltwater addition after the formation of WW was negligible near the GIS. The apparently lower basal melting rate may have also resulted from weaker intrusion of mCDW into the cavity of the GIS compared with that in the DIS. 3.4 Improvement on the estimation of submarine glacial meltwater We postulate that the rapid increase in the sea ice concentration in March and April and resultant brine rejection promote pWW formation, the properties of which do not change markedly when sea ice concentrations are close to the maximum (≥90% from June to August). Consequently, the SMW fractions observed in early January 2011 reﬂect the amount of SMW introduced over the past 4 – 7 months, depending on the timing of pWW formation. The basal melting rate was estimated to be in the range of 53 – 94 Gt yr-1 by combining an SMW amount of 31.2 km3 during a duration of 4 – 7 months. If only the amount of SMW in the deep layer is taken into consideration, the melting rate 3.4 Improvement on the estimation of submarine glacial meltwater The circle at the bottom of Station 7 highlights a large discrepancy between the SMW fractions estimated by the NG-OMPA and CT methods. Given the lower DHe and DNe at Station 7 (Figure 2), compared with those at Station 10, it is likely that the SMW fraction at Station 7 is lower than that at Station 10. The fractions of ventilated SMW (VMW) calculated by the NG-OMPA are shown as orange lines. The fractions of glacial meltwater derived from excess Ne are depicted as purple lines. FIGURE 5 Comparison of SMW fractions calculated by the NG-OMPA and CT methods. Red lines and shaded areas show the averages and the uncertainties of NG-OMPA method. The uncertainties are one standard deviation of 10,000 fractions calculated by Monte Carlo simulation with perturbed values. SMW fractions calculated by the CT method with different pairs of tracers are shown as teal lines. The circle at the bottom of Station 7 highlights a large discrepancy between the SMW fractions estimated by the NG-OMPA and CT methods. Given the lower DHe and DNe at Station 7 (Figure 2), compared with those at Station 10, it is likely that the SMW fraction at Station 7 is lower than that at Station 10. The fractions of ventilated SMW (VMW) calculated by the NG-OMPA are shown as orange lines. The fractions of glacial meltwater derived from excess Ne are depicted as purple lines. 08 Frontiers in Marine Science frontiersin.org 10.3389/fmars.2022.951471 Shin et al. and deep layers of the water column by integrating the vertical proﬁle of the SMW fractions at each site with the corresponding depth intervals. The boundary between the two layers was set to 300 m according to St-Laurent et al. (2017). The researchers regarded the upper layer as subject to deep convection that replenished it with nutrients and dissolved iron in winter. Furthermore, they argued that iron, which supports high primary production in summer, is mainly supplied to the upper layer by buoyancy-driven overturning circulation, the so-called “meltwater pump” (St-Laurent et al., 2017; St- Laurent et al., 2019). The mean fractions of the upper and deep layers at each sampling site were assumed to be representative of the water column adjacent to the site. The eastern and western boundaries of the water columns roughly followed the ridges ﬂanking the DGT (see the polygons in Figure 1). frontiersin.org 3.5 Calculation of basal melting rate from the SMW fractions To convert the SMW fractions to the amount of meltwater, we calculated the mean fractions at each station for the upper Frontiers in Marine Science 09 frontiersin.org Shin et al. 10.3389/fmars.2022.951471 becomes 38 – 67 Gt yr-1. However, we favor the estimates that include SMW amounts from both the upper and deep layers because the depths of approximately 200 m, where the SMW fraction in the upper layer is high, are similar to the depths with a strong outﬂow of melt-laden overturned water from the DIS (Randall-Goodwin et al., 2015; Kim et al., 2016b). This suggests that 29% (8.9 km3 out of 31.2 km3) of the SMW produced in the DGT is upwelled to the upper layer. This upwelled SMW is eventually brought to the surface layer by deep convection in winter and replenishes the surface layer with iron, which is in turn available for primary production in the subsequent summer. The estimates of the basal melting rate were mostly ascribed to the DIS because the SMW amount of 1.9 km3 in the water column adjacent to the GIS (Figure 1) was only 6% of the total amount of SMW. either as falling snow or melts of glacial ice. While SMW dominated the sum of the SMW and VMW fractions (METOMPA) in the deep layer, VMW became the major component of METOMPA at the depths <200 m (Figures 5, 6 and Table 2). The METOMPA fractions did not exceed 0.6%, except in the upper layer where the values were as high as 1.5%. It is intriguing that the METOMPA fractions were much less than the meteoric water fraction estimated by the d18O (MET18O; Meredith et al., 2008; Randall-Goodwin et al., 2015). The latter fractions were obtained by assuming the often-adopted three source waters: MET, SIM, and mCDW (refer to Supplementary Material for the method). Most of the fractions of MET18O fell within a narrow range of 1.5 – 2% from the surface to >400 m depths (Figure 6). We speculate that the large discrepancy between METOMPA and MET18O results from the fact that the d18O method calculates meteoric fractions accumulated relative to the mCDW. In contrast, the NG-OMPA, which includes pWW as a source water, estimates the meteoric water accumulated since the previous pWW formation. 3.5 Calculation of basal melting rate from the SMW fractions This speculation seems plausible, given the fact that if mCDW is replaced with pWW, the d18O method gives meteoric water fractions similar to METOMPA (MET18O (pWW) in Figure 6) This also suggests that the inventory of meteoric water in the DGT (MET18O) is much larger than the amount of meteoric water accumulated since the previous pWW formation (METOMPA). The four times larger inventory (Table 2) may be ascribed to the combined effects of high meteoric water content at the surface and periodic delivery of the surface water to the deep layer by deep convection in winter. The negative SIM fraction from the d18O method (SIM18O in Figure 6) implies that brine produced by sea ice formation is delivered to the deep layer in winter. Westward- ﬂowing coastal currents seem to play an important role in keeping the surface layer rich in meteoric water (1 – 2%) by Although our estimates have a relatively large range owing to the uncertain timing of pWW formation, the melting rates are in the range of previous estimates for the DIS. For example, Jenkins et al. (2018) demonstrated a large temporal variation in the melting rate in the range of 20 – 90 Gt yr-1 from 16 years of CTD observations in front of the DIS. They estimated a melting rate of 55 ± 15 Gt yr-1 in 2011. Similarly, Rignot et al. (2013) estimated a melting rate of 45 ± 4 Gt yr-1 using 2003 – 2008 satellite data. Because melts near the surface are subject to gas exchange and classiﬁed as VMW in the NG-OMPA, the basal melting rate calculated from SMW alone should be considered a lower bound. 3.6 Comparison of meteoric water fractions from the NG-OMPA and d18O methods Both the SMW and VMW from the NG-OMPA constitute meteoric water, i.e., freshwater originated from the atmosphere, FIGURE 6 Comparison of MET fractions calculated by the NG-OMPA (METOMPA = SMW + VMW) and d18O methods. The MET fractions calculated using the d18O method with mCDW as a source water, in addition to SIM and MET, are shown in black. If pWW, instead of mCDW, is considered as a source water, the differences in the fractions calculated by the d18O (shown as orange lines) and NG-OMPA methods (shown as red lines) become smaller. SIM18O are the fraction of sea ice meltwater calculated using d18O method. Negative fractions of SIM18O indicate sea ice formation. FIGURE 6 Comparison of MET fractions calculated by the NG-OMPA (METOMPA = SMW + VMW) and d18O methods. The MET fractions calculated using the d18O method with mCDW as a source water, in addition to SIM and MET, are shown in black. If pWW, instead of mCDW, is considered as a source water, the differences in the fractions calculated by the d18O (shown as orange lines) and NG-OMPA methods (shown as red lines) become smaller. SIM18O are the fraction of sea ice meltwater calculated using d18O method. Negative fractions of SIM18O indicate sea ice formation. 10 Frontiers in Marine Science frontiersin.org Shin et al. 10.3389/fmars.2022.951471 TABLE 2 Meltwater in the upper (<300 m) and deep layers of the Dotson-Getz Trough. SMW and VMW stand for submarine meltwater and ventilated meltwater respectively ater in the upper (<300 m) and deep layers of the Dotson-Getz Trough. SMW and VMW stand for submarine meltwater and water, respectively. p y Volume of water (km3) SMW (km3) VMW (km3) METOMPA (km3) MET18O (km3) Upper 11800 8.9 48.7 57.6 254.9 Deep 13700 22.3 2.6 24.9 83.4 Total 25500 31.2 51.3 82.5 338.3 METOMPA and MET18O are the amounts of meteoric water calculated by the NG-OMPA and d18O methods, respectively. carrying upstream meltwater. The meltwater laden on coastal currents ﬂows along the ice shelves year-round, and part of it spreads along the edges of the DGT, forming a clockwise circulation (St-Laurent et al., 2017). Assuming a volume transport of 4 – 5 Sv for the coastal current (St-Laurent et al., 2019) and a volume of 1.18 × 104 km3 for the seawater in the upper layer (Table 2), the upper layer of the DGT is replaced every month with upstream water. Data availability statement The datasets for this study can be found in the Korea Polar Data Center (http://kpdc.kopri.re.kr) with the data ID of KOPRI- KPDC-00001633. Funding This work was supported by grant from Korea Polar Research Institute PE22110 and PE21140 funded by the Ministry of Oceans and Fisheries. DS and DH were partially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2020R1A2C1009440). The meteoric fractions obtained from the NG-OMPA, assumed to be the sum of SMW and VMW, were four-fold lower than those obtained using the d18O method with mCDW as one of the source waters. While the meteoric fractions determined using NG-OMPA represent locally produced fractions since the previous WW formation, the fraction from the d18O method appeared to represent the amount of meteoric water accumulated over multiple years, which was likely strongly inﬂuenced by upstream meltwater. To better understand the distribution of meteoric water and its potential impact on bottom water formation in downstream regions, it is 3.6 Comparison of meteoric water fractions from the NG-OMPA and d18O methods Although it is often implicitly assumed that MET is proportional to the melting rate of adjacent ice shelves, it appears that MET18O represents accumulation relative to mCDW, integrating meltwaters from a larger space and time compared with SMW estimated using CT or NG– OMPA methods. necessary to collect information on meltwater transport to and from the upper layer, under the strong inﬂuence of westward- ﬂowing coastal currents, and the propagation of surface meltwater to the deep layer by vertical mixing in winter. Thus, separation of VMW from SMW using NG-OMPA and comparison with MET from the d18Omethod is an important tool for distinguishing upstream meltwater from locally produced waters. Author contributions In this study, noble gases were used as additional tracers in the OMPA method to separate ventilated and submarine meltwaters in the Dotson-Getz Trough in the Amundsen Sea.The increased number of tracers provided better constraints on the distribution of glacial meltwater on the surface. We demonstrated that noble gases are an invaluable set of tools that can be used for verifying and correcting the meltwater distributions depicted using the CT method. Clear identiﬁcation of SMW and VMW allowed us to provide a reasonable basal melting rate of 53 – 94 Gt yr-1 for ice shelves in the DGT. Information on the timing of winter water formation is important for reducing the uncertainty regarding the melting rate. This information may be obtained from overwinter time-series observations of the physical and chemical tracers used in the NG-OMPA. DH and TR contributed to conception and design of the study. 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https://openalex.org/W4321790127	https://researchonline.jcu.edu.au/77792/1/Sheel%20et%20al.%20%282023%29.pdf	English	null	Parents’ Evaluation of Developmental Status and Strength and Difficulties Questionnaire as Screening Measures for Children in India: A Scoping Review	Pediatric Reports	2,023	cc-by	14,732	Review Parents’ Evaluation of Developmental Status and Strength and Difﬁculties Questionnaire as Screening Measures for Children in India: A Scoping Review Hina Sheel , Lidia Suárez and Nigel V. Marsh * School of Social and Health Sciences, James Cook University, 149 Sims Drive, Singapore 387380, Singapore * Correspondence: nigel.marsh@jcu.edu.au Abstract: Due to the limited availability of suitable measures, screening children for developmen- tal delays and social–emotional learning has long been a challenge in India. This scoping review examined the use of the Parents’ Evaluation of Developmental Status (PEDS), PEDS: Developmen- tal Milestones (PEDS:DM), and the Strength and Difﬁculties Questionnaire (SDQ) with children (<13 years old) in India. The scoping review was conducted following the Joanna Briggs Institute Protocol to identify primary research studies that examined the use of the PEDS, PEDS:DM, and SDQ in India between 1990 and 2020. A total of seven studies for the PEDS and eight studies for the SDQ were identiﬁed for inclusion in the review. There were no studies using the PEDS:DM. Two empirical studies used the PEDS, while seven empirical studies used the SDQ. This review represents the ﬁrst step in understanding the use of screening tools with children in India. Keywords: PEDS; SDQ; screening; children; India; scoping review Developmental disability (DD) is a broad spectrum of impairments or a lack of devel- opmental features appropriate to a child’s age and vital for their growth [1]. DD is usually present at birth and negatively impacts the individual’s physical, intellectual, and/or social development. DD results in impairments in the ability to learn, reason, and solve problems, and it also impairs adaptive behaviour, which consists of social and life skills [2]. The Diagnostic and Statistical Manual of Mental Disorders, 5th edition, text revision (DSM-5-TR) characterised DD as an Intellectual Developmental Disorder (IDD), where an individual lacks in general mental abilities and adaptive functioning [3]. The International Classiﬁcation of Diseases, Eleventh Revision (ICD-11) considered DD as a neurodevelopmental disorder that arises during the developmental period and involves difﬁculties in intellectual, motor, language, or social functions [4]. Citation: Sheel, H.; Suárez, L.; Marsh, N.V. Parents’ Evaluation of Developmental Status and Strength and Difﬁculties Questionnaire as Screening Measures for Children in India: A Scoping Review. Pediatr. Rep. 2023, 15, 175–196. https://doi.org/ 10.3390/pediatric15010014 Academic Editor: Sabrina Bonichini The target population included new-borns, children in Anganwadi centres (rural childcare centres across India), and government schools [13]. However, the annual progress report of the scheme for 2018–2019 provided scant information on the tools used for screening purposes. Mukherjee et al. [11] concluded that the number of children identiﬁed for delay and disability has increased since the inception of the scheme. However, some states, such as Maharashtra and Odisha, faced issues with implementation, infrastructure constraints, and limited resources [14]. Furthermore, the scheme does not cater to private schools in India, which constitute 49% of children in urban areas and 21% in rural areas [15]. Developmental screening “is a brief assessment procedure designed to identify chil- dren who should receive a more intensive diagnosis or assessment” [16]. Developmental surveillance monitors the child’s progress by gathering information on the child’s devel- opment from multiple sources and determining whether the rate and extent of a child’s development elicits concerns [17]. The World Health Organization (WHO) has emphasised the importance of screening children for any form of disability, explicitly highlighting the relevance of interventions that promote young children’s development [18]. Screening tools developed in India, such as the Baroda Development Screening Test (BDST), Developmental Assessment Scale for Indian Infants (DASII), and Trivandrum Developmental Screening Chart (TDSC), are linguistically and culturally reasonable. Nonetheless, their psychometric properties are suboptimal, and their use has been restricted to a speciﬁc population given that health professionals initially developed them for community services [10,19]. Most low- and medium-income countries (LMIC) use tools developed in Western countries to screen children for DD and SEL. However, there are three main limitations to using these screening tools. First, most screening tools measuring DD in children aged 0–8 years are developed in Western and high-income countries. They require extensive training, which is not readily available in LMIC due to limited funds to purchase tools and training costs [20]. Second, tools developed in Western countries lack psychometri- cally valid translations to use in other cultures [21,22]. Third, most screening tools are copyrighted and require permission to translate them into other languages for schools and clinics to use; this is often expensive [23]. Screening tools that have been adapted and translated for use in LMIC include the Bayley Scale of Infant and Toddler Developmental Screening test (BSITDS; ref. [24]), Ages and Stages Questionnaire (ASQ; ref. [25]), Guide for Monitoring Child Development (GMCD; ref. Academic Editor: Sabrina Bonichini Received: 27 December 2022 Revised: 11 February 2023 Accepted: 13 February 2023 Published: 24 February 2023 g g Social–emotional learning (SEL) is a child’s ability to understand themselves and others, regulate emotions and attention, and engage with others [5]. Individuals with DD may have difﬁculties with social relationships when compared to neurotypical peers and have differences in their reading of neurotypical nonverbal and subtle social cues [6]. Therefore, they usually have impairments in SEL too. In India, 2.5–3.4% of children had various developmental problems diagnosed us- ing screening tools. The most common forms were developmental delay, speech delay, global delay, gross motor delay, and hearing impairment [7]. In addition, in India, a review of recent studies showed that the prevalence of mental health problems in school- going children varies from 6.33% to 43.1%. Speciﬁcally, the prevalence of behavioural and emotional problems among orphans and other vulnerable children ranges from 18.3% to 64.53%. In children with typical development, it was reported to range between 8.7% and 18.7% [8]. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/pediatrrep Pediatr. Rep. 2023, 15, 175–196. https://doi.org/10.3390/pediatric15010014 Pediatr. Rep. 2023, 15 176 The recent research literature in India has revealed multiple issues within universal developmental and SEL surveillance and screening [9,10]. To begin with, parents are un- aware that screening services exist, nor are they aware of why those services are necessary. Health care is given priority only when there is an acute illness. Furthermore, the popu- lation of doctors who serve the needs of Indian children is heterogeneous, with varying skills. If parents express concerns, they often receive inaccurate information without proper evaluation [10]. Postgraduate paediatric courses in India lack formal training in develop- mental and SEL screening and assessment [10]. Moreover, in India, paediatricians make clinical judgments based on unstructured probing of developmental milestones, and India needs more developmental paediatricians [10,11]. In 2013, the Indian government launched the Rashtriya Bal Swasthya Karyakram, also known as the ‘Child Health Screening and Early Intervention Services scheme’, which caters speciﬁcally to government schools [12]. The scheme aims at early identiﬁcation and early intervention for children from birth to 18 years old to cover the four Ds: defects at birth; deﬁciencies; diseases; and development delays. This includes disability. Academic Editor: Sabrina Bonichini [26]), and Parent Evaluation of Developmental Status (PEDS; ref. [27]) for assessing DD. The Eyberg Child Behaviour Inventory (ECBI, ref. [28]), Child Behaviour Checklist (CBC; ref. [29]), Children Emotional Adjustment Scale (CEAS; ref. [30]), and Strength and Difﬁculties Questionnaire (SDQ; ref. [31]) have been adapted and translated for assessing SEL. Pediatr. Rep. 2023, 15 177 In comparison to the PEDS, PEDS:DM, and SDQ, the other screening tools reviewed such as the BSITDS, Denver Developmental Screening Test (DDST), ASQ-3, GMCD, ECBI, and CEAS have several limitations [22,24–26,28,30,32]. First, these scales’ psychometric properties have been questioned for LMIC because research outcomes from the Western world cannot be applied to LMIC [33,34]. Second, tools such as the BSITDS and GMCD require professional training, which is time consuming and costly [35]. Finally, scales such as the ASQ-3 and BSITDS-III need parents and clinicians to attempt multiple developmental tasks with the child before ﬁlling in the questionnaire, which may hinder the evaluation due to the longer administration time and the child’s level of comfort with the activity and the environment [36,37]. Among the screening tools for DD and SEL, the PEDS and SDQ are probably the most appropriate for use in India because they are less costly and cater to a wider age range (com- pared to tools such as GMCD and CEAS). The PEDS and SDQ are also easily accessible, do not require extensive training for administration, and have proven psychometric properties for use in LMIC [31,38]. Some preliminary studies have validated PEDS in LMIC such as Thailand [39], Bhutan [40], Tehran [41], and India [42,43], and the SDQ has been validated in Nigeria [44], Vietnam [45], Turkey [46], Thailand [47], and India [48–50]. However, the PEDS studies carried out in India were challenged by the PEDS developer for its scoring procedure and the gold standard tool the study used for its cross-validation [42,43,51]. Furthermore, these studies did not use Parents’ Evaluation of Developmental Status: De- velopmental Milestone (PEDS:DM). Philips Owen et al. [49] translated and validated the SDQ in the regional language (Malayalam) instead of the Indian national language of Hindi. Michelson et al. [48] and Singh et al. [50] used the Hindi version of the SDQ in their respective studies. Nevertheless, these studies validated the Hindi SDQ on adolescents and did not consider children. 2. Objective and Research Question for the Scoping Review Scoping reviews are used in healthcare research to map the scope and depth of a concept in a speciﬁc research area and to identify the sources and types of evidence available [56]. This scoping review’s primary objective is to determine the extent to which two developmental screening tools (PEDS and PEDS:DM) and one SEL screening tool (SDQ) have been used with children in India. This review aims to do the following: ﬁrst, increase awareness among parents and professionals in health and education about the relevance of screening children for DD and SEL. Incorporating screening tools during doctor visits and school enrolments may result in earlier and more rigorous assessment and intervention. Second, it aims to promote the use of valid, reliable, and accessible low-cost tools in LMIC such as India. Third, since PEDS, PEDS:DM, and SDQ meet the criteria, the study aims to determine whether these tools are validated for use in India. The scoping review addresses the following three research questions: (1) what is the published evidence for the use of the PEDS, PEDS:DM, and SDQ in screening children aged 0–12 years for DD and SEL in India, (2) what are the demographic characteristics of the studies’ participants, and (3) what conclusions have been drawn from the empirical research using the PEDS and SDQ screening tools in India? 3. Inclusion and Exclusion Criteria This scoping review was completed using the PRISMA extension for scoping reviews and the Joanna Briggs Institute (JBI) Protocol for evidence synthesis (Appendix B) [57]. 3.2. Concepts Studies included in this review had to use the PEDS, PEDS:DM, or SDQ as screening tools. Only studies written and published in English and between the years 1990 and 2020 were considered for this review. 3.1. Population The study included children aged 0–12 years and living in India. Exclusion criteria included studies conducted on people older than 12 years. Academic Editor: Sabrina Bonichini Thus, fewer children with developmental disabilities were missed in addition to correctly identifying children with no delays or disabilities [53]. false negative results. Thus, fewer children with developmental disabilities were missed in addition to correctly identifying children with no delays or disabilities [53]. The SDQ screening measure evaluates children’s mental health problems in the age range of 2–16 years [54]. The SDQ is completed by parents and teachers and comprises 25 questions under ﬁve domains: emotional symptoms, conduct problems, hyperactiv- ity/inattention, peer relation problems, and prosocial behaviour [31]. This screening tool comprises a three-point rating scale ranging from not true, somewhat true, and certainly true. The scoring for the SDQ comprises the total difﬁculties score, which is obtained by summing the scores for all scales except the prosocial scale. The resulting scores range from 0 to 40. The cut-off points for SDQ scores are categorised as normal, borderline, and abnormal [54]. The SDQ has good psychometric properties. The tool was administered to 10,435 British participants by their parents, teachers, and self-evaluation. The internal consistency of the tool was 0.73, the test–retest reliability was 0.62, and the sensitivity and speciﬁcity of the scale were 95% and 35%, respectively [54]. The SDQ reported high sensitiv- ity, i.e., the tool correctly identiﬁed individuals 95% of the time with mental health problems. However, the SDQ inaccurately identiﬁed participants with no mental health problems as false positives, as reported with low speciﬁcity. There have been recent attempts to examine the usefulness of the SDQ for children with DD [55]. Academic Editor: Sabrina Bonichini Therefore, there is limited research on whether these measures have been translated, adapted, and validated with Indian children [21,47]. The PEDS [27] is a surveillance and screening tool for children aged 0 to 8 years. The tool elicits and addresses parents’ concerns about development, behaviour, and mental health. The tool comprises one form with 10 questions across 10 categories (expressive language, receptive language, social–emotional, behavioural, ﬁne motor, gross motor, self- help, school, cognitive, and health). The questions in the PEDS elicit parents’ perspectives of their child’s development as high/medium/low risk. The response options include yes, no, and a little. The scoring for the PEDS includes columns for each age range and identiﬁes which concerns predict problems and which do not. The PEDS interpretation form houses an algorithm to decide whether to refer, screen further, observe, counsel parents, or reassure them on the results obtained [27]. PEDS has sound psychometric properties and was re-standardised and revalidated in 2013 [52]. The interrater reliability was 0.95, and the test–retest reliability was 0.88. The validity of the PEDS ranges from 0.84–0.99 when compared with later deﬁcits and diagnoses [52]. p g The PEDS:DM is a new measure that can be used with the PEDS or by itself. The PEDS:DM comprises six to eight items per age and aims to predict the developmental status of children accurately. Each item on the PEDS:DM addresses different domains (ﬁne motor, gross motor, expressive language, receptive language, self-help, social–emotional, and, for older children, reading and math). The age-appropriate items are presented on a single page within a laminated book that includes essential visual stimuli. Parents answer the PEDS:DM items via a multiple-choice format in fewer than 5 min. A single scoring template that is built into the binder is used to determine whether the milestones are met or unmet. Furthermore, the PEDS:DM uses the same evidence-based decision regarding the results as the PEDS. The PEDS:DM is reported to have good psychometric properties, with internal consistency across all domains being 0.98. The test–retest reliability was 0.98 and 0.99, and the interrater reliability is reported to range from 0.82 to 0.96 across subtests. The concurrent, discriminant, and criterion-related validity for PEDS:DM is satisfactory compared with other similar disability and screening tools. In addition, the speciﬁcity and sensitivity of the scale are 80% and 85%, respectively, indicating that PEDS:DM reports few Pediatr. Rep. 2023, 15 178 false negative results. 4.4. Eligibility Stage The second screening stage involved screening titles and abstracts to determine the use of the PEDS and SDQ in India. At this stage, 38 articles from the PEDS collection and 132 articles from the SDQ collection were removed for not meeting the inclusion criteria. The ﬁrst and third authors conducted 100% of the screening. Overall, an interrater reliability of 98% was obtained between the two reviewers on the agreement of including 20 full-text articles for the PEDS and 29 articles for the SDQ for review. The 2% disagreement between the reviewers was resolved by reviewing and discussing the articles again. The reviewers agreed to not include these articles in the scoping review. 3.4. Types of Sources Primary research studies, systematic reviews, meta-analyses, experimental studies and epidemiological (grey literature) research were included in this scoping review. 3.3. Context The context of this review was limited to studies conducted in India. However, the setting across India could vary from children in schools to orphanages or institu- tional homes. Pediatr. Rep. 2023, 15 179 4.3. Screening Stage The ﬁrst author (HS) carried out the initial screening and intentionally maintained the screening process as inclusive. In this stage, Endnote was used to accumulate all articles (61 articles for the PEDS and 184 articles for the SDQ) identiﬁed through the different databases and removed duplicate articles. The PEDS:DM is a new measure recommended to be used with the PEDS. However, the PEDS:DM was not mentioned in any articles in the initial screening. Therefore, it was eliminated from the PRISMA diagram. Relevant titles and articles that mentioned DD and SEL were retained. The ﬁrst and third authors (HS and NVM) then conducted 100% of the screening using the inclusion and exclusion cri- teria. The JBI System for the Uniﬁed Management, Assessment, and Review of Information (JBI SUMARI) was used to gather and screen all the articles for the PEDS and SDQ. 4.2. Identiﬁcation Stage The identiﬁcation stage involved identifying relevant studies published between 1990 and 2020 using databases such as the Web of Science, Scopus, PEDS and SDQ websites, and Google Scholar for grey literature. Different variations of keywords were included. For the PEDS, the keywords were Parent Evaluation of Developmental Status; PEDS; Parent Evaluation of Developmental Status: Developmental Milestones; PEDS:DM; children; and India. For the SDQ, the keywords included Strength and Difﬁculties Questionnaire; SDQ; children; and India. The grey literature was also searched using Google Scholar. The literature included consensus, opinion, and position papers. A total of 61 articles for the PEDS (PEDS website = 28, Web of Science = 3, Scopus = 8, and Google Scholar = 22) and 184 articles for the SDQ (SDQ website = 13, Web of Science = 22, Scopus = 29, and Google Scholar = 120) were identiﬁed for possible inclusion in the scoping review. The identiﬁcation stage processes were conducted by the ﬁrst author (HS). 4.1. Pre-Identiﬁcation Process The pre-identiﬁcation process consisted of identifying and reﬁning the research ques- tion. In the current scoping review, three questions were developed to explore whether the PEDS and SDQ screening tools have been used with the population of India, the participants’ demographic characteristics, and the ﬁndings obtained from these studies. 4.2. Identiﬁcation Stage 4. Search Strategy 4.1. Pre-Identiﬁcation Process 4.5. Final Screening Stage EVIEW 7 Figure 2. PRISMA diagram of studies included in the comprehensive scoping review for the SDQ. 5. Data Extraction Process The following information was extracted from the seven articles for the PEDS and Records identified through database searching (n = 64) Additional records identified through other sources (n = 120) Records after duplicates removed (n = 161) Full-text articles assessed for eligibility (n = 29) Studies included (n = 8) Full text articles excluded, with reasons (n = 21) Studies did not indicate the use of SDQ Studies did not cater to children and India Records excluded (n = 132) Identification Records screened (n = 161) Screening Eligibility Included Figure 2. PRISMA diagram of studies included in the comprehensive scoping review for the SDQ. Figure 1. PRISMA diagram of studies included in the comprehensive scoping review for the PEDS. Records identified through database searching (n = 39) Additional records identified through other sources (n = 22) Records after duplicates removed (n = 58) Full-text articles assessed for eligibility (n = 20) Studies included (n = 7) Full text articles excluded, with reasons (n = 13) Studies did not indicate the use of PEDS Studies did not cater to children and India Records excluded (n = 38) Identification Records screened (n = 58) Screening Eligibility Included Figure 1. PRISMA diagram of studies included in the comprehensive scoping review for the PEDS. VIEW 7 Additional records identified through other sources (n = 22) Records identified through database searching (n = 39) Records after duplicates removed (n = 58) Records excluded (n = 38) Records screened (n = 58) Full text articles excluded, with reasons (n = 13) Studies did not indicate the use of PEDS Studies did not cater to children and India Full-text articles assessed for eligibility (n = 20) Included EW Studies included (n = 7) (n = 7) Figure 1. PRISMA diagram of studies included in the comprehensive scoping review for the PEDS. Figure 1. PRISMA diagram of studies included in the comprehensive scoping review for the PE Figure 1. PRISMA diagram of studies included in the comprehensive scoping review for the PEDS. Figure 1. PRISMA diagram of studies included in the comprehensive scoping review for the PEDS. Figure 1. PRISMA diagram of studies included in the comprehensive scoping review for the PEDS. Figure 1. PRISMA diagram of studies included in the comprehensive scoping review for the PEDS. 4.5. Final Screening Stage At this stage, only studies such as empirical research, systematic reviews, literature reviews, and grey literature (dissertation, opinion pieces, etc.) that mentioned the use of the PEDS and SDQ in India were incorporated into the scoping review. For the review decision process, the ﬁrst author (HS) reviewed all the full-text articles. p The reviewer excluded full-text articles that: (1) did not use the PEDS or SDQ in India, (2) used the PEDS or SDQ on adolescents, or (3) did not provide sufﬁcient information on Pediatr. Rep. 2023, 15 180 if, and if so how, the tools were translated to Hindi. As a result, 13 full-text articles were removed from the PEDS collection, and 21 articles were removed from the SDQ collection. A total of seven articles (six peer reviewed and one grey literature) were included in the ﬁnal review for the PEDS, and eight articles (six peer reviewed and two grey literature) were included in the ﬁnal review for the SDQ (Figures 1 and 2). ( ) Q , ( ) p if, and if so how, the tools were translated to Hindi. As a result, 13 full-text articles were removed from the PEDS collection, and 21 articles were removed from the SDQ collection. A total of seven articles (six peer reviewed and one grey literature) were included in the final review for the PEDS, and eight articles (six peer reviewed and two grey literature) were included in the final review for the SDQ (Figures 1 and 2). if, and if so how, the tools were translated to Hindi. As a result, 13 full-text articles were removed from the PEDS collection, and 21 articles were removed from the SDQ collection. A total of seven articles (six peer reviewed and one grey literature) were included in the ﬁnal review for the PEDS, and eight articles (six peer reviewed and two grey literature) were included in the ﬁnal review for the SDQ (Figures 1 and 2). ( ) Q , ( ) p if, and if so how, the tools were translated to Hindi. As a result, 13 full-text articles were removed from the PEDS collection, and 21 articles were removed from the SDQ collection. 4.5. Final Screening Stage A total of seven articles (six peer reviewed and one grey literature) were included in the final review for the PEDS, and eight articles (six peer reviewed and two grey literature) were included in the final review for the SDQ (Figures 1 and 2). if, and if so how, the tools were translated to Hindi. As a result, 13 full-text articles were removed from the PEDS collection, and 21 articles were removed from the SDQ collection. A total of seven articles (six peer reviewed and one grey literature) were included in the ﬁnal review for the PEDS, and eight articles (six peer reviewed and two grey literature) were included in the ﬁnal review for the SDQ (Figures 1 and 2). ( ) Q , ( ) p if, and if so how, the tools were translated to Hindi. As a result, 13 full-text articles were removed from the PEDS collection, and 21 articles were removed from the SDQ collection. A total of seven articles (six peer reviewed and one grey literature) were included in the final review for the PEDS, and eight articles (six peer reviewed and two grey literature) were included in the final review for the SDQ (Figures 1 and 2). , Q A total of seven articles (six peer reviewed and one grey literature) were included in the ﬁnal review for the PEDS, and eight articles (six peer reviewed and two grey literature) were included in the ﬁnal review for the SDQ (Figures 1 and 2). A total of seven articles (six peer reviewed and one grey literature) were included in the final review for the PEDS, and eight articles (six peer reviewed and two grey literature) were included in the final review for the SDQ (Figures 1 and 2). Figure 1. PRISMA diagram of studies included in the comprehensive scoping review for the PEDS. Records identified through database searching (n = 39) Additional records identified through other sources (n = 22) Records after duplicates removed (n = 58) Full-text articles assessed for eligibility (n = 20) Studies included (n = 7) Full text articles excluded, with reasons (n = 13) Studies did not indicate the use of PEDS Studies did not cater to children and India Records excluded (n = 38) Identification Records screened (n = 58) Screening Eligibility Included Figure 1. PRISMA diagram of studies included in the comprehensive scoping review for the PEDS. 7. PEDS and PEDS:DM The scoping review for the PEDS found seven full-text articles on the use of the PEDS for DD in India. However, only two studies were empirical research, and none used the PEDS:DM, either alone or with the PEDS. The participants belonged to north India and were between 6 and 60 months of age. The conclusion drawn was that the PEDS could detect concerns among parents regarding their child’s developmental milestones. The number of PEDS studies varied across years. No studies were reported for 1990–2000, three (43%) were reported for 2001–2005, one (14%) was reported for 2006– 2010, two (29%) were reported for 2011–2015, and one (14%) was reported for 2016–2020. Three (42%) of the studies were text- and opinion-based evidence, two (29%) were empirical studies, and two (29%) were systematic reviews. y Malhi and Singhi [42,43] reported the results from two empirical studies using the PEDS. These two studies did not use the PEDS:DM and aimed to identify the range of concerns that parents have about their children’s development and its relationship to the child’s developmental status. The ﬁrst study included 55 parent–child dyads recruited through outpatient paediatric care in a tertiary care teaching hospital in Chandigarh. The second study recruited 79 parent–child dyads from the same hospital and city in India. The age range of the children was 6–60 months. The ﬁrst study concluded that 38% of the parents indicated no concerns, while 20% raised non-signiﬁcant developmental problems. Among these children, 91% passed the developmental screening. The second study concluded that parents’ concerns regarding their children’s developmental milestones were moderately sensitive predictors of DD in children aged 2 to 5 years. The authors suggested that since the PEDS’s speciﬁcity (34.8%) and sensitivity (65%) were lower in LMIC than in the United States, the tool is not recommended as an alternative to standardised screening measures. Instead, it may be used as a pre-screening tool in an outpatient setting to identify those children who require more in-depth developmental screening [43]. In response to the conclusions of Malhi and Singhi [41,42], Glascoe [51] wrote a letter to the editors indicating that their ﬁndings may not be accurate for two reasons. First, there was a lack of clarity regarding their scoring of the PEDS. Second, their use of the Developmental Proﬁle-II as the criterion measure was problematic, as it tends to both under- and over-detect developmental concerns among children [51]. Poon et al. 4.5. Final Screening Stage Figure 2. PRISMA diagram of studies included in the comprehensive scoping review for the SDQ. Records identified through database searching (n = 64) Additional records identified through other sources (n = 120) Records after duplicates removed (n = 161) Full-text articles assessed for eligibility (n = 29) Studies included (n = 8) Full text articles excluded, with reasons (n = 21) Studies did not indicate the use of SDQ Studies did not cater to children and India Records excluded (n = 132) Identification Records screened (n = 161) Screening Eligibility Included Figure 2. PRISMA diagram of studies included in the comprehensive scoping review for the SDQ. Additional records identified through other sources (n = 120) Records identified through database searching (n = 64) Records after duplicates removed (n = 161) Records excluded (n = 132) Records screened (n = 161) Full text articles excluded, with reasons (n = 21) Studies did not indicate the use of SDQ Studies did not cater to children and India Full-text articles assessed for eligibility (n = 29) Studies included (n = 8) Figure 2. PRISMA diagram of studies included in the comprehensive scoping review for the SDQ. Figure 2. PRISMA diagram of studies included in the comprehensive scoping review for the SDQ. Pediatr. Rep. 2023, 15 181 5. Data Extraction Process The following information was extracted from the seven articles for the PEDS and the eight articles for the SDQ: (1) author and date of publication, (2) type of article, (3) type of study, (4) where the study was conducted in India, (5) aim of the study, (6) the sample size and age range, (7) the setting in which the PEDS and SDQ were used, and (8) the main ﬁndings of the study (Appendix A). 6. Results and Discussion of the Scoping Review The aim of this review was to examine the amount of published evidence on the use of the PEDS, PEDS:DM, and SDQ for screening children for DD and SEL in India, to explore the demographic characteristics of the studies’ participants, and to report the conclusions drawn from the empirical research using the PEDS and SDQ screening tools. 7. PEDS and PEDS:DM [58] provided an opinion piece discussing the prevalence of DD in children. This article emphasised the beneﬁts of early identiﬁcation using developmental screening and surveillance. The authors believed that it is necessary to listen to parents’ concerns with regularity, integrate routine screening with health maintenance visits, refer patients to paediatricians and therapists early, and provide early intervention services and therapies that have proven effective independent of the medical diagnosis. The authors reported that the PEDS has been translated into more than 10 different languages and completed by parents. Mukherjee et al. [10] also found the PEDS to be reliable in developing countries. However, the authors concluded that there is limited research from India. Pediatr. Rep. 2023, 15 182 Marlow et al. [37] conducted a systematic review of DD and autism spectrum disorder (ASD) screening tools and provided DD and ASD screening recommendations for LMIC. The review included children aged 0–7 years, studies published in English, the tools used for screening purposes, studies that included at least one of the developmental domains, and provided information on the measure’s performance. The review conﬁrmed that the PEDS has been translated for use in India and that it can detect DD among children in LMIC. Woolfenden et al. [59] aimed to understand the use of the PEDS in evaluating parental concerns of children with developmental risk and associated risk factors. Their systematic review’s inclusion criteria speciﬁed primary observational studies with available prevalence data. Their review found that the PEDS reported 13% of parents indicating their child as high developmental risk and 19% of parents reporting moderate to low developmental risks. However, these evaluations depended on the children’s body weights, socioeconomic conditions, and access to medical care, which provided variation in the quality of studies included in the systematic review. Furthermore, comparing the PEDS to other measures of developmental risk such as the DDST, ASQ, and the Australian Early Developmental Index showed the same conﬁdence interval around the pooled prevalence estimates of high and moderate developmental risks. Therefore, the two systematic reviews concluded that there is a substantial literature on the prevalence of parental concern for developmental risk in children. However, most of the ﬁndings were ﬂawed due to the methodological issues of a small sample size and the use of an inappropriate measure to screen children for developmental delay. Furthermore, only eight studies used the PEDS in LMIC, including India. Of the parents who expressed one or more signiﬁcant developmental concerns about their child, 47.8% of these children failed the screening. In addition, 43% of the parents whose children failed developmental screening expressed medical concerns, 35.7% reported expressive language concerns, and 28% indicated global/cognitive concerns. 38% of parents indicated no concerns, and 20% raised non-signiﬁcant developmental concerns about their child’s development. From these children, 90.6% passed the development screening. Parents’ concerns about the developmental milestones of their child were moderately sensitive predictors of DD in children between 2 and 5 years. 7. PEDS and PEDS:DM Table 1 summarises the studies conducted using PEDS in India. The table reports the authors, the article type, the design of the study, the city/state where the study was conducted, the aim of the study, the population setting (size and age range), the measures used, the results, and the key ﬁndings of the study. Pediatr. Rep. 2023, 15 183 Table 1. Summary of studies on Parent Evaluation of Developmental Status (PEDS) use in India. Population Author Article Type Design City in India Aim/Purpose Size Age Range Setting and Measures Used Main Results Key Findings Marlow, Servili, and Tomlinson, 2019 Peer reviewed Systematic review Chandigarh, India (Reference to Malhi and Singhi, 2001 study) Identify current screening instruments for DD and ASD, create screening proﬁles, and provide recommendation for screening in LMIC. A sample of more than 300 participants for each instrument 0–7 years Search Strategy of the tools (2014–2017); Inclusion and exclusion criteria and speciﬁc criteria for screening instruments. The review identiﬁed 10 screening tools suitable to screen children in LMIC for ASD and 7 screening tools for DD. PEDS is adapted and able to detect DD in LMIC. Woolfenden et al., 2014 Peer reviewed Systematic review Chandigarh, India (Reference to Malhi and Singhi, 2001 study) To understand the worldwide prevalence of parental concerns measured by PEDS that indicated developmental risks and associated risk factors. 20 to 54,602 Less than 1 month to 7 years and 11 months Search Strategy of PEDS; Inclusion and exclusion criteria for study participants and review process. 14% of parents raised concerns associated with a high risk of developmental problems, and 19% raised concerns about a moderate risk for developmental problems. Eight studies of PEDS were conducted in low- and medium-income countries (including India). Malhi and Singhi, 2001 Peer reviewed Diagnostic test accuracy Chandigarh, India To identify the range of concerns that parents have about their child’s development and its relationship to the child’s developmental status. 55 parent–child dyads 6 to 60 months Patients recruited through outpatient paediatric care in a tertiary care teaching hospital. 38% of parents indicated no concerns, and 20% raised non-signiﬁcant developmental concerns about their child’s development. From these children, 90.6% passed the development screening. Of the parents who expressed one or more signiﬁcant developmental concerns about their child, 47.8% of these children failed the screening. The authors advised against using the PEDS as a substitute for standardized developmental screening measures because its speciﬁcity and sensitivity were lower than those reported by the US. The PEDS can be used as a pre-screening tool to ﬁnd children who might need comprehensive developmental screening in outpatient settings. 7. PEDS and PEDS:DM In addition, 43% of the parents whose children failed developmental screening expressed medical concerns, 35.7% reported expressive language concerns, and 28% indicated global/cognitive concerns. Malhi and Singhi, 2002 Peer reviewed Diagnostic test accuracy Chandigarh, India To identify the range of concerns parents have about their child’s development and evaluate the relationship between parent concern and the child’s developmental status. 79 parent–child dyads 24 to 60 months Patients recruited through outpatient paediatric care in a tertiary care teaching hospital; Two questionnaires used: PEDS and Developmental Proﬁle II. Parents’ concerns about the developmental milestones of their child were moderately sensitive predictors of DD in children between 2 and 5 years. The authors advised against using the PEDS as a substitute for standardized developmental screening measures because its speciﬁcity and sensitivity were lower than those reported by the US. The PEDS can be used as a pre-screening tool to ﬁnd children who might need comprehensive developmental screening in outpatient settings. Table 1. Summary of studies on Parent Evaluation of Developmental Status (PEDS) use in India. The authors advised against using the PEDS as a substitute for standardized developmental screening measures because its speciﬁcity and sensitivity were lower than those reported by the US. The PEDS can be used as a pre-screening tool to ﬁnd children who might need comprehensive developmental screening in outpatient settings. 184 Pediatr. Rep. 2023, 15 Population Article Type Design City in India Aim/Purpose Size Age Range Setting and Measures Used Main Results Key Findings Grey literature Letter to the authors N.A. 79 parent–child dyads 24 to 60 months Patients recruited through outpatient paediatric care in a tertiary care teaching hospital; Two questionnaires used: PEDS and Developmental Proﬁle II. Parents’ concerns about the developmental milestones of their child were moderately sensitive predictors of DD in children between 2 and 5 years. The author’s letter noted that the scoring method utilized in the paper was not clear. Given that DP-II has a propensity to overidentify developmental issues, the concurrent test utilized to evaluate the accuracy of PEDS is questionable. PEDS would gain from the use of a different scoring system. Peer reviewed Text and opinion study The paper discusses the prevalence of DD in children and recent literature regarding the beneﬁts of early identiﬁcation and beneﬁts of developmental screening and surveillance. Not speciﬁed N.A. The author’s letter noted that the scoring method utilized in the paper was not clear. Given that DP-II has a propensity to overidentify developmental issues, the concurrent test utilized to evaluate the accuracy of PEDS is questionable. PEDS would gain from the use of a different scoring system. PEDS has been used in India. The tool has been translated to over 10 different languages and is completed by parents. PEDS has been found reliable in developing countries. However, there is limited research from India. 7. PEDS and PEDS:DM The review’s key conclusions stated that it is important to pay attention to parents’ worries while maintaining regular surveillance, integrating routine screening, making early referrals to paediatricians and therapists, and offering early intervention services and therapies that have been proven to be successful regardless of the medical diagnosis. PEDS has been used in India. The tool has been translated to over 10 different languages and is completed by parents. Peer reviewed Text and opinion Study India The aim of the article was to review existing tools for children under the age of ﬁve that were validated in India and to provide a purposed paradigm for developmental screening in ofﬁce practice. Not speciﬁed Under the age of 5 years N.A. Tools developed in India lack psychometric properties and were developed by healthcare workers, and the screening tools developed in the US are costly and not easily accessible. PEDS has been found reliable in developing countries. However, there is limited research from India. Note: PEDS was administered in English for all studies. Parents’ concerns about the developmental milestones of their child were moderately sensitive predictors of DD in children between 2 and 5 years. 8. SDQ From the 184 abstracts and titles screened for the SDQ scoping review, only eight full- text articles met the inclusion criteria (seven empirical studies and one literature review). The participants were recruited from different parts of India, and the children were up to 12 years old. The studies concluded that the SDQ was able to differentiate between different groups of children on the basis of their total difﬁculties score. No studies were conducted from 1990 to 2000, one (13%) study was conducted in both 2001–2005 and 2006–2010, two (25%) studies were conducted in 2011–2015, and four (50%) studies were conducted in 2016–2020. There was one (13%) study each in the format of a case-control study and a literature review, and there were three (38%) studies each using cross-sectional and longitudinal designs. g g Of the three cross-sectional studies conducted in India using the SDQ, one study used a community sample [60], and two studies used clinical samples [61,62]. Bele et al. [60] assessed the prevalence of emotional and behavioural difﬁculties among children liv- ing in urban slums in Andhra Pradesh, India. Their study evaluated 370 children aged 5–10 years using the SDQ completed by parents. They concluded that for the children, residing in urban slums was signiﬁcantly associated with behaviour problems. Boys had a higher risk of mental health difﬁculties than girls. Factors such as low nutrition, low socioeconomic status, ﬁnancial constraints, and conﬂicts in the family were predictors of behaviour problems among the children. Two cross-sectional studies examined the psychological health of children with acute lymphoblastic leukaemia and congenital heart disease [61,62]. Chari and Hirisave [61] assessed 40 children (20 children with acute lymphoblastic leukaemia and 20 children from a healthy group) aged 4 to 8 years in terms of psychiatric disturbance using the SDQ completed by parents. The study was carried out at the paediatric oncology ward at Kidwai memorial institute of Oncology, Bangalore, Karnataka. The results showed that children with acute lymphoblastic leukaemia demonstrated more disruptive behaviour and peer problems than healthy children. Kiron [62] examined 242 children aged 10 years and below for the psychosocial impact of congenital heart disease. They used the Malayalam version of the SDQ, which was completed by parents at the Sree Chitra Tirunsal Institute of Medical Science and Technology, Kerala. Key Findings Pediatr. Rep. 2023, 15 185 8. SDQ They reported that children aware of their congenital heart disease had a higher total difﬁculty score on the SDQ than children unaware of their congenital heart disease. The children with awareness of their congenital heart disease also exhibited more behaviour problems and less prosocial behaviour than the children without awareness of their congenital heart disease [62]. The three longitudinal studies were carried out across several LMIC, including India. One was carried out in India alone [63], one in India and Vietnam [64], and one in Cambodia, Kenya, Tanzania, Ethiopia, and India [65]. The scoping review included these multi-country studies, as they met the inclusion criteria of including participants from India. Malhotra et al.’s [63] study in Chandigarh, India, aimed to establish the incidence of psychological difﬁculties in 727 school children aged 4–11 years. At the six-year follow up, children with psychological disorders were compared to children with no psychological disorders on socio-demographic factors and psychological variables. The ﬁndings, based on parent reports, found no signiﬁcant differences between the two groups on age, gender, or psychological parameters such as temperament, parental handling, life events, and IQ. Trinh’s [64] study compared the mental health impact of child labour in India and Vietnam. The study was of 1934 children aged 7–9 years assessed over a period of 15 years on parent-completed SDQ. The study concluded that the effect of child labour on the ﬁve dimensions of the SDQ was not uniform across the two countries. In Vietnam, children who participated in the labour market were likely to have worse conduct problems, hyperactivity, peer problems, and reduced prosocial behaviour compared to those who did not work. In India, child labour and mental health symptoms were signiﬁcantly correlated with hyperactivity and reduced prosocial behaviour. Pediatr. Rep. 2023, 15 186 Huynh et al.’s [65] longitudinal study compared the psychological wellbeing of 2837 orphans and separated children in the age range of 6–12 years over 36 months in ﬁve LMIC (Cambodia, India, Kenya, Tanzania, and Ethiopia). The study used the self- report version of the SDQ and translated the questionnaire to its native languages in the ﬁve countries. The ﬁndings revealed no meaningful difference in the SDQ total difﬁculties score across care settings (residential versus community-based) or between orphaned and separated children in residential care settings. A total of 20 of the 186 children that were monitored had a psychological condition. In terms of age, gender, and psychological (temperament, parental handling, life stress, and IQ) factors at baseline, children with the disorder at follow-up did not vary from those without it. Children scoring above a cut-off score on the SDQ (≥14) were clinically examined by a psychiatrist at home or at the clinic. In the ﬁve scales of the SDQ, peer problems and prosocial behaviour were found to be signiﬁcantly impacted by working in both countries. SDQ to measure child mental health and child participation in the labour market was assessed by understanding if the child has undertaken any activity to earn money. 8. SDQ Population r Article Type Design City in India Aim/Purpose Size Age Range Setting and Measures Used Outcome 2013 * Peer reviewed Cross-sectional study Gauthaminagar in Karimnagar district of Andhra Pradesh To estimate the prevalence of emotional and behavioural disorders using standardised instruments among children in urban slums. N = 370 5–10 years Emotional and behavioural problems among children were evaluated using the Strength and Difﬁculties Questionnaire (SDQ), and depression was assessed using Patient Health Questionnaire (PHQ-9). On at least one SDQ domain, 22% of the children scored abnormally. The children’s behavioural issues and poorer academic achievement were found to be signiﬁcantly correlated. SD va un comp vari total affec a et al., * Peer reviewed Prevalence study/Longitudinal study Chandigarh, India To establish the incidence of psychiatric disorders in school children in India. N = 873 4–11 years Rutter B (teachers rating), Childhood Psychopathology Measurement Schedule, Temperament Measurement Scale, Parent Handling Questionnaire, Parent Interview Schedule, Life event scale for Indian children, and SDQ. A total of 20 of the 186 children that were monitored had a psychological condition. In terms of age, gender, and psychological (temperament, parental handling, life stress, and IQ) factors at baseline, children with the disorder at follow-up did not vary from those without it. Chi cut (≥ exam at 20 * Peer reviewed Longitudinal study India and Vietnam To study the mental health impact of child labour. N = 978 children in Vietnam and 956 children in India 7–9 years SDQ to measure child mental health and child participation in the labour market was assessed by understanding if the child has undertaken any activity to earn money. Child labour did not uniformly affect the ﬁve dimensions of the SDQ. Compared to children who did not work, those who participated in the labour market in Vietnam were more likely to experience conduct problems, hyperactivity, peer issues, and less prosocial behaviour. The outcomes for working children in Vietnam were noticeably lower regarding peer issues and less prosocial behaviour. Hyperactivity and a decline in prosocial behaviour were signiﬁcantly linked with child labour and mental health symptoms in India. In SDQ pro fou imp Table 2. Summary of studies on the Strength and Difﬁculties Questionnaire (SDQ) use in India. 8. SDQ In this study, orphans were deﬁned as children who had lost one or both parents, and separated children were deﬁned as children separated from their parents with no expectation of return. This study demonstrated that in under-resourced societies in LMIC, orphaned and separated children’s overall wellbeing may depend on the quality of care rather than the type of care setting itself [65]. A prospective case-control study was conducted in Mumbai, Maharashtra, to study the impact of neurobehavioral disorders in children with and without epilepsy [66]. The children, aged 5–12 years, were classiﬁed on the etiologic classiﬁcation: epilepsy, epilepsy control, irregular school attendance, and school dropouts. The 222 children with epilepsy were matched with 226 non-epileptic children on age, gender, and socioeconomic status. The parent version of the SDQ was administered in the Indian language to screen for neurobehavioral disorders. The authors reported that 63% of the children with epilepsy had emotional problems and abnormal conduct scores, high hyperactivity, poor peer relations, and poor pro-sociality, leading to low school attendance. The SDQ total difﬁculties score was abnormal in 39%, borderline in 16%, and normal in 45% of the cases for children with epilepsy. It was abnormal for 8%, borderline for 3%, and normal for 89% of the cases for children without epilepsy. A literature review (grey literature) by Galab et al. [67] focused on childhood poverty in Andhra Pradesh, India, and how the national policies have impacted the state. The report used the parent and translated version (Telegu) of the SDQ to assess the mental health problems of children aged 1–8 years. The report concluded that nearly 20% of children were classiﬁed as abnormal and 20% as borderline However, the author cautioned that the SDQ had not been validated for use in Andhra Pradesh and that local normative data were not available [55]. Table 2 summarises the studies conducted using SDQ in India. The table reports the authors, the article type, the design of the study, the city/state where the study was conducted, the aim of the study, the population setting (size and age range), the measures used, the results, and the key ﬁndings of the study. Pediatr. Rep. 2023, 15 187 Table 2. Summary of studies on the Strength and Difﬁculties Questionnaire (SDQ) use in India. Child labour did not uniformly affect the ﬁve dimensions of the SDQ. Compared to children who did not work, those who participated in the labour market in Vietnam were more likely to experience conduct problems, hyperactivity, peer issues, and less prosocial behaviour. The outcomes for working children in Vietnam were noticeably lower regarding peer issues and less prosocial behaviour. Hyperactivity and a decline in prosocial behaviour were signiﬁcantly linked with child labour and mental health symptoms in India. SDQ scores and mean values for affected and unaffected groups were compared, and a signiﬁcant variance was found in the total problems score in the affected group (borderline The Young Lives results reported that nearly 20% of children were classiﬁed as abnormal and 20% as borderline. However, the authors recommended that these results should be interpreted with caution since the SDQ has not been validated in Andhra Pradesh and normative data is not available. Screening of cases and controls with the SDQ-P (parent version) was conducted, and the total difﬁculties score was abnormal in 39% of cases and 7.9% of controls, and it was normal in 44.5% of cases and 88.9% of controls. Previously, the SDQ had not been validated in Andhra Pradesh, and normative data were unavailable. The study was the ﬁrst to use the SDQ in Andhra Pradesh, and the ﬁndings suggest that child mental health issues may be a potential problem, especially in the rural areas of Andhra Pradesh, where the prevalence of abnormal cases was over 20%. On the SDQ, there were signiﬁcant differences between groups in total difﬁculties, conduct, and peer problems. However, median scores were in the normative range. Children with ALL demonstrated more disruptive behaviours and peer problems than healthy children. Children with ALL (acute lymphoblastic leukaemia) were reported on SDQ to display more behavioural disturbances. Key Findings SDQ scores and mean values for affected and unaffected groups were compared, and a signiﬁcant variance was found in the total problems score in the affected group (borderline d b l ) On at least one SDQ domain, 22% of the children scored abnormally. The children’s behavioural issues and poorer academic achievement were found to be signiﬁcantly correlated. Child labour did not uniformly affect the ﬁve dimensions of the SDQ. Compared to children who did not work, those who participated in the labour market in Vietnam were more likely to experience conduct problems, hyperactivity, peer issues, and less prosocial behaviour. The outcomes for working children in Vietnam were noticeably lower regarding peer issues and less prosocial behaviour. Hyperactivity and a decline in prosocial behaviour were signiﬁcantly linked with child labour and mental health symptoms in India. Pediatr. Rep. 2023, 15 188 Table 2. Cont. Population r Article Type Design City in India Aim/Purpose Size Age Range Setting and Measures Used Outcome Key Fin 2016 Peer reviewed Prospective case control study Mumbai, India To study the prevalence, type, and impact of neurobehavioral disorders in children with and without epilepsy. N = 222 5–12 years SDQ was assessed in four groups: epilepsy, epilepsy control, irregular school attendance, and school dropout. The study revealed that 14.4% of children with epilepsy during schooling had learning problems, and 10.3% had behavioural problems compared to non-epileptics. In addition, 63% of the people with epilepsy had emotional difﬁculties and abnormal conduct scores. High hyperactivity, poor peer relations, and poor pro-social behaviour led to low school attendance in 35% of epileptic patients. Screening o controls with (parent ver conducted, a difﬁculties abnormal in 3 and 7.9% of co was normal cases and 88.9 nd 020 * Peer reviewed Cross-sectional study Bangalore, India To examine the psychological health of young children undergoing treatment for acute lymphoblastic leukaemia. N = 40 4–8 years SDQ to assess psychiatric disturbances, feeling cards to examine the subject’s current emotional state, and teddy bear’s picnic to examine personal construct. Children with ALL (acute lymphoblastic leukaemia) were reported on SDQ to display more behavioural disturbances. SDQ to assess psychiatric disturbances, feeling cards to examine the subject’s current emotional state, and teddy bear’s picnic to examine personal construct. The SDQ was used to assess the mental health of children of 8 years of age. Children not aware of their CHD had signiﬁcantly lower levels of problems compared to children who were aware and had experienced CHD. In addition, children in the ﬁrst group were higher in prosocial behaviour compared to the second group. Child psychosocial well-being across different levels of quality of care showed negligible differences between residential- and community-based care settings, suggesting the critical factor in child well-being is quality of care rather than environment of care. On being assessed with the total difﬁculties score, children who were aware of their congenital heart disease were at substantial risk of clinically signiﬁcant problems compared to the other group. Key Findings On the SDQ signiﬁcant d between gro difﬁculties, c peer problem median score normative ran with ALL de more disruptiv and peer pro healthy c 2003 Grey literature Literature review Andhra Pradesh The report provides a brief literature on childhood poverty in Andhra Pradesh in India and explains how national policies have impacted childhood poverty in that state. N = 3000 1–8 years The SDQ was used to assess the mental health of children of 8 years of age. The Young Lives results reported that nearly 20% of children were classiﬁed as abnormal and 20% as borderline. However, the authors recommended that these results should be interpreted with caution since the SDQ has not been validated in Andhra Pradesh and normative data is not available. Previously, th not been va Andhra Pra normative unavailable. T the ﬁrst to us Andhra Prad ﬁndings sugg mental health a potential especially in th of Andhra Pra the prevalence cases was To study the prevalence, type, and impact of neurobehavioral disorders in children with and without epilepsy. SDQ was assessed in four groups: epilepsy, epilepsy control, irregular school attendance, and school dropout. SDQ was assessed in four groups: epilepsy, epilepsy control, irregular school attendance, and school dropout. Grey literature Literature review Andhra Pradesh The report provides a brief literature on childhood poverty in Andhra Pradesh in India and explains how national policies have impacted childhood poverty in that state. N = 3000 1–8 years The SDQ was used to assess the mental health of children of 8 years of age. Pediatr. Rep. 2023, 15 189 Population Author Article Type Design City in India Aim/Purpose Size Age Range Setting and Measures Used Outcome Key Findings Huynh et al., 2019 Peer reviewed Longitudinal study Five low- and middle-income countries: Battambang District, Cambodia; Nagaland and Hyderabad, India; Bungoma District, Kenya; Kilimanjaro Region, Tanzania; and Addis Ababa, Ethiopia. Children were enrolled between 2006 and 2008 and followed biannually To examine the psychological wellbeing of orphans and separate children in under-resourced societies in LMIC and to associate quality of care with children’s psychosocial wellbeing. N = 2013 (923 resi- dential care and 1090 community- based sample) 6–12 years at baseline; 36 months of follow-up data Quality of care was assessed using the Child Status Index, and child’s psychosocial wellbeing was assessed using the SDQ. Key Findings Child psychosocial well-being across different levels of quality of care showed negligible differences between residential- and community-based care settings, suggesting the critical factor in child well-being is quality of care rather than environment of care. When the authors controlled the orphan’s gender, status, and age, components of quality of care predicted SDQ total difﬁculties better than care setting. Mean SDQ total difﬁculties scores across “high” and “low” quality of care showed differences between care settings to be minimal. There were no meaningful differences in SDQ total difﬁculties across care settings. Orphans and separated children (OCS) in residential care settings had higher SDQ total difﬁculties scores than in community-based settings. Kiron, 2012 * Grey literature Cross-sectional study (Project 2) Sree Chitra Tirunal Institute of Medical Science and Technology, Kerala, India To analyse whether those children who grow up without being aware of their congenital heart disease have any psychosocial advantage over those children who grow up being aware that they have undergone a major interventional procedure for their congenital heart disease. N = 242 (only 147 parents responded to SDQ) Less than 10 years The SDQ was used to ascertain the impact of CHD in children. On being assessed with the total difﬁculties score, children who were aware of their congenital heart disease were at substantial risk of clinically signiﬁcant problems compared to the other group. Children not aware of their CHD had signiﬁcantly lower levels of problems compared to children who were aware and had experienced CHD. In addition, children in the ﬁrst group were higher in prosocial behaviour compared to the second group. Note: * demonstrates SDQ administered in English, demonstrates SDQ administered in Hindi and * demonstrates SDQ administered in the regional languages. The SDQ was used to ascertain the impact of CHD in children. Quality of care was assessed using the Child Status Index, and child’s psychosocial wellbeing was assessed using the SDQ. 9.1. Limitations of the Existing Research The limited number of studies identiﬁed in this scoping review were conducted in different parts of India and examined the use of the PEDS and SDQ with children. Empirical research on the use of the PEDS in India is scarce (Table 1). The PEDS:DM was not used together with the PEDS in any of the studies. The studies that reported using translated versions of the PEDS in India provided limited to no information on the psychometric properties of the translated versions. Marlow et al. [37] noted that Malhi and Singhi’s [42,43] study translated PEDS to Hindi. However, the original article does not provide this information. The SDQ has been used more frequently than the PEDS in India (Table 2). However, most of the studies did not consider teacher evaluation and lacked transparency in the translation process of the SDQ. Two studies (grey literature) translated the SDQ to the relevant regional language [62,67]. Two studies indicated that they had translated the SDQ to the Native or Indian language without explicitly saying which language [65,66], and three studies did not translate the SDQ to Hindi [60,61,64]. Only one study explicitly indicated that they had used the Hindi version of the SDQ in their research [63]. The psychometric properties of the translated questionnaire in the regional and national languages of India must be considered with caution. The recommended cut-off value for the UK cannot be assumed to be suitable for use in countries where cultural differences exist [37]. 9.2. Strengths, Limitations, and Implications of this Scoping Review Strengths of this review are its comprehensive search strategy and its addressing of a broad research question related to the use of two screening tools in the population of India. The review process evaluated the quality of the studies that met the inclusion criteria for the PEDS and the SDQ. However, the review only assessed papers in English that were published within a certain year range. Future studies could possibly include studies in other languages used in India. Key Findings When the authors controlled the orphan’s gender, status, and age, components of quality of care predicted SDQ total difﬁculties better than care setting. Mean SDQ total difﬁculties scores across “high” and “low” quality of care showed differences between care settings to be minimal. There were no meaningful differences in SDQ total difﬁculties across care settings. Orphans and separated children (OCS) in residential care settings had hi h SDQ l higher SDQ total difﬁculties scores than in community-based settings. Children not aware of their CHD had signiﬁcantly lower levels of problems compared to children who were aware and had experienced CHD. In addition, children in the ﬁrst group were higher in prosocial behaviour compared to the second group. On being assessed with the total difﬁculties score, children who were aware of their congenital heart disease were at substantial risk of clinically signiﬁcant problems compared to the other group. The SDQ was used to ascertain the impact of CHD in children. Cross-sectional study (Project 2) glish, demonstrates SDQ administered in Hindi and * demonstrates SDQ administered in the regional languages. Pediatr. Rep. 2023, 15 190 9. Summary The key ﬁndings addressed the three aims of the scoping review. First, there is scant published literature on the use of the PEDS in India. Most of the literature exists in the form of text- and opinion-based evidence that emphasised the lack of screening in India and the limited use of the PEDS as a tool to screen children for DD. Furthermore, only one study brieﬂy mentioned the use of translated versions of PEDS questionnaires [37]. Overall, the articles highlighted the importance of early identiﬁcation of children with DD and listening to parents’ concerns through regular surveillance and screening. The SDQ compared mental health across different LMIC and was administered more frequently than PEDS to screen children in India. Second, the characteristics of the participants included in the PEDS study were children aged 6–60 months with typical development living in Chandigarh, India. The SDQ studies comprised children aged 0–12 years from clinical and community samples recruited from different parts of the country. Third, empirical studies using PEDS reported the tool as having below acceptable sensitivity and speciﬁcity. The SDQ studies reported that the tool was effective in differenti- ating groups of individuals based on SEL and behavioural concerns. Since India is a diverse country with many regional languages, the studies that used the SDQ catered to different population types and translated the questionnaire to regional and national languages. 10. Conclusions In terms of screening children for DD and SEL concerns, India lacks both a literature base and evidence of practice. Evidence on the use of the PEDS and SDQ suggests that these screening tools have not been widely used with Indian children. Therefore, the translation and administration of the PEDS, PEDS:DM, and SDQ will ensure that these screening tools are relevant and applicable to the Indian population. Furthermore, concurrent use of these Pediatr. Rep. 2023, 15 191 tools will provide a better understanding of the relationship of DD with SEL concerns among children. It is important that research is undertaken and published to address the current gap in local literature and practice. 11. Implications for Practice Due to the limited availability of suitable measures, screening children for develop- mental delay and social–emotional learning is a challenge in India. A scoping review was conducted on the use of the Parent’s Evaluation of Developmental Status (PEDS) and the Strength and Difﬁculties Questionnaire (SDQ) with children in India. There is an absence of research demonstrating the complementary use of both measures to provide a holistic screening of children. Author Contributions: H.S. wrote the manuscript under the supervision of L.S. and N.V.M. H.S. and N.V.M. conducted the data analysis. All authors contributed to and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. 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. Research Ethics: This is a literature review; research ethics approval was not required. Appendix A Table A1. Data Extraction Table. Background Information Author and Date of Publication Article Type Type City/State Aim of the Study Sample Number of Participants Age Range Setting and Measures used Results Main Results Key Findings Pediatr. Rep. 2023, 15 192 Appendix B Table A2. Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist. SECTION ITEM PRISMA-ScR CHECKLIST ITEM REPORTED ON PAGE # TITLE Title 1 Identify the report as a scoping review. Page 1 ABSTRACT Structured summary 2 Provide a structured summary that includes (as applicable): background, objectives, eligibility criteria, sources of evidence, charting methods, results, and conclusions that relate to the review questions and objectives. Page 2 INTRODUCTION Rationale 3 Describe the rationale for the review in the context of what is already known. Explain why the review questions/objectives lend themselves to a scoping review approach. Page 5 Objectives 4 Provide an explicit statement of the questions and objectives being addressed with reference to their key elements (e.g., population or participants, concepts, and context) or other relevant key elements used to conceptualize the review questions and/or objectives. Page 7 METHODS Protocol and registration 5 Indicate whether a review protocol exists; state if and where it can be accessed (e.g., a Web address); and if available, provide registration information, including the registration number. OSF Eligibility criteria 6 Specify characteristics of the sources of evidence used as eligibility criteria (e.g., years considered, language, and publication status), and provide a rationale. Page 8 Information sources * 7 Describe all information sources in the search (e.g., databases with dates of coverage and contact with authors to identify additional sources) as well as the date the most recent search was executed. Page 8 Search 8 Present the full electronic search strategy for at least one database, including any limits used, such that it could be repeated. Page 8–10 Selection of sources of evidence † 9 State the process for selecting sources of evidence (i.e., screening and eligibility) included in the scoping review. Page 8–10 Data charting process ‡ 10 Describe the methods of charting data from the included sources of evidence (e.g., calibrated forms or forms that have been tested by the team before their use, and whether data charting was done independently or in duplicate) and any processes for obtaining and conﬁrming data from investigators. Appendix A Page 10–13 Data items 11 List and deﬁne all variables for which data were sought and any assumptions and simpliﬁcations made. Page 13 Critical appraisal of individual sources of evidence § 12 If done, provide a rationale for conducting a critical appraisal of included sources of evidence; describe the methods used and how this information was used in any data synthesis (if appropriate). - Synthesis of results 13 Describe the methods of handling and summarizing the data that were charted. Page 11–12 RESULTS Selection of sources of evidence 14 Give numbers of sources of evidence screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally using a ﬂow diagram. Page 16–18 and 23–25 Characteristics of sources of evidence 15 For each source of evidence, present characteristics for which data were charted, and provide the citations. Page 16–18 and 23–25 Critical appraisal within sources of evidence 16 If done, present data on critical appraisal of included sources of evidence (see item 12). - Appendix B Table A2. Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist. Pediatr. Rep. 2023, 15 193 Table A2. Cont. SECTION ITEM PRISMA-ScR CHECKLIST ITEM REPORTED ON PAGE # Results of individual sources of evidence 17 For each included source of evidence, present the relevant data that were charted that relate to the review questions and objectives. Page 16–18 and 23–25 Synthesis of results 18 Summarize and/or present the charting results as they relate to the review questions and objectives. Page 26 DISCUSSION Summary of evidence 19 Summarize the main results (including an overview of concepts, themes, and types of evidence available), link to the review questions and objectives, and consider the relevance to key groups. Page 13–15 and 19–22 Limitations 20 Discuss the limitations of the scoping review process. Page 26–27 Conclusions 21 Provide a general interpretation of the results with respect to the review questions and objectives, as well as potential implications and/or next steps. Page 27–28 FUNDING Funding 22 Describe sources of funding for the included sources of evidence as well as sources of funding for the scoping review. Describe the role of the funders of the scoping review. N.A. JBI = Joanna Briggs Institute; PRISMA-ScR = Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews. Appendix A * Where sources of evidence (see second footnote) are compiled from, such as bibliographic databases, social media platforms, and Web sites. † A more inclusive/heterogeneous term used to account for the different types of evidence or data sources (e.g., quantitative and/or qualitative research, expert opinion, and policy documents) that may be eligible in a scoping review as opposed to only studies. This is not to be confused with information sources (see ﬁrst footnote). ‡ The frameworks by Arksey and O’Malley (6) and Levac and colleagues (7) and the JBI guidance (4, 5) refer to the process of data extraction in a scoping review as data charting. § The process of systematically examining research evidence to assess its validity, results, and relevance before using it to inform a decision. This term is used for items 12 and 19 instead of “risk of bias” (which is more applicable to systematic reviews of interventions) to include and acknowledge the various sources of evidence that may be used in a scoping review (e.g., quantitative and/or qualitative research, expert opinion, and policy document). From: Tricco, A.C.; Lillie, E.; Zarin, W.; O’Brien, K.K.; Colquhoun, H.; Levac, D.; Moher, D.; Peters, M.D.J.; Horsley, T.; Weeks, L.; et al. PRISMA Extension for Scoping Reviews (PRISMAScR): Checklist and Explanation. Ann. Intern. Med. 2018, 169, 467–473. https://doi.org/10.7326/M18-0850. [68]. Table A2. Cont. References 1. Zablotsky, B.; Black, L.I.; Maenner, M.J.; Schieve, L.A.; Danielson, M.L.; Bitsko, R.H.; Boyle, C.A. Prevalence and trends of developmental disabilities among children in the United States: 2009–2017. Pediatrics 2019, 144, e20190811. [CrossRef] [PubMed] 2. Sutherland, G.; Couch, M.A.; Iacono, T. Health issues for adults with developmental disability. Res. 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https://openalex.org/W3199645825	https://iiste.org/Journals/index.php/JEES/article/download/57024/58888	English	null	Assessment of Indigenous Knowledge of Farmers on Soil Fertility Management: In Case of Bule Hora Woreda, West Guji Zone, Bule Kegna Kebele	null	2,021	cc-by	6,817	1. INTRODUCTION Agriculture remains the backbone of the Ethiopia economy and so deserves critical attention. Soil fertility was a fundamental in determining the productivity of all farming system. People of world depend up on soil to produce food crop. Where the soil was fertile and where the society has the means to improve fertility good crop can be grown. As mentioned in Taffa (2002) economic sustainability develops from good soil used intelligently and protect the soil erosion are unnecessary wastage of rain fall by excessive run off. In Ethiopia soil fertility problem was the results of human failures to understood and manage the soil .In the most part of agrarian country the soil fertility problem was the accumulation effects of deforestation, over grazing, burning of forests, clearing of ground cover (Taffa, 2002). According to FAO (2001), the decline of soil productivity was attributed to high population preserve which intern leads to deforestation, over grazing and misses used of land. Problem of soil fertility was perceived as the greatest threat to sustainability of agricultural production in the developing countries. According to Taffa (2002),the role of indigenous knowledge that contributes soil fertility management through practiced of physical ,biological and mechanical way have central and cuties role at grass root level .Indigenous knowledge plays a crucial role in conserving of natural resource ,forest, soil etc. through applying different kinds of soil fertility management practice. In this study, local or indigenous knowledge was defined as skills and practices of the people living in a certain area generated by their own and their ancestors' experience as well as those originating from elsewhere which has been internalized by the local people. Soil fertility management refers to any strategy employed by farmers to maintain and possibly increase soil fertility for sustaining crop productivity through optimizing all possible sources of plant nutrients needed for crop growth and appropriate to each crop system and specific ecological and socioeconomic situation. The Indigenous knowledge plays crucial role in improved of soil fertility specifically at the Guji zone, south of Ethiopia particular in Bule Hora Woreda. The soil fertility problem in this area might be due to depletion of natural resource, mis-management of natural resource, poor farming system and failure of practiced, which result in high erosion, depletion of nutrient from plant root, deterioration of physical soil surface and less productive soil. Journal of Environment and Earth Science Journal of Environment and Earth Science Journal of Environment and Earth Science ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.11, No.8, 2021 www.iiste.org www.iiste.org Abstract The study was conducted in Bule Kenga kebele to assess the indigenous knowledge of community on soil fertility management. The main objective of this study was to assess the Indigenous knowledge of farmers on soil fertility management in the study area. The data was collected through household survey, interviews, questionnaire and observation. To do so, one samples kebeles were used. Accordingly, the majority of the respondents revealed that indigenous soil fertility management together with natural resource management was very significant, at community level. Local farmers know their own strength, forces knowledge, and technique to conserve soil even hilly lands. The major indigenous knowledge of soil fertility management in the study area was crop rotation, Organic fertilizer, Agroforestry, intercropping, and green manure. As a result the, majority of respondents (57%) reported that they had knowledge on the soil fertility management whereas 43% had no knowledge on soil fertility management. As the result shows, out of the total respondents, 60%, 26%, 9%, and 6 % of the respondents said role of indigenous knowledge of soil fertility improvement were good, better, best and bad, respectively. The traditional land management practices improved cropland productivity through addition of organic matter to the soil, adding nitrogen, maintaining organic matter and plant nutrients, and improving soil structures increasing water infiltration and reducing run off. Lastly local knowledge ought not to be ignored because it has own attribution to reduce the soil fertility problems which in line with the current study area DOI: 10.7176/JEES/11-8-03 Publication date:August 31st 2021 Assessment of Indigenous Knowledge of Farmers on Soil Fertility Management: In Case of Bule Hora Woreda, West Guji Zone, Bule Kegna Kebele KOAT MACHAR1 Abdissa Debela1* 1* Department of Natural Resource Management, College of Agricultural Sciences (CAS), Bule Hora University (BHU), P.O. Box: 144, Oromia, Ethiopia 1Graduate student from Department of Natural Resource Management at Bule Hora University, Oromia, Ethiopia 4. RESULT AND DISCUSSION The results from the household survey, key informant interviews with the representatives of the community and focus group discussion with the farmers of the Bule kenga are discussed under this chapter. 1.3.2. Specific objectives 1.3.2. Specific objectives To assess farmers perception for soil fertility improvement in the study area id if i di il f ili i d h i bl i l 3.2. Specific objectives To assess farmers perception for soil fertility improvement in the study area p j To assess farmers perception for soil fertility improvement in the study area To identify indigenous soil fertility management practiced that can promote to sustainable ag production. 3.1. 4 Methods of data collection Survey was conducted to assessed Indigenous Knowledge of Farmers on Soil Fertility Management status in Bule Haro Woreda, West Guji Zone. To achieve the objective mentioned the above, data were collected through primary and secondary source. Primary data was collected from respondents by interview, questionare, focus group discussion and personal and direct observation of the study area. Secondary data was collected from book, research reports, internet and both publishes and unpublished materials and annual report of the kebele office. 1.3.1 General objectives 1.3.1 General objectives The General objective of the study was to assess the Indigenous knowledge of farmers on soil fertility management 3.1.3 Sampling size and technique p g q Bule Kenga Kebele was selected from Bule Hora Woreda purposively based on their involvement in different crop production activities, presence of soil fertility problem such as deforestation, farming system and soil erosion. Households were purposively selected based on their involvement in crop production activities. This was because of the fact that it could have been meaningless to interview farmers non-participating on crop production activities farmers about soil fertility management system in the area. The total household of the kebele is 600, (Bule hora Woreda agricultural office 2018). Because of time limitation, lack of finance and large number of households, 35 households of the respondents was selected by used purposively sampling techniques from 600 household of the Bule Kenga kebele. 1. INTRODUCTION The focus of this study, therefore, was on the indigenous knowledge perspective of soil fertility management. 16 Journal of Environment and Earth Science www.iiste.org ste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.11, No.8, 2021 1.3. Objectives of the study 1.3.1 General objectives The General objective of the study was to assess the Indigenous knowledge of farmers on soil fertility management 3.1. 1. Location and Climatic condition 3.1. 1. Location and Climatic condition The Bule hora district was one of the districts in West Guji zone, Oromia region. It is located in south from Yabello and 467km in from Addis Ababa, and in the west by southern nation and nationalities, people and north by the Ganale Dorya Rivers which separate it from Bale zone and on the east by the Somalia region. Total areas coverage was about 6021 square kilometer with an altitude range from 500-2800 m.a.s.l level and it have the 32 kebele. Annual rainfall of the district range between 750-1500mm per year and it has three agro-ecology zone, were the bada dare (Wayina dega), Bada “dega” and Gamojii (kola). Most of the kebele were found in the high land climatic zone and average temperature range from 15 to 30 and agriculture was the main sources of income for the farming communities (West Guji zone agricultural office, 2018). 3.1.2 Demographic Information and Land use system Population of Bule Hora Woreda belongs at most to five ethnic groups: namely Guji Oromo, Burji, Amara, Gurage and koira. According to the current Housing and census data appointed the total population of Bule Hora Woreda was 266,150 of which 134,603 and 131,547 was male and female respectively (Bule hora agricultural office, 2018). The rural settlement pattern was more of scattered and even in some places of the ‘kola ‘area no constant settlement pattern. However, a few farmers were seen settled at areas where social service like market and the like are situated and this created rural village. According to the Agricultural Office of the Bule Hora Woreda, (2018), some of the dominant crops that were produced in the Woreda include maize, enset, teff, barley, wheat, soybean and beans. These crops were used for home consumption. The Woreda also produces some cash crops such as coffee, chat, inset and different varieties of fruits. Livestock was the main economic based of the community of the areas, which of the most economic activities were livestock production and agricultural production (Bule hora Woreda agricultural and rural development, 20018). 3. MATERIAL AND METHOD 3.1 Description of the study area 3.1 Description of the study area 4.1. Personal Data (household Characteristics) 4.1.1. Household Size As the table 2 shows, from the field survey, the majority of 51.84% of the respondent lies in the age between 35-60, 22.8% of the respondents lies in the age between 25-35, 20% of the respondents lies in the age between 15-25, and the 5.7% of the respondents lies in the age between 7-12. According to the respondent, most of the household heads (37%) were in the age category between 25-35 years. Farmers in this age group (25-35) are to have a good understanding of the problem of soil fertility. As explored through interview, farmers of these age groups to have a good understanding on the problem of soil-water conservation, soil fertility management and usually interested in implementing soil fertility management practices than the other age group. Knowledge and skills developed during long periods of farming is of vital importance to the practice of sustainable agriculture g Age was one of the major factors that could play a significant role in the study area for the soil fertility management activities. As the table 2 shows, from the field survey, the majority of 51.84% of the respondent lies in the age between 35-60, 22.8% of the respondents lies in the age between 25-35, 20% of the respondents lies in the age between 15-25, and the 5.7% of the respondents lies in the age between 7-12. According to the respondent, most of the household heads (37%) were in the age category between 25-35 years. Farmers in this age group (25-35) are to have a good understanding of the problem of soil fertility. As explored through interview, farmers of these age groups to have a good understanding on the problem of soil-water conservation, soil fertility management and usually interested in implementing soil fertility management practices than the other age group. Knowledge and skills developed during long periods of farming is of vital importance to the practice of sustainable agriculture p g Table 2. Distribution of Farmers by age Age Number of Farmers Percentage 7-12 2 5.7% 15-25 7 20% 25-35 8 22.8% 35-60 18 51.84% >60 - - Total 35 100 4.1.3 Education Status The study has identified four educational levels in the study district: illiterate, can read and write, elementary schooling and secondary schooling. 4.1. Personal Data (household Characteristics) 4.1.1. Household Size From the survey result, about 20% of the household heads had no formal education, 11.5% of the respondent can read and write, 60% have primary schooling and 8.5 % secondary schooling as shown in the table3. Educational level of the community was one of the determining factors for management of the soil fertility. Table3.Educational back ground of respondents Educational Back ground Number of Farmers Percentage Illiterate 7 20% Only read and write 4 11.5% Secondary school 3 8.5% Primary school 21 60% Total 35 100% Source: from my survey 2018 As the questionnaire survey indicates, the majority of 42% of the respondents were protestant followed by orthodox (22%), Muslims (15%), catholic (15%) and other religion (6%) as shown in figure1. As response from group-discussion, indicate that having this different religion in one kebele have no any influence on the practice of soil fertility management Table 2. Distribution of Farmers by age Age Number of Farmers Percentage 7-12 2 5.7% 15-25 7 20% 25-35 8 22.8% 35-60 18 51.84% >60 - - Total 35 100 4.1.3 Education Status The study has identified four educational levels in the study district: illiterate, can read and write, elementary schooling and secondary schooling. From the survey result, about 20% of the household heads had no formal education, 11.5% of the respondent can read and write, 60% have primary schooling and 8.5 % secondary schooling as shown in the table3. Educational level of the community was one of the determining factors for management of the soil fertility. Table3.Educational back ground of respondents Educational Back ground Number of Farmers Percentage Illiterate 7 20% Only read and write 4 11.5% Secondary school 3 8.5% Primary school 21 60% Total 35 100% Source: from my survey 2018 As the questionnaire survey indicates, the majority of 42% of the respondents were protestant followed by orthodox (22%), Muslims (15%), catholic (15%) and other religion (6%) as shown in figure1. As response from group-discussion, indicate that having this different religion in one kebele have no any influence on the practice of soil fertility management 4.1.3 Education Status The study has identified four educational levels in the study district: illiterate, can read and write, elementary schooling and secondary schooling. 4.1. Personal Data (household Characteristics) 4.1.1. Household Size From the survey result, about 20% of the household heads had no formal education, 11.5% of the respondent can read and write, 60% have primary schooling and 8.5 % secondary schooling as shown in the table3. Educational level of the community was one of the determining factors for management of the soil fertility. Table3.Educational back ground of respondents Educational Back ground Number of Farmers Percentage Illiterate 7 20% Only read and write 4 11.5% 4.1.3 Education Status The study has identified four educational levels in the study district: illiterate, can read and write, elementary schooling and secondary schooling. From the survey result, about 20% of the household heads had no formal education, 11.5% of the respondent can read and write, 60% have primary schooling and 8.5 % secondary schooling as shown in the table3. Educational level of the community was one of the determining factors for management of the soil fertility. The study has identified four educational levels in the study district: illiterate, can read and write, elementary schooling and secondary schooling. From the survey result, about 20% of the household heads had no formal education, 11.5% of the respondent can read and write, 60% have primary schooling and 8.5 % secondary schooling as shown in the table3. Educational level of the community was one of the determining factors for management of the soil fertility. management of the soil fertility. Table3.Educational back ground of respondents Educational Back ground Number of Farmers Percentage Illiterate 7 20% Only read and write 4 11.5% Secondary school 3 8.5% Primary school 21 60% Total 35 100% Source: from my survey 2018 As the questionnaire survey indicates, the majority of 42% of the respondents were protestant followed by orthodox (22%), Muslims (15%), catholic (15%) and other religion (6%) as shown in figure1. As response from group-discussion, indicate that having this different religion in one kebele have no any influence on the practice of soil fertility management management of the soil fertility. Table3.Educational back ground of respondents Educational Back ground Number of Farmers Percentage Illiterate 7 20% Only read and write 4 11.5% Secondary school 3 8.5% Primary school 21 60% Total 35 100% management of the soil fertility. 4.1. Personal Data (household Characteristics) 4.1.1. Household Size Family was one of the social institutions that has play vital role in the process of socialization and performing collective work. As shown in table 1, the family size less than 3 accounts for 25.7%. The majority of the respondents (28.6%) were in the categories of 4-6 family members. The respondent with family size between 7and 9 accounts for 25.7 %, and the remaining 20 % had family size above 10. The nature and size of family affects the soil fertility. As clearly known soil and water conservation structure and soil fertility management 17 Journal of Environment and Earth Science www.iiste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.11, No.8, 2021 need more labor intensive so, households with larger family size make easily SWC structures and soil fertility management implement on their farm land. According to the respondents, large family size was very important for soil conservation measures because having a small number of children requires additional labor from out of family to construct and maintain soil conservation structures and managing of soil fertility such as applied manure, compost. The result consistent with the assertion that the size of households was the major constraints to the adoption of the soil fertility management practices (Franzel, 1999). T bl 1 F il i f d Journal of Environment and Earth Science ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.11, No.8, 2021 need more labor intensive so, households with larger family size make easily SWC structures and soil fertility management implement on their farm land. According to the respondents, large family size was very important for soil conservation measures because having a small number of children requires additional labor from out of family to construct and maintain soil conservation structures and managing of soil fertility such as applied manure, compost. The result consistent with the assertion that the size of households was the major constraints to the adoption of the soil fertility management practices (Franzel, 1999). Family size Number of Farmers Percentage <3 9 25.7% 4-6 10 28.5% 7-9 9 25.7% >10 7 20% Total 35 100 (Source: own field survey 2018) 4.1.2. Age-Sex characteristics 4.1.2. Age-Sex characteristics Age was one of the major factors that could play a significant role in the study area for the soil fertility management activities. 4.1. Personal Data (household Characteristics) 4.1.1. Household Size As shown in the figure2, 54% of the respondents replied that the current agricultural production is increasing when compared to the previous agricultural production. From the basis of reflection it is concluded that due to different indigenous knowledge of soil fertility practices like green manure, agro forestry, and composting and animal manure enhance the productivity of the soil. Consequently the combined integrated biophysical conservation method applying in the area intensively are collectively promotes sustainable agricultural production and this finding agreement with the Scronth and Sinclair (2002), suggested that the use of tree in agroforestry can improve the nutrient balance of the site, both by reducing unproductive nutrient loss from erosion and by increasing nutrient input, through nitrogen fixation that can improve the soil fertility and water, increasing the biological activities in the soil by providing the biomass and suitable microclimate. Journal of Environment and Earth Science ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.11, No.8, 2021 Figure1. Religion of the respondents Figure1. Religion of the respondents 4.2. Farmers’ perception on soil fertility management 4.2. Farmers’ perception on soil fertility management From the total 35 respondents as it shown in the table4, 20% of the respondent had comment on the deforestation, 68.57% of the respondent comment on the soil erosion, 5.7% of the respondents perceived on soil poor farming practices and 5.7% of respondent comment on expansion of the absence of crop rotation. Most of respondents characterized fertile soil in dark rich in biodiversity and infertile soils are red, poor in biodiversity, and less productive. Farmers stated as they always harvest good yield from fertile soil. It is the wearing always, detachment and removal of soil from one place to another and it is deposition at another through the forcing of striking, moving water and blowing wind (Hanhum, 1986) g g g ( ) Table4. Farmer’s perception on soil fertility depletion Table4. Farmer’s perception on soil fertility depletion Status No of respondents Percentage (%) Soil erosion 24 68.57% crop rotation 2 5.7% Deforestation 7 20% Poor farming practices 2 5.7% Total 35 100% Source: from my survey 2018. As shown in the figure2, 54% of the respondents replied that the current agricultural production is increasing when compared to the previous agricultural production. 4.1. Personal Data (household Characteristics) 4.1.1. Household Size From the basis of reflection it is concluded that due to different indigenous knowledge of soil fertility practices like green manure, agro forestry, and composting and animal manure enhance the productivity of the soil. Consequently the combined integrated biophysical conservation method applying in the area intensively are collectively promotes sustainable agricultural production and this finding agreement with the Scronth and Sinclair (2002), suggested that the use of tree in agroforestry can improve the nutrient balance of the site, both by reducing unproductive nutrient loss from erosion and by increasing nutrient input, through nitrogen fixation that can improve the soil fertility and water, increasing the biological activities in the soil by providing the biomass and suitable microclimate. Poor farming practices Total 4.1. Personal Data (household Characteristics) 4.1.1. Household Size Table3.Educational back ground of respondents Educational Back ground Number of Farmers Percentage Illiterate 7 20% Only read and write 4 11.5% Secondary school 3 8.5% Primary school 21 60% Total 35 100% Source: from my survey 2018 As the questionnaire survey indicates, the majority of 42% of the respondents were protestant followed by orthodox (22%), Muslims (15%), catholic (15%) and other religion (6%) as shown in figure1. As response from group-discussion, indicate that having this different religion in one kebele have no any influence on the practice of soil fertility management Source: from my survey 2018 Source: from my survey 2018 As the questionnaire survey indicates, the majority of 42% of the respondents were protestant followed by orthodox (22%), Muslims (15%), catholic (15%) and other religion (6%) as shown in figure1. As response from group-discussion, indicate that having this different religion in one kebele have no any influence on the practice of soil fertility management y y As the questionnaire survey indicates, the majority of 42% of the respondents were protestant followed by orthodox (22%), Muslims (15%), catholic (15%) and other religion (6%) as shown in figure1. As response from group-discussion, indicate that having this different religion in one kebele have no any influence on the practice of soil fertility management 18 Journal of Environment and Earth Science www.iiste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.11, No.8, 2021 Figure1. Religion of the respondents 4.2. Farmers’ perception on soil fertility management From the total 35 respondents as it shown in the table4, 20% of the respondent had comment on the deforestation, 68.57% of the respondent comment on the soil erosion, 5.7% of the respondents perceived on soil poor farming practices and 5.7% of respondent comment on expansion of the absence of crop rotation. Most of respondents characterized fertile soil in dark rich in biodiversity and infertile soils are red, poor in biodiversity, and less productive. Farmers stated as they always harvest good yield from fertile soil. It is the wearing always, detachment and removal of soil from one place to another and it is deposition at another through the forcing of striking, moving water and blowing wind (Hanhum, 1986) Table4. Farmer’s perception on soil fertility depletion Status No of respondents Percentage (%) Soil erosion 24 68.57% crop rotation 2 5.7% Deforestation 7 20% Poor farming practices 2 5.7% Total 35 100% Source: from my survey 2018. Source: from my survey 2018. As shown in the figure2, 54% of the respondents replied that the current agricultural production is increasing when compared to the previous agricultural production. From the basis of reflection it is concluded that due to different indigenous knowledge of soil fertility practices like green manure, agro forestry, and composting and animal manure enhance the productivity of the soil. Consequently the combined integrated biophysical conservation method applying in the area intensively are collectively promotes sustainable agricultural production and this finding agreement with the Scronth and Sinclair (2002), suggested that the use of tree in agroforestry can improve the nutrient balance of the site, both by reducing unproductive nutrient loss from erosion and by increasing nutrient input, through nitrogen fixation that can improve the soil fertility and water, increasing the biological activities in the soil by providing the biomass and suitable microclimate. Figure2. Shown an indigenous knowledge of farmers on status of agriculture productivity Figure2. Shown an indigenous knowledge of farmers on status of agriculture productivity Figure2. Shown an indigenous knowledge of farmers on status of agriculture productivity 19 Journal of Environment and Earth Science www.iiste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.11, No.8, 2021 Vol.11, No.8, 2021 4.2.1 .Indigenous knowledge of farmers on soil fertility depletion This deal with the attitudes of farmer towards soil conservation and also deal with the knowledge of local people have on soil fertility depletion. As show in the table5 below 74.3% of the respondents had knowledge on the soil fertility depletion and 25.7% of the respondents had no knowledge on soil fertility depletion ,the role of indigenous knowledge for soil fertility depletion in the study area as the same the other researcher said before and this finding agreement with the (Toflg, 2001). Because the indigenous of the farmer were very important for the soil fertility management practices such as manure, crop rotation, intercropping and leaf litter to some extents. Table5. Knowledge on soil fertility depletion Have knowledge on soil fertility depletion Number of respondent Percentage (%) YES 26 74.3% NO 9 25.7% Total 35 100 Source: from my survey 2018. 4.2.2. Attitude of farmers towards soil fertility improvement Most farmers have developed good attitude towards soil fertility improvement and some farmer have medium interest towards soil fertility improvement practices. Source: from my survey 2018. Role of indigenous knowledge for soil fertility management Status Farmers Percentage (%) Good 21 60% Better 9 25.71% Best 3 8.57% Bad 2 5.71% Total 35 100% Source: from my survey 2018 The majority 22.85% of the respondents were replied that it is good for soil fertility management practices such as, crop rotation and 8.57% used the crop residues and other soil fertility management practices were Farmers Indigenous Knowledge on soil fertility management practices 4.4. Farmers Indigenous Knowledge on soil fertility management practices Regarding soil fertility management as shown in table, majority of respondents (57%) reported that they had knowledge on the soil fertility management whereas, the remaining 43% had no knowledge on soil fertility management. Different finding showed that the indigenous knowledge on soil fertility improvement plays a great role. According to Young (1989), the use of trees, different agro forestry practice, use of terrace, counter bund, mulching, green manure are highly significant on soil fertility improvement. Thus, the finding of this study also agreed that the role of indigenous knowledge on soil fertility improvement contribute a major role in improving physical soil structures, maintain and increase soil fertility, conserving different biodiversity and enhancing vegetation and forest maintenance. Table7. Knowledge on the soil fertility management Have knowledge on the soil fertility management Respondent Percentage (%) YES 22 62.8% NO 13 37.14% Total 35 100% Source: from my survey 2018. As shown in the table7, out of the total respondents, 60%, 25.71%, 8.57%, and 5.71 % of the respondents said role of indigenous knowledge of soil fertility improvement were good, better, best and bad, respectively Table8. Role of indigenous knowledge for soil fertility management Status Farmers Percentage (%) Good 21 60% Better 9 25.71% Best 3 8.57% Bad 2 5.71% Total 35 100% Source: from my survey 2018 The majority 22.85% of the respondents were replied that it is good for soil fertility management practices such as, crop rotation and 8.57% used the crop residues and other soil fertility management practices were Source: from my survey 2018. Knowledge on soil fertility depletion Have knowledge on soil fertility depletion Number of respondent Percentage (%) YES 26 74.3% NO 9 25.7% Total 35 100 Source: from my survey 2018. 4.2.2. Attitude of farmers towards soil fertility improvement Most farmers have developed good attitude towards soil fertility improvement and some farmer have medium interest towards soil fertility improvement practices. The table6 shown that, most of the respondents were 54.28% low perceptions towards using soil fertility management practices, this were consistent with the previous study (Muhammed, 2007) found that labor problem, economic problem and lack of awareness were major factors that hinder farmers from performing soil fertility management practices . Table6. Farmer perceptions towards soil fertility improvement Variable Respondents Percent Low 19 54.28% Medium 9 25.71% High 7 20% Total 35 100% Source: from my survey 2018 4.4. Farmers Indigenous Knowledge on soil fertility management practices Regarding soil fertility management as shown in table, majority of respondents (57%) reported that they had knowledge on the soil fertility management whereas, the remaining 43% had no knowledge on soil fertility management. Different finding showed that the indigenous knowledge on soil fertility improvement plays a great role. According to Young (1989), the use of trees, different agro forestry practice, use of terrace, counter bund, mulching, green manure are highly significant on soil fertility improvement. Thus, the finding of this study also agreed that the role of indigenous knowledge on soil fertility improvement contribute a major role in improving physical soil structures, maintain and increase soil fertility, conserving different biodiversity and enhancing vegetation and forest maintenance. Table7. Knowledge on the soil fertility management Have knowledge on the soil fertility management Respondent Percentage (%) YES 22 62.8% NO 13 37.14% Total 35 100% Source: from my survey 2018. As shown in the table7, out of the total respondents, 60%, 25.71%, 8.57%, and 5.71 % of the respondents said role of indigenous knowledge of soil fertility improvement were good, better, best and bad, respectively Table8. Source: from my survey 2018. The table6 shown that, most of the respondents were 54.28% low perceptions towards using soil fertility management practices, this were consistent with the previous study (Muhammed, 2007) found that labor problem, economic problem and lack of awareness were major factors that hinder farmers from performing soil fertility management practices . Table6. Farmer perceptions towards soil fertility improvement Variable Respondents Percent Low 19 54.28% Medium 9 25.71% High 7 20% Total 35 100% Source: from my survey 2018 4.2.1 .Indigenous knowledge of farmers on soil fertility depletion hi d l i h h i d f f d il i d 4.2.1 .Indigenous knowledge of farmers on soil fertility depletion 4.2.1 .Indigenous knowledge of farmers on soil fertility depletion This deal with the attitudes of farmer towards soil conservation and also deal with the knowledge of local people have on soil fertility depletion. As show in the table5 below 74.3% of the respondents had knowledge on the soil fertility depletion and 25.7% of the respondents had no knowledge on soil fertility depletion ,the role of indigenous knowledge for soil fertility depletion in the study area as the same the other researcher said before and this finding agreement with the (Toflg, 2001). Because the indigenous of the farmer were very important for the soil fertility management practices such as manure, crop rotation, intercropping and leaf litter to some extents. 4.2.1 .Indigenous knowledge of farmers on soil fertility depletion This deal with the attitudes of farmer towards soil conservation and also deal with the knowledge of local people have on soil fertility depletion. As show in the table5 below 74.3% of the respondents had knowledge on the soil fertility depletion and 25.7% of the respondents had no knowledge on soil fertility depletion ,the role of indigenous knowledge for soil fertility depletion in the study area as the same the other researcher said before and this finding agreement with the (Toflg, 2001). Because the indigenous of the farmer were very important for the soil fertility management practices such as manure, crop rotation, intercropping and leaf litter to some extents. T bl 5 K l d il f tilit d l ti g g ( g, ) g y p the soil fertility management practices such as manure, crop rotation, intercropping and leaf litter to some extents. Table5. 5. Conclusion and Recommendation Based on the finding of the study, soil fertility depletion recognized in the Bule Kenga Kebele was mostly resulted from human activities that could accelerate soil erosion in the study area. Topography, deforestation, improve land use system, over grazing, in appropriate tillage are the main cause of soil erosion. The clearing of forest and disturbance of its ecosystem exposed the fertile soil for erosion and this in turn decrease the fertility of the soil. Farmers have developed conservation and land management system on own knowledge which is the basis for sustainable agriculture and natural resource management. Apparently, indigenous soil fertility practices are many various biophysical, agro economic and different soil fertility management practices are considered as building block in designing the new ones. The study area revealed different indigenous soil fertility management practices which are used for sustainable SFM practices while conserving soil, land, and natural resource by indigenous community dwelling in the area. According to in the study area, respondent indicated that traditional indigenous soil fertility management is every aspect as contributes to sustainable agricultural productivity. Source: from my survey 2018. Ethiopian farmers use household refuse and leaf litter to improve soil fertility that was agreement with the (Murdoch book, 2004). 17.14%, 8.57% and 5.71% for agroforestry, green manure and composting, respectively while mulching, tillage and intercropping were 8.57%, 5.71%, 2.85% and 8.57%, respectively. There were a number of soil fertility management practices that are important for the improving the fertility of the soils (Morgan, 1996). These include organic fertilizer, inorganic fertilizer, terracing, intercropping and others. The majority of the respondents use other soil fertility management practices include cover crop, mixed cropping, and others, while inorganic fertilizer had not been well available due to lack of awareness and lack of capital to apply practices. Indigenous soil fertility management practices were very important to improve soil fertility and increasing of the agricultural productivity. In the study area, some farmers used cow dung to add fertility to the soil where farmers feel that the soil exhausted they add cow dung to its. This was more frequently used in the areas when the mixed farming was more dominant. Ethiopian farmers use household refuse and leaf litter to improve soil fertility that was agreement with the (Murdoch book, 2004). Figure3. Shows methods of soil fertility management in the study area Figure3. Shows methods of soil fertility management in the study area Source: from my survey 2018. y y The majority 22.85% of the respondents were replied that it is good for soil fertility management practices such as, crop rotation and 8.57% used the crop residues and other soil fertility management practices were 20 Journal of Environment and Earth Science www.iiste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.11, No.8, 2021 17.14%, 8.57% and 5.71% for agroforestry, green manure and composting, respectively while mulching, tillage and intercropping were 8.57%, 5.71%, 2.85% and 8.57%, respectively. There were a number of soil fertility management practices that are important for the improving the fertility of the soils (Morgan, 1996). These include organic fertilizer, inorganic fertilizer, terracing, intercropping and others. The majority of the respondents use other soil fertility management practices include cover crop, mixed cropping, and others, while inorganic fertilizer had not been well available due to lack of awareness and lack of capital to apply practices. Indigenous soil fertility management practices were very important to improve soil fertility and increasing of the agricultural productivity. In the study area, some farmers used cow dung to add fertility to the soil where farmers feel that the soil exhausted they add cow dung to its. This was more frequently used in the areas when the mixed farming was more dominant. Ethiopian farmers use household refuse and leaf litter to improve soil fertility that was agreement with the (Murdoch book, 2004). Journal of Environment and Earth Science www.iiste.org 17.14%, 8.57% and 5.71% for agroforestry, green manure and composting, respectively while mulching, tillage and intercropping were 8.57%, 5.71%, 2.85% and 8.57%, respectively. There were a number of soil fertility management practices that are important for the improving the fertility of the soils (Morgan, 1996). These include organic fertilizer, inorganic fertilizer, terracing, intercropping and others. The majority of the respondents use other soil fertility management practices include cover crop, mixed cropping, and others, while inorganic fertilizer had not been well available due to lack of awareness and lack of capital to apply practices. Indigenous soil fertility management practices were very important to improve soil fertility and increasing of the agricultural productivity. In the study area, some farmers used cow dung to add fertility to the soil where farmers feel that the soil exhausted they add cow dung to its. This was more frequently used in the areas when the mixed farming was more dominant. Recommendation To meet the comprehensive, integrate, holistic and sustainable agricultural production, the following points should not be undermined. To meet the comprehensive, integrate, holistic and sustainable agricultural production, the following points should not be undermined. The relatively less practiced physical and biological soil conservation measures should be applied properly and wisely. The relatively less practiced physical and biological soil conservation measures should be applied properly and wisely. p p y y Community of indigenous knowledge of SFM should get adequate, tangible, technical and material support from government and non-governmental organization to improve or run physical and biological soil fertility conservation measures. Community of indigenous knowledge of SFM should get adequate, tangible, technical and material support from government and non-governmental organization to improve or run physical and biological soil fertility conservation measures. 21 www.iiste.org ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online) Vol.11, No.8, 2021 Thus, indigenous soil fertility management practice in all direction should be encouraged without neglecting the combined, integrated, holistic and sustainable agriculture experts, managers in study area. Thus, indigenous soil fertility management practice in all direction should be encouraged without neglecting the combined, integrated, holistic and sustainable agriculture experts, managers in study area. g g , g , g p , g y Generally the food we eat and most of minerals we use for different purpose are coming from soil directly or indirectly .So, maintenance of soil fertility and conservation of the precious soil resource is very important. g g g g p g y Generally the food we eat and most of minerals we use for different purpose are coming from soil directly or indirectly .So, maintenance of soil fertility and conservation of the precious soil resource is very important. 6. REFERENCES (2001) Soil Fertility Management in support of Food Security in sub-Saharan Africa, Food and Agricultur son, N.W.(1981).Soil Conservation B.T Bats ford LTD London. FAO (2001) Soil Fertility Management in support of Food Security in sub-Saharan Africa, Food and Agriculture. Hand son, N.W.(1981).Soil Conservation B.T Bats ford LTD London. Hunch,(1998) Degradation and Soil Conservation in Ethiopia high land a paper presented at the first International Work shop on Africa Maintain and High lands Addis Abe be October 13-27 Hunch,(1998) Degradation and Soil Conservation in Ethiopia high land a paper presented at the first International Work shop on Africa Maintain and High lands Addis Abe be October 13-27 H l h k d A R B d 2009 S il C i M d C l P 44 45 I di P d ut honker and Anson R.Betrand, 2009 .Soil Conservation Measure and Control Page 44-45.India-Perd University University Hans Humin, 1986 Guide lines for Development agent in Soil Conservation in Ethiopia. Hans Humin, 1986 Guide lines for Development agent in Soil Conservation in Ethiopia. Krubel Melese (2002).Impact Assessment of Biological Conservation Measure on the Soil and Socio-economic in Gergel area Tigray. Krubel Melese (2002).Impact Assessment of Biological Conservation Measure on the Soil and Socio-economic in Gergel area Tigray. Morgan, R.P.C,(1993) Soil Erosion and Soil Conservation. Murdoch Book (2004) Vege tation gardening, growing and Harvesting Vegetable PP. chwab and Glenn (1993).Soil and Water Conservations Engineering (4thEd)New York, John and Son. ( ) g g ( ) , Taffa Tulu (2002).Soil and Water Conservation for Sustainable Development and .Addis Ababa. Mega publishing enterprise. Taffa Tulu (2002).Soil and Water Conservation for Sustainable Development and .Addis Ababa. Mega publishing enterprise. Tofinga M (2001).An over view of traditional Farming System in the pacific Islands proceeding of IRETA work shop, Alafual campus, Journal of pacific Agriculture. Tofinga M (2001).An over view of traditional Farming System in the pacific Islands proceeding of IRETA work shop, Alafual campus, Journal of pacific Agriculture. p p p g Tom ado Tanah,(2oo2).Teaching Material for principle and practices of GDP Production. Tom ado Tanah,(2oo2).Teaching Material for principle and practices of GDP P Young, A.(1989).Agroforestry for Soil Conservation CAB International UKs. Young, A.(1989).Agroforestry for Soil Conservation CAB International UKs. 22
https://openalex.org/W2906876974	https://europepmc.org/articles/pmc6339216?pdf=render	English	null	Consumer’s Willingness to Pay a Premium for Organic Fruits in China: A Double-Hurdle Analysis	International journal of environmental research and public health/International journal of environmental research and public health	2,019	cc-by	10,488	Keywords: organic fruits; China; double-hurdle model; premium; willingness to pay Keywords: organic fruits; China; double-hurdle model; premium; willingness to pay International Journal of Environmental Research and Public Health Lijia Wang 1,2, Jianhua Wang 3,* and Xuexi Huo 4 1 State Key Laboratory of Grassland Agro-Ecosystems, Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, Lanzhou University, Lanzhou 730020, China; wanglijia@lzu.edu.cn 1 State Key Laboratory of Grassland Agro-Ecosystems, Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, Lanzhou University, Lanzhou 730020, China; wanglijia@lzu.edu.cn 2 College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China 3 School of Business, Jiangnan University, Wuxi 214122, China 4 College of Management and Economics, Northwest A&F University, Yangling, Xi’an 712100, China; xuexihuo@nwsuaf.edu.cn 2 College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China 3 School of Business, Jiangnan University, Wuxi 214122, China 4 College of Management and Economics, Northwest A&F University, Yangling, Xi’an 712100, China; xuexihuo@nwsuaf.edu.cn * Correspondence: jianhua.w@jiangnan.edu.cn; Tel.: +86-182-5154-9736 * Correspondence: jianhua.w@jiangnan.edu.cn; Tel.: +86-182-5154-9736 Consumer’s Willingness to Pay a Premium for Organic Fruits in China: A Double-Hurdle Analysis Lijia Wang 1,2, Jianhua Wang 3,* and Xuexi Huo 4 Int. J. Environ. Res. Public Health 2019, 16, 126; doi:10.3390/ijerph16010126 Received: 8 November 2018; Accepted: 27 December 2018; Published: 5 January 2019 Abstract: The aim of the paper was to assess how consumers evaluate organic labeled fruits and to what extent they are willing to pay a premium for fresh fruits with organic labels. A double-hurdle model is applied to data obtained by interviewing 407 fresh fruit consumers in nine Chinese cities. Willingness-to-pay a premium was modeled as a function of a series of demographic, socio-economic variables, plus fruit attributes, perceptions of fruit safety, and risk attitudes. Results indicate that the most important factors inﬂuencing willingness to pay a premium involved positive attitudes toward organic label, attention to fruit safety, the perception of importance of fruit attributes. Moreover, the more income consumers earn, the more likely they would be willing to pay a premium for organic fresh fruits. The recorded consumer interest in safety and quality of fresh fruits reveals that a promising market for organic fruits could be developed by an adequate knowledge on organic label and an effective market monitoring system. 1. Introduction In 2004, General Administration of Quality Supervision, Inspection, and Quarantine of PRC (AQSIQ) promulgated “Management measures of organic products certification”, and established national standards of organic products (GB/T 19630.1‐19630.4‐2005) in 2005. The Organic Products Regulations define specific requirements for organic products to be labeled as organic or that bear the organic agricultural product legend (logo) (Figure 1). Figure 1. China Organic Product Certification Mark. Source: https://www.organic‐bio.com/en/labels. Figure 1. China Organic Product Certiﬁcation Mark. Source: https://www.organic-bio.com/en/labels. Figure 1. China Organic Product Certification Mark Source: https://www organic‐bio com/en/labels Figure 1. China Organic Product Certiﬁcation Mark. Source: https://www.organic-bio.com/en/labels. With the passage of the National Organic Program (NOP) in 2002 creating uniform USDA standards, the organic food industry has increased tremendously in terms of size and product scope [9] According to Research Institute of Organic Agriculture (FiBL), by 2014 the land area of organic agriculture in China had reached 1.9 million hectares. This is just behind Australia (22.7 million hectares), Argentina (3.1 million hectares) and the United States of America (2.0 million hectares). In 2016, the organic culture area reached 2.3 million hectares, compared to 1.6 million hectares in 2015. Therefore, China occupies the third place in the world regarding the area dedicated to organic agriculture [10]. Meanwhile, the sales volume of organic products in domestic China has been continuously increasing [11]. By the end of 2015, the sales volume of organic products in domestic China had reached 129.9 billion RMB, 10.95 thousand production enterprises received certification which was officially approved by National Standard GB/T19630 Organic Products [12] With the passage of the National Organic Program (NOP) in 2002 creating uniform USDA standards, the organic food industry has increased tremendously in terms of size and product scope [9] According to Research Institute of Organic Agriculture (FiBL), by 2014 the land area of organic agriculture in China had reached 1.9 million hectares. This is just behind Australia (22.7 million hectares), Argentina (3.1 million hectares) and the United States of America (2.0 million hectares). In 2016, the organic culture area reached 2.3 million hectares, compared to 1.6 million hectares in 2015. Therefore, China occupies the third place in the world regarding the area dedicated to organic agriculture [10]. Meanwhile, the sales volume of organic products in domestic China has been continuously increasing [11]. 1. Introduction By the end of 2015, the sales volume of organic products in domestic China had reached 129.9 billion RMB, 10.95 thousand production enterprises received certiﬁcation which was ofﬁcially approved by National Standard GB/T19630 Organic Products [12]. which was officially approved by National Standard GB/T19630 Organic Products [12]. Although the organic market in China is not fully developed, the consumption of organic food has started to grow in large cities [13]. With growing numbers of consumers concerned about health, organic fruits which contribute many nutritional benefits to human health and to overcome disease hazards [14], have started to become popular among Chinese consumers. However, consumption of organic fresh fruits has not risen to the same degree, due to the extremely higher price [11]. The sales volume of organic fruits and nuts reached 8.6 billion RMB, only accounts for 6.6% of the total organic products in domestic China in 2015 [12]. Yin et al. found that 79% of Chinese consumers perceived organic food as expensive and only 16% judged the price as reasonable [8] Although the organic market in China is not fully developed, the consumption of organic food has started to grow in large cities [13]. With growing numbers of consumers concerned about health, organic fruits which contribute many nutritional beneﬁts to human health and to overcome disease hazards [14], have started to become popular among Chinese consumers. However, consumption of organic fresh fruits has not risen to the same degree, due to the extremely higher price [11]. The sales volume of organic fruits and nuts reached 8.6 billion RMB, only accounts for 6.6% of the total organic products in domestic China in 2015 [12]. Yin et al. found that 79% of Chinese consumers perceived organic food as expensive, and only 16% judged the price as reasonable [8]. organic food as expensive, and only 16% judged the price as reasonable [8]. Results of the study should help organic fruits producers and retailers to adjust marketing strategies according to market demand. Understanding consumers’ perception and attitude toward organic fruits would provide insight for policymakers in formulating regulation to restore consumer confidence. 1. Introduction An outbreak of Escherichia coli O157:H7 in 2006 prompted the U.S. Food and Drug Administration (FDA) to issue warnings about the safety of fresh bagged spinach and recommend consumers not to eat it [1]. In 1998, the US FDA developed guidelines to reduce microbial food hazards, particularly in the context of fresh fruits and vegetables [2]. In Europe, the “Euro-leaf” certiﬁcation for organic food was launched in 1 July 2010. The use of the logo is mandatory for pre-packaged foods [3]. Organic fruits and vegetables which are free from chemicals have rapidly emerged as an important food industry in the developed countries [4]. In China, a large number of consumers panicked about all kinds of dairy products, particularly milk powder, after the 2008 melamine milk crisis. During the decade from January 2001 to January 2010, up to 1460 food quality safety incidents occurred [5]. The food scandals made food consumption in China very risky and consumers began to search for alternative safe food consumption options which ultimately led consumers toward organic food consumption as well [6]. The Chinese government has approved a series of tougher food safety laws and regulations to keep the food supply safe [7]. In 2010 and 2011, the Special Operation against Quality Safety Problems of Agricultural Products led by the Ministry of Agriculture and the Special Operation Combating Illegal Food Additives carried out by the State Administration of Food and Drug Safety has achieved positive results. Meanwhile, organic food, green food, non-harmful products and good agricultural Int. J. Environ. Res. Public Health 2019, 16, 126; doi:10.3390/ijerph16010126 www.mdpi.com/journal/ijerph 2 of 14 2 of 14 Int. J. Environ. Res. Public Health 2019, 16, 126 I t J E i R P bli H lth 2019 16 practices (GAP) have been successively put into use to confront the increasing severity of food safety and environmental concerns [8]. Organic products have developed quickly in China. In 2004, General Administration of Quality Supervision, Inspection, and Quarantine of PRC (AQSIQ) promulgated “Management measures of organic products certiﬁcation”, and established national standards of organic products (GB/T 19630.1-19630.4-2005) in 2005. The Organic Products Regulations deﬁne speciﬁc requirements for organic products to be labeled as organic or that bear the organic agricultural product legend (logo) (Figure 1). agricultural practices (GAP) have been successively put into use to confront the increasing severity of food safety and environmental concerns [8]. Organic products have developed quickly in China. 2. Literature Review The increase in the organic food market size has led to a number of studies on understanding consumers concerns, behavior and their readiness to pay for organic food which directly inﬂuences the organic market. Price premium, the excess prices paid over and above the “fair” price that is justiﬁed by the “true” value of the product [15], may be indicators of consumer’s demand for the products [16]. Thus, willingness-to-pay (WTP) for organic products can be a good predictor of organic food demand [17]. Historically, issues concerning consumers’ WTP price premium for organic food have been extensively discussed. Studies had shown that age, income, education, regional difference, attitude towards certiﬁed traceability system were signiﬁcant factors affecting consumer WTP a price premium for certiﬁed food [18,19]. Nilsson et al. found that concerns about food safety had signiﬁcantly affected consumers’ WTP for price premiums [20]. Schott and Bernard concluded that farm size affected consumer WTP for milk products [21]. In a survey of cities in China, Zheng et al. found that consumers were willing to pay a premium for organic attributes, taking soymilk as an example [13]. A more recent survey study disclosed that Indian consumers were willing to pay relatively more for environmentally certiﬁed production practices than their Chinese or UK counterparts [22]. Van Loo et al. pointed out that consumers were willing to pay a premium of 1.193 $/lb (34.8%) for the general organic label and 3.545 $/lb (103.5%) for the USDA organic label [23]. Little research has been dedicated to analyzing Chinese consumer preferences for organic fresh produce, particularly on their willingness to pay a premium for certiﬁed fresh fruits. From a pioneering study in Japan, He et al. revealed that Japanese consumers were willing to pay 64,300 yen per year for food with higher safety guarantees imported from China on average [24]. Huang and Lee reported that Chinese respondents were willing to pay $22.0 extra per year to buy organic certiﬁed agricultural standards milk [25]. A variety of methodological approaches are applied to study consumers’ WTP for premium organic food. A number of studies have designed choice experiments to elicit WTP values from respondents [26–28]. Choice experiments allow respondents to choose among products instead of rating or ranking them, which likely simulates real purchasing situations more closely [29]. But other scholars argue that choice experiments provide several hypothetical purchasing scenarios which may lead to hypothetical bias [30]. 1. Introduction Overall, taking into consideration of the rising interest of consumers for healthy quality fruits, the article aims to understand consumers’ WTP a premium price for organic fresh fruits and elicit factors affecting their payment behavior Results of the study should help organic fruits producers and retailers to adjust marketing strategies according to market demand. Understanding consumers’ perception and attitude toward organic fruits would provide insight for policymakers in formulating regulation to restore consumer conﬁdence. Overall, taking into consideration of the rising interest of consumers for healthy quality fruits, the article aims to understand consumers’ WTP a premium price for organic fresh fruits and elicit factors affecting their payment behavior. elicit factors affecting their payment behavior. The paper is structured into five sections. The next section reviews the literature on consumer’s WTP for organic food and various research approaches. Section 3 covers the econometric model. This is followed by a description of survey design, sampling procedure. The descriptive statistical results are illustrated in Section 4. The empirical results including WTP a premium choice strategy and its impact on the premium are reported in Section 5. Discussions are finally presented along with the key recommendations for action The paper is structured into ﬁve sections. The next section reviews the literature on consumer’s WTP for organic food and various research approaches. Section 3 covers the econometric model. This is followed by a description of survey design, sampling procedure. The descriptive statistical results are illustrated in Section 4. The empirical results including WTP a premium choice strategy and its impact on the premium are reported in Section 5. Discussions are ﬁnally presented along with the key recommendations for action. Int. J. Environ. Res. Public Health 2019, 16, 126 3 of 14 2. Literature Review Therefore, a combination of sensory evaluation and experimental auctions was employed to analyze consumers’ WTP [31–33]. Besides, other methods such as binary probit analysis [34], interval regression method [18] were applied as well. To date, the use of the double hurdle model to identify important consumer characteristics that are associated with WTP for organic food has increased. Katare et al. used the double-hurdle model to evaluate the impact of information on consumers’ WTP for nano-packaged food products [35]. Shi et al. employed a double hurdle count data model to explore the different consumer structures in the fresh and frozen blueberry markets [36]. Studies carried out in the African food market showed that consumers were willing to pay a premium for food-safety [1]. However, this approach has seen little use to study Chinese consumers’ WTP for organic fresh fruits. 3. Methods: Double-Hurdle Model In the study, a substantial number of participants prefer not to pay a premium for organic fruits and submit a zero answer implying a zero WTP. Assuming that for each individual the decision whether or not to pay a premium price for organic fresh fruits and the decision about the amount paid to organic fruits are made independently, the double-hurdle model introduced by Cragg is applied to identify the factors that inﬂuence respondent’s WTP [37]. Because a hurdle model is a modiﬁed count model in which the two processes generating the zeros and the positives are not constrained to be the same [38]. Consumers in our research make two decisions with respect to the WTP: whether willing to pay a premium price for organic fresh fruits, and how much a positive amount is paid by consumers for the Int. J. Environ. Res. Public Health 2019, 16, 126 4 of 14 organic fruit. Let Cj be thejth j-th consumer’s premium for the organic fruit, then the probability of the consumer choosing not to pay a premium price (Cj = 0) can be written as: Prob(Cj = 0) = Φ(−α1′Xj) (1) (1) where Φ is the standard normal density function, Xj represents a vector for consumer jth socio-economic demographics, attitude towards fruit safety and perception of the importance degree of various fruit attributes, α1 is a vector of coefﬁcients. The second hurdle determines the effect of independent variables on Cj given that Cj > 0. It is with respect to how much to pay for organic fresh fruits given that the WTP consumer has decided to buy and pay. The distribution of Cj conditional on being positive is truncated at zero with mean α2Xj and variance σ2. The form of the second hurdle function is then: L(Cj Cj > 0) = (1/σ)Φ (Cj −α2′Xj)/σ Φ(α2′Xj/σ) (2) (2) where α2 denotes a vector of coefﬁcients. The hypothesis that the two stages are independent decisions is tested by a likelihood ratio statistic. The two models can be estimated simultaneously when the error terms in the two models are assumed to be correlated, or separately when they are assumed to be independent [39]. Several research has used the same set of independent variables in the two models (hurdles), especially when data are limited [40]. 3. Methods: Double-Hurdle Model In this study, the same set of independent variables in the binomial probability model and truncated-at-zero count model are employed while the two models are estimated separately largely due to data limitation. In order to interpret the percentage changes in the probability that a consumer is willing to pay a premium for organic fresh fruits due to a unit change of the explanatory variable of interest given others being constants, marginal effects are calculated in the binary Probit model [41]. With regard to dummy explanatory variables, the marginal effect evaluates the probability changes caused by a discrete change from 0 to 1 [42]. 4.1. Survey Design Overall, from the perspective of economic development, the nine cities could represent the developed developing and undeveloped regions in China; from a geographical view they Overall, from the perspective of economic development, the nine cities could represent the developed, developing and undeveloped regions in China; from a geographical view, they distributed in central, northern, southern, western parts of China. Overall, from the perspective of economic development, the nine cities could represent the developed, developing and undeveloped regions in China; from a geographical view, they distributed in central, northern, southern, western parts of China. l, northern, southern, western parts of China. Figure 3. Location of the sample cities in China. Figure 3. Location of the sample cities in China. Figure 3. Location of the sample cities in China. Figure 3. Location of the sample cities in China. Figure 3. Location of the sample cities in China. A self‐completion paper questionnaire was used to obtain the data, coupled with a face‐to‐face interview. In each city, five chain supermarkets located in different parts of the city were selected, the survey was conducted from 9 to 11 am, and 3 to 5 pm in the weekend so that the collected consumers could be more representative. To guarantee the respondents were all fruit consumers, the interviews standing in front of the supermarket were required to question consumer who came from the supermarket with a receipt showing that he/she bought a kind of fruit from the supermarket. Finally 50 urban consumers participated in the survey and a total of 450 questionnaires were A self‐completion paper questionnaire was used to obtain the data, coupled with a face‐to‐face interview. In each city, five chain supermarkets located in different parts of the city were selected, the survey was conducted from 9 to 11 am, and 3 to 5 pm in the weekend so that the collected consumers could be more representative. To guarantee the respondents were all fruit consumers, the interviews standing in front of the supermarket were required to question consumer who came from the supermarket with a receipt showing that he/she bought a kind of fruit from the supermarket. Finally, 50 urban consumers participated in the survey and a total of 450 questionnaires were collected during May–August 2014. 4.1. Survey Design Public Health 2019, 16, x 5 of 14 160 Kg Grain Meat and eggs F h t bl D i are characterized as developed cities in China; Zhengzhou is an average city in central China; Taiyuan and Xi’an are characterized as developing cities in northern and northwestern China; Lanzhou and Guiyang are regarded as undeveloped cities located in western and southwestern China, respectively. Int. J. Environ. Res. Public Health 2019, 16, x 5 of 14 Int. J. Environ. Res. Public Health 2019, 16, x 5 of 14 160 Kg Grain Meat and eggs F h t bl D i Figure 2. Per Capita Consumption of Major Food of Urban Households, 1990–2012. Note: The data are compiled on the basis of the integrated household income and expenditure survey of the National Bureau of Statistics of the People’s Republic of China including urban and rural 0 20 40 60 80 100 120 140 160 1990 1995 2000 2005 2010 2011 2012 Kg Grain Meat and eggs Fresh vegetables Dairy Figure 2. Per Capita Consumption of Major Food of Urban Households, 1990–2012. Note: The data are compiled on the basis of the integrated household income and expenditure survey of the National Bureau of Statistics of the People’s Republic of China, including urban and rural households. Figure 2. Per Capita Consumption of Major Food of Urban Households, 1990–2012. Note: The data are compiled on the basis of the integrated household income and expenditure survey of the National Bureau of Statistics of the People’s Republic of China, including urban and rural households. 0 20 40 60 80 100 120 140 1990 1995 2000 2005 2010 2011 2012 g y Figure 2. Per Capita Consumption of Major Food of Urban Households, 1990–2012. Note: The data are compiled on the basis of the integrated household income and expenditure survey of the National Bureau of Statistics of the People’s Republic of China including urban and rural Figure 2. Per Capita Consumption of Major Food of Urban Households, 1990–2012. Note: The data are compiled on the basis of the integrated household income and expenditure survey of the National Bureau of Statistics of the People’s Republic of China, including urban and rural households. are compiled on the basis of the integrated household income and expenditure survey of the National Bureau of Statistics of the People’s Republic of China, including urban and rural households. households. 4.1. Survey Design Ensuring the quality of fruit is of extreme importance in China as fruits have been becoming the major constituent of the Chinese diet due to their high nutritional value. The consumption of fresh fruits in China has increased during the last two decades due to the rising recognition of the nutritional value of these agricultural products. According to the China Statistical Yearbook (1991–2015), the Chinese consumed an estimated 56.1 kg of fresh fruits per urban household in 2012, up from 41.1 kg in 1990 (Figure 2), whereas 100.1 kg per urban household of fresh vegetables were consumed by Chinese consumers in 2014, down from a high of 138.7 kg in 1990. In particular, according to the National Bureau of Statistics food availability data, urban Chinese on average consumed more fresh fruit (48.1 kg per household) than meat, eggs, and nuts (41.9 kg in total) in 2014. Fresh apples can be used as an illustrative case to interpret consumers’ WTP a premium for organic fruits for the following reasons. China is one of the largest fresh apple producers and exporters in the world [43]. Apples are an important source of different vitamins and rich in nutrients [44]. According to the Chinese Statistical Yearbook (2007–2018), the consumption of fresh apples has increased from 25.12 million tons in 2006 to 43.50 million tons in 2017. The growth rate of fresh apple consumption has reached 70% in the past 11 years in China. To gain a general picture of urban consumers’ willingness to pay a premium for organic fruits across major regions of China, the research was conducted in nine cities distributed in different parts of China. The nine cities partially represent the diverse types of cities in China (Figure 3): Beijing is the political and cultural capital city of China; Nanjing, Ningbo, and Wuhan, located in southern China, 5 of 14 Int. J. Environ. Res. Public Health 2019, 16, 126 are characterized as developed cities in China; Zhengzhou is an average city in central China; Taiyuan and Xi’an are characterized as developing cities in northern and northwestern China; Lanzhou and Guiyang are regarded as undeveloped cities located in western and southwestern China, respectively. Int. J. Environ. Res. Public Health 2019, 16, x 5 of 14 Int. J. Environ. Res. 4.1. Survey Design The 450 original survey answers were entered into computer files for further analysis and 407 respondents were eligible for the statistical analysis after the data A self-completion paper questionnaire was used to obtain the data, coupled with a face-to-face interview. In each city, ﬁve chain supermarkets located in different parts of the city were selected, the survey was conducted from 9 to 11 am, and 3 to 5 pm in the weekend so that the collected consumers could be more representative. To guarantee the respondents were all fruit consumers, the interviews standing in front of the supermarket were required to question consumer who came from the supermarket with a receipt showing that he/she bought a kind of fruit from the supermarket. Finally, 50 urban consumers participated in the survey and a total of 450 questionnaires were collected during May–August 2014. The 450 original survey answers were entered into computer ﬁles for further analysis, and 407 respondents were eligible for the statistical analysis after the data cleaning. Finally, 50 urban consumers participated in the survey and a total of 450 questionnaires were collected during May–August 2014. The 450 original survey answers were entered into computer files for further analysis, and 407 respondents were eligible for the statistical analysis after the data files for further analysis, and 407 respondents were eligible for the statistical analysis after the data cleaning. A series of questions about respondents’ attitudes are questioned. All interviewees were asked A series of questions about respondents’ attitudes are questioned. All interviewees were asked a number of close-ended questions divided into three parts. The ﬁrst part mainly concerned respondent’s 6 of 14 Int. J. Environ. Res. Public Health 2019, 16, 126 socio-demographic characteristics including age, educational level, gender, occupation, family size, monthly income, working conditions, and location of residence, etc. The second section was dedicated to acquiring information on consumers’ preferences to fruit-speciﬁc attributes covering sales price, taste, appearance, color, variety, wrapping, province of origin, and nutritional value applying a ﬁve-point Likert scale. These statements were designed to test consumers’ attitudes toward the importance of fresh fruit attributes in their payment decisions. In the ﬁnal section, respondents’ perception and knowledge of organic label, their judgment on safety food, and trust in current monitoring the situation of food quality safety issues by the State Council, ministries, and local governments at all level were investigated. 4.2. Description of Variables Table 1 contains a description of the variables used in the study, along with their associated summary statistics. ble 1 contains a description of the variables used in the study, along with their associated ry statistics. Table 1 contains a description of the variables used in the study, along with their associated summary statistics. Table 1. Summary statistics for empirical variables. Variable Description Variable Scale Mean Std. Dev. WTP Willingness to pay a premium 1 = Willing to pay; 0 = Unwilling to pay 0.61 0.49 MONE The premium pay for organic fresh fruit yuan/kg 0.69 0.85 Socio-economics characteristics SEX Gender 1 = male; 0 = female 0.46 0.50 AGE Age Years 38.96 14.66 EDU Education a Years of schooling 16.04 2.97 SIZE Household size Number of individuals 3.07 0.93 INCM Monthly income 1–5 b 2.37 1.52 OCCP Occupation 1 = Well educated; 0 = Blue collar 0.78 0.41 DEVE Location of residence 1 = developed city; 0 = otherwise 0.49 0.50 PPRI Purchase price yuan/kg 7.58 3.93 CFRE Consumption frequency 1–5 c 3.57 0.97 Importance attach to fruit attributes PRIC Sales price 1–5 d 3.51 0.89 WRAP Wrapping 2.30 0.87 APPE Appearance 4.00 0.76 NUTR Nutritional value 3.10 1.08 EABU Purchase convenience 3.41 0.84 PREG Province of origin 2.46 1.23 ORLB Organic label 2.41 1.01 VATY Variety 3.60 0.93 TAST Taste 4.35 0.83 Attitudes toward fruit safety WSAF Worried fruit safety 1–5 c 1.81 0.65 QUTI Notice of organic label 2.30 1.01 ATTE Concerns about fruit safety 3.23 0.80 CRED Credence in organic label 4.39 0.94 FREQ Frequency of buying unsafely fruit which causing diarrhea or other diseases 2.83 0.94 POSB Possibility of buying fruits produced by companies occurred safety incident 1.70 1.23 Note: a 6 = primary school, 9 = middle school, 12 = high school, 16 = bachelor degree, 19 = master degree, 22 = PhD; b 1 = less than 3000 yuan, 2 = (3000, 4000) yuan, 3 = (4000, 5000) yuan, 4 = (5000, 6000) yuan, 5 = equal and more than 6000 yuan; c 1 = very low, 2 = low, 3 = moderate, 4 = high, 5 = very high; d 1 = not importance at all, 2 = not important, 3 = moderate, 4 = important, 5 = very important. Table 1. Summary statistics for empirical variables. 4.1. Survey Design Particularly, in order to ﬁgure how much a positive amount consumer willing to pay for organic fruit, the question “if you are willing to purchase organic fruits, how much more you are willing to pay for organic apples compared to non-organic apples with similar size and variety?” is indicated in the questionnaire. 4.2. Description of Variables More than half of the respondents (54%) were female, which was conﬁrmed by previous literature [45,46]. This is reasonable because females are the primary shoppers for household products 7 of 14 7 of 14 e iou Int. J. Environ. Res. Public Health 2019, 16, 126 Int. J. Environ. Res. Public Health 2019, 16, x h h lf f h in China. Of the 407 respondents, 61.4% expressed WTP price premium for organic fruits, with an average premium of 0.69 yuan/kg. Moreover, the average purchase price of organic fresh apples was reported as 7.58 yuan/KGg, thus the price premium consumers willing to pay was approximately equal to 10% of the average purchase price. The average age of fruit consumer was around 39, with 16 years of formal education obtaining bachelor degree. Typically, 78% of participants had a well-educated job, with monthly income between 3000 and 5000 yuan on average. In the questionnaire, consumers were asked to rate the degree they noticed the organic label when purchasing fruits. Although most of the respondents showed highly credence in organic label, they often do not pay much attention to the label when buying fruit. Therefore, noticing an organic label only scores 2.30, but belief in the same label is 4.39. literature [45,46]. This is reasonable because females are the primary shoppers for household products in China. Of the 407 respondents, 61.4% expressed WTP price premium for organic fruits, with an average premium of 0.69 yuan/kg. Moreover, the average purchase price of organic fresh apples was reported as 7.58 yuan/KGg, thus the price premium consumers willing to pay was approximately equal to 10% of the average purchase price. The average age of fruit consumer was around 39, with 16 years of formal education obtaining bachelor degree. Typically, 78% of participants had a well‐educated job, with monthly income between 3000 and 5000 yuan on average. In the questionnaire, consumers were asked to rate the degree they noticed the organic label when purchasing fruits. Although most of the respondents showed highly credence in organic label, they often do not pay much attention to the label when buying fruit. Therefore, noticing an organic label only scores 2.30, but belief in the same label is 4.39. 4.3. Attitudes toward Fruit Safety 4.3. Attitudes toward Fruit Sa To assess consumer’s attitude towards fruit safety issues, a ﬁve-digit Likert scale was used, where 1 = very low and 5 = very high. Give the past research in this area [47–50], the following statements as explanatory variables in the matter of consumer’s WTP decision making are included. Figure 4 depicts the differences of consumers’ attitudes toward fruit safety between WTP and unwilling to pay (UWTP). To assess consumer’s attitude towards fruit safety issues, a five‐digit Likert scale was used, where 1 = very low and 5 = very high. Give the past research in this area [47–50], the following statements as explanatory variables in the matter of consumer’s WTP decision making are included. Figure 4 depicts the differences of consumers’ attitudes toward fruit safety between WTP and unwilling to pay (UWTP). 32.0% 45.2% 0.0% 15.6% 18.0% 71.2% 33.8% 75.8% 1.3% 15.9% 12.1% 73.9% 16.0% 18.0% 79.6% 31.2% 9.2% 7.6% 9.6% 1.9% 54.8% 41.4% 3.8% 7.0% 0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% WSAF QUTI CRED ATTE FREQ POSB UWTP‐High WTP‐High UWTP‐Low WTP‐Low Figure 4. Comparison of attitudes toward fruit safety between WTP and UWTP consumers. Note: For the purpose of presentation, the five digit‐scale categories were combined into three: “high” in the figure combines the frequencies of “very high” and “high” responses; “low” combines the 32.0% 45.2% 0.0% 15.6% 18.0% 71.2% 33.8% 75.8% 1.3% 15.9% 12.1% 73.9% 16.0% 18.0% 79.6% 31.2% 9.2% 7.6% 9.6% 1.9% 54.8% 41.4% 3.8% 7.0% 0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% WSAF QUTI CRED ATTE FREQ POSB UWTP‐High WTP‐High UWTP‐Low WTP‐Low Figure 4. Comparison of attitudes toward fruit safety between WTP and UWTP consumers. Note: For the purpose of presentation, the ﬁve digit-scale categories were combined into three: “high” in the ﬁgure combines the frequencies of “very high” and “high” responses; “low” combines the frequencies of “very low” and “low” responses. Figure 4. Comparison of attitudes toward fruit safety between WTP and UWTP consumers. Note: For the purpose of presentation, the five digit‐scale categories were combined into three: “high” in the figure combines the frequencies of “very high” and “high” responses; “low” combines the Figure 4. Comparison of attitudes toward fruit safety between WTP and UWTP consumers. 4.3. Attitudes toward Fruit Safety 4.3. Attitudes toward Fruit Sa Note: For the purpose of presentation, the ﬁve digit-scale categories were combined into three: “high” in the ﬁgure combines the frequencies of “very high” and “high” responses; “low” combines the frequencies of “very low” and “low” responses. frequencies of very low and low responses. In terms of fruit safety, less than 10% of consumers in both groups expressed great worry about fruit safety. 41.4% of UWTP respondents and 31.2% of WTP ones declared high attention to fruit safety issues, respectively. Concerning the notice of organic label when purchasing fruits, 16.0% of WTP consumers paid attention to the organic label compared to 9.6% of UWTP consumers, more than half of the interviewees in both group expressed no concern. 75.8% of UWTP respondents told us that they had a lower frequency of purchasing unsafely fruits, compared with 45.2% of WTP interviewees. The result suggests a positive relation between consumers’ WTP and their frequency In terms of fruit safety, less than 10% of consumers in both groups expressed great worry about fruit safety. 41.4% of UWTP respondents and 31.2% of WTP ones declared high attention to fruit safety issues, respectively. Concerning the notice of organic label when purchasing fruits, 16.0% of WTP consumers paid attention to the organic label compared to 9.6% of UWTP consumers, more than half of the interviewees in both group expressed no concern. 75.8% of UWTP respondents told us that they had a lower frequency of purchasing unsafely fruits, compared with 45.2% of WTP interviewees. The result suggests a positive relation between consumers’ WTP and their frequency of buying unsafely fruits. In addition, nearly 80.0% of WTP consumers had confidence in organic label whereas only 54.8% of UWTP ones expressed the same attitudes. This data reflects that the basic knowledge of organic label and the trust in organic label may significantly affect consumers’ WTP a premium for organic fresh fruits. Int. J. Environ. Res. Public Health 2019, 16, 126 8 of 14 4.4. Perception of Importance of Fruit Attributes To obtain the information on the perception of importance on fruit attributes that affect consumers WTP, an ordinal scale ranging from 1 = not important at all to 5 = very important was used. Consumer evaluations of the importance of fruit attributes were quite different between the two groups (Table 2). Taste and appearance were regarded as the most important attributes in both groups. Purchase convenience and the variety of fruits are perceived as the second most important attributes by WTP consumers (60.4%). The respondents in the WTP group rated the nutritional value (47.6%) comparatively highly while 18.5% of UWTP consumers perceived nutrition as important. On the other hand, substantial consumers in the UWTP category evaluated sales price as “very important” (72.0%). This might be partly the reason for their unwillingness to pay a higher price for organic fruits. In addition, wrapping appears to be less acute for all interviewees. Table 2. Perception of Importance on fruit attributes. (Percent of respondents in each category) a. Item Willing to Pay (N = 250) Unwilling to Pay (N = 157) Unimportant Moderate Important Unimportant Moderate Important Taste 3.2 11.6 85.2 4.5 7.0 88.5 Appearance 3.6 12.8 83.6 3.2 17.2 79.6 Purchase convenience 8.8 30.8 60.4 18.5 45.9 35.7 Variety 10.4 29.2 60.4 15.3 25.5 59.2 Nutritional Value 19.6 32.8 47.6 39.5 42.0 18.5 Sales price 16.8 40.0 43.2 13.4 14.6 72.0 Province of origin 49.2 22.0 28.8 62.4 28.7 8.9 Organic label 42.0 34.0 24.0 75.8 23.6 0.6 Wrapping 49.2 44.4 6.4 63.7 33.1 3.2 Note: a For the purpose of presentation, the ﬁve digit-scale categories were combined into three: “important” in this table combines the frequencies of “very important” and “important” responses; “unimportant” combines the frequencies of “not important at all” and “not important” responses. Table 2. Perception of Importance on fruit attributes. (Percent of respondents in each category) a. Note: a For the purpose of presentation, the ﬁve digit-scale categories were combined into three: “important” in this table combines the frequencies of “very important” and “important” responses; “unimportant” combines the frequencies of “not important at all” and “not important” responses. 5. Empirical Analysis The determinants of WTP identified through the double-hurdle model are reported in Table 3. The first two columns illustrate the effects of the fruit-specific attributes and socio-economic characteristics on the probability that a consumer express willingness to pay a premium for organic fresh fruits, while the determinants of the amount paid for organic fruits are presented in the last two columns. Marginal effects evaluated at the means for each of the explanatory variables are contained in Table 3. Table 3. Estimation results of the double-hurdle model. Independent Variables First Hurdle Probability of Paying Second Hurdle Amount Paid Coef. (Std. Err.) Marginal Effect Coef. (Std. Err.) Elasticity a Socio-economic characteristics SEX −0.1355 (0.2026) −0.0492 0.0939 (0.1964) 0.0838 AGE −0.0034 (0.0097) −0.0012 −0.0769 (0.0498) −0.0687 EDU 0.1018 (0.0510) 0.0369 0.0339 (0.0107) * 0.0271 SIZE −0.1286 (0.1049) −0.0467 −0.0521 (0.1086) −0.0465 INCM 0.3769 (0.1249) * 0.1368 0.1990 (0.0680) * 0.1777 OCCP 0.6892 (0.2409) * 0.2622 0.2168 (0.3441) −0.1136 DEVE 0.6381 (0.2070) * 0.2292 0.6895 (0.2129) * 0.6156 PPRI 0.1633 (0.0265) * 0.0593 0.1258 (0.0270) * 0.1123 CFRE 0.2471 (0.0843) * 0.0897 0.0887 (0.0896) 0.0792 Table 3. Estimation results of the double-hurdle model. 9 of 14 Int. J. Environ. Res. Public Health 2019, 16, 126 Table 3. Cont. Table 3. Cont. Table 3. Cont. Independent Variables First Hurdle Probability of Paying Second Hurdle Amount Paid Coef. (Std. Err.) Marginal Effect Coef. (Std. Err.) Elasticity a Importance attach to fruit attributes PRIC 0.0323 (0.1242) 0.0117 0.0899 (0.2868) 0.0803 WRAP −0.0979 (0.0711) −0.0355 0.0930 (0.1166) 0.0830 APPE 0.3205 (0.1130) * 0.1163 0.3702 (0.1245) * 0.3305 NUTR 0.3255 (0.1020) * 0.1181 0.0100 (0.1086) 0.0089 EABU 0.0398 (0.1004) 0.0144 0.3458 (0.1241) 0.3088 PREG −0.0255 (0.0771) −0.0093 0.0917 (0.1064) 0.0819 ORLE 0.5193 (0.1272) * 0.1884 0.2698 (0.0801) *** 0.2409 VATY 0.0544 (0.0978) 0.0198 0.0959 (0.1011) 0.0856 TAST 0.2071 (0.1146) * 0.0752 0.1635 (0.1287) 0.1460 Attitudes toward fruit safety WSAF 0.0442 (0.1429) 0.0160 −0.0189 (0.1422) −0.0169 QUTI 0.0457 (0.0994) 0.0166 −0.0289 (0.0967) −0.0258 ATTE 0.1536 (0.1208) * 0.0557 0.3014 (0.0999) * 0.2691 CRED 0.3678 (0.0951) 0.1334 0.3946 (0.1238) 0.3523 FREQ 0.2429 (0.1249) * 0.0881 0.0845 (0.1824) 0.0754 POSB −0.0470 (0.0746) −0.0170 −0.1596 (0.3433) −0.1425 Observations (n) 407 250 Wald χ2 162.12 72.82 Log pseudo-likelihood −168.18 −214.01 Sigma b 0.8954 Note: Standard errors which are robust are reported in parentheses. 5. Empirical Analysis Marginal effects of changing the explanatory variables are evaluated at the mean of the explanatory variables; a The elasticity is calculated at the sample mean; b Sigma is the error variance; * significant at the 10% level; significant at the 5% level; * significant at the1 percent level. Note: Standard errors which are robust are reported in parentheses. Marginal effects of changing the explanatory variables are evaluated at the mean of the explanatory variables; a The elasticity is calculated at the sample mean; b Sigma is the error variance; * significant at the 10% level; significant at the 5% level; * significant at the1 percent level. 5.1. WTP a Premium Choice Strategy A cursory look the results displayed in Table 3 emphasizes the importance of consumer’s education, income, occupation, location of residence, purchase price, and fruits consumption frequency in explaining the probability of paying a premium for organic fresh fruits by Chinese consumers. All the above mentioned statistically signiﬁcant variables have positive contributions to consumers’ WTP. Respondent’s educational background plays an important role in WTP, while the magnitude is small (3.69%). Better educated consumers are more likely to buy organic fruits at a higher price. Our ﬁndings have nonetheless corresponded with those of Chelang’a et al. [46] and Kapoor and Kumar [51], as the literature shows that high-income respondents have greater probabilities to be willing to purchase organic foods [45]. Likewise, the estimated results show that consumers living in developed provinces with decent jobs and better salary are more likely to pay a premium for organic fruits. The result is in line with the ﬁndings from Nandi et al. [52]. Besides, the WTP likelihood is higher for participants who frequently buy expensive fruits. A plausible explanation is that consumers expending higher on fruits may pay more attention to the quality and have a better income to afford the price. This ﬁnding is reinforced by the fact that the coefﬁcient on income and occupation being positive and statistically signiﬁcant at the 1% level. Consumers attaching more importance to fruit appearance, nutritional value, and organic label will show a higher WTP a premium for organic fresh fruits. The coefﬁcients are positive and statistically signiﬁcant at the 1% level. This is in consist with the ﬁndings from Tobia et al. and Wang and Huo that consumers who are more concerned about nutrition and certiﬁcate have higher willingness to pay a premium for organic food products [53,54]. Surprisingly, sales price showed a statistically insigniﬁcant impact on consumers’ WTP, probably because their incomes have reached the level to allow them to concentrate on quality and safety instead of cheap price. Similarly, wrapping, province of origin, 10 of 14 10 of 14 Int. J. Environ. Res. Public Health 2019, 16, 126 variety and purchase convenience are unimportant attributes to consumers’ WTP. During the survey we noticed that more than 80% of the participants would value wrapping as an important attribute only when the fruit was purchased as a visiting gift. 5.2. WTP a Premium Choice and Its Impact on the Premium Table 3 presents the parameter estimates and elasticity for the determinants affecting premium paid for organic fresh fruits. The elasticity reﬂects changes in the explanatory variables on the level of the amount paid by those consumers who are willing to pay a premium. As stated earlier, variables referring to consumer’s education background, income, and location of residence play a positive and signiﬁcant role on the amount paying for organic fresh fruit. In other words, respondents with more schooling years, higher salary, and live in a developed province in China observe an increased payment in the premium for organic produce of 0.03%, 0.18%, and 0.11%, respectively. Misra and Ott pointed out that consumers with more formal education probably had a better understanding of the importance of organic label on fresh produce [56]. Govindasamy and Italia discovered that a better educated consumer had a higher degree of conﬁdence in the quality certiﬁcate [57]. According to the survey data, people living in developed provinces, i.e., Beijing, Nanjing, Wuhan, were willing to pay 0.69 yuan/kg more for organic fresh fruits than those living in less developed regions such as Lanzhou and Guiyang. Other fruit attributes that have a positive impact on the premium paid by consumers who have shown a WTP are the appearance and the organic label of fruits. Hamzaoui-Essoussi and Zahaf recognized that organic label for fruit was not only perceived as a guarantee for fruit quality, but also as an assurance of the fruit safety [58]. That is, consumers who show a higher degree of conﬁdence in the label have a signiﬁcantly greater probability of WTP a premium for organic fruit and are prepared to pay more than those taking organic label unimportant. Moreover, although the impact of other fruit attributes is positive, the contribution is small and the statistical result is insigniﬁcant. The effective amount paid for organic fresh fruit is also signiﬁcantly inﬂuenced by participants’ attention to fruit safety issues. Consumers who concentrated more on food safety issues were willing to pay 0.30 yuan/kg more for organic fresh fruits than those paid less attention to food safety. Possible implications suggest that the frequent outbreaks of serious food safety incidence indeed arouse consumers WTP a premium for fresh fruits with organic labels. 5.1. WTP a Premium Choice Strategy These results are crucial for fruit growers and agro-ﬁrms to conduct cost and beneﬁt analysis during fruit planting process or before starting any consumer promotion programs for the certiﬁed policy. The signiﬁcantly positive sign for the ATTE (the concern to food safety) variable indicates that the probability of consumer WTP a premium for organic fresh fruits increases as concerns about safety issues on fresh produce increase. Consumers who frequently purchase unsafety food show a higher probability to pay a premium. This result makes sense that failure purchase experiences might stimulate higher passion of consumers on buying safety food with a premium. Wu et al. found similar results [55]. 6. Discussion Previous consumer survey works based on WTP analysis gave inconsistent results on the impact of consumer characteristics on food safety assuming that consumers’ WTP is equal to their actual payment behavior. Using the ﬁeld survey data from nine cities in China, this paper assesses consumer’s willingness to pay a premium for organic fresh fruits and investigates their paying behavior for the organic fruits, which are important for fresh producers and quality certiﬁcate institutions. Generally, it is believed that Chinese consumers have a complex attitude towards organic product largely depending on their educational background, residential location, monthly income, and attention to fruit safety. Occupation and consumption frequency signiﬁcantly affects consumers’ WTP premium for organic fruits, while the inﬂuence on the amount paid by consumers for organic fruits is statistically insigniﬁcant. Income is not only signiﬁcant in the participation stage, but also signiﬁcant in the consumption stage. Thus, economic reasons are part of the cause of the participation 11 of 14 Int. J. Environ. Res. Public Health 2019, 16, 126 and consumption of organic fresh fruits whereas social position and consumption habits are not the reasons for consumption. From the ﬁndings, we can construct a proﬁle of the consumer most probably to purchase organic fresh fruits at a premium price. Better educated individuals with higher income and living in developed cities would be more likely to exhibit a greater WTP a premium for organic fresh fruits. As an implication for organic fresh fruit dealers, these ﬁndings indicate that there exist target consumer segments for the organic fresh fruit sector in China, particularly in the developed cities. That is, marketing strategies targeted at higher income, and better-educated consumers can be effective in attracting new consumers and eliciting more purchases for organic fresh fruits. A notable result is that the degree of perception on fruit safety and the frequency of purchasing unsafely fruit are a far more compelling reason motivating a positive willingness to pay a premium for organic fresh fruit than conﬁdence in quality certiﬁcate. It appears that the unfavorable purchase experience would be more useful to inspire consumers’ payment behavior on organic fresh fruits at a premium price than simply have trust in organic label. A further marketing implication drawn from the paper pertains to the importance of certiﬁcation regarding the safety guarantee system through promulgating regulations and optimizing the management system. 6. Discussion The asymmetric nature of information leads to market distortion of food quality safety problems. The recorded consumer interest in safety and quality of fresh fruits reveals that a promising market for organic fresh fruits could be developed by an adequate knowledge on organic label and an effective market supervision system. Therefore, the provision of educational programs to help consumers correctly and clearly understating various kinds of food quality certiﬁcates by both public administrations and private sectors might be an effective strategy to up consumer WTP and to rebuild his/her conﬁdence in fresh produce certiﬁed by a regulating institution. The ﬁndings have some limitations in terms of being able to generalize. Further research is necessary so as to enlarge the samples size to make the results be more preventative and to go ahead with design improvements to mitigate social desirability bias. Social desirability bias might lead to a greater possibility of indicating a preference for paying a higher price premium for organic apples compared to respondents’ true action. Consumers might overstate the premium to provide a socially-desirable answer. The study tried to pay more attention to identify the impact factors on consumers’ WTP premium for organic fruits, the social desirability bias is thus to be acceptable in this study. But a better method, such as non-hypothetical experiments [59] should be applied in future research. Moreover, the double-hurdle model limited in that it assumes that the shocks to the participation and consumption processes are independent, which is not always a realistic assumption [60]. The model would be improved in the future study. Author Contributions: L.W. contributed to the research design, data collection and analysis, and drafted the manuscript; J.W. and X.H. revised the manuscript. Funding: We thank the three anonymous reviewers for their constructive comments on an earlier draft. Financial support was provided by the Key Projects of the National Natural Science Foundation of China (Grants 71673115), the Belt and Road Special Project of Lanzhou University (Grants 2018ldbrzd006), the China Agriculture Research System (Grants CARS-28), and the Chinese Center for Strategic Research of Grassland Agriculture Development (Grants 2016-XZ-38). Conﬂicts of Interest: The authors declare no conﬂict of interest. 1. Alphonce, R.; Alfnes, F. Eliciting consumer WTP for food characteristics in a developing context: Application of four valuation methods in an African market. J. Agric. Econ. 2017, 123–142. [CrossRef] 2. U.S. Food & Drug Administration. Guidance for Industry: Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and Vegetables; Ofﬁce of Food Safety, Division of Plant and Dairy Food Safety (HFS-317), Center for Food Safety and Applied Nutrition (CFSAN), U.S. Food & Drug Administration: Washington, DC, USA, 1998. References 1. Alphonce, R.; Alfnes, F. 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https://openalex.org/W2025170204	https://zenodo.org/records/1789630/files/article.pdf	English	null	Note on the Solubility of Benzidine Sulfate in Water	The journal of industrial and engineering chemistry/Journal of industrial and engineering chemistry	1,920	public-domain	2,099	sI:sIIl.%RY The desirability of investigating the possible eflect of sulfur conipounds on the properties of liihric,ating oils is pointed out. l h e disadvantages of the methods now in general use for the determination of sulfur are indicated. ,. A new procedure, which gives results at least as acciirate as those by the bomh calorimeter, is de- scribed. rll'l'ARATGS A sheet of coppcr foil 4 in. X i j in. is rolled up in siicii fashion that no two points of its surface c.ome in contact. I t is desirable that the copper sheet be rolled uniformly so that there will be a space of about three- sistcenths inch between any one turn of the roll and the next turn. The sheet muy be cut so that at one corner a tab three-eighths inch square projects. A hole is punched in this tab so that the coil may be lifted from the Wiley tube qith a wire hook. The end x i t h this tab should be rolled first, and the tab project from one end of the coil. The solvent is transferred to the tall beahcr, filter- ing if necessary to eliminate the copper sulfide, which xi11 doubtless partially flake off. The coil, the Wiley tube, ani1 the filter are waslied with smali quantities of pctrolcum ethcr, the combined filtrate and wash- ings gently evaporated to dryness at very low heat, and the residue rveighcd. By L. S. Busbnelt and H. Smith Clark By L. S. Busbnelt and H. Smith Clark F X ~ P O R T SVLP~WR COMPANY, Fxrs~onr. TEXA Received November 2s. 1919 By L. S. Busbnelt and H. Smith Clark F X ~ P O R T SVLP~WR COMPANY, Fxrs~onr. TEXA Received November 2s. 1919 F X ~ P O R T SVLP~WR COMPANY, Fxrs~onr. TEXAS Received November 2s. 1919 The presence of very small amounts of oil in sulfur is objected to by manidacturers of certain sulfur products. Tlic sulfur may contain carbonaceous residues from burned oil, and the method here de- scribed is not intended to include these. The estimation of oil by simple extraction as ordi- narily made with a volatile solvent-such as sulfuric ether or petroleum ether-will not suffice in thc case of sulfur, becausc, while the sulfur is only slightly soliil~le, there is usually such a small amount of oil pres- cnt (from 0.001 per cent to 0.32 per cent, or more) that Lhc catrnct contains considerably more sulfur than oil. in t.liis method extraction is made as usual, and the solvcnt purified from sulfur by boiling it in a Wilcy coniiuuous extractor in which has been placed a roll of wpper foil. l'hc coppcr coil is cleaned with dilute nitric acid, rpashed with water, then with nlcoliol, dried with ether, and placed in the Wiley tuhe. Tlic petroleum ether is then boilcd until all of the suliur has been deposited on the coppcr as coppcr sulfide. 1 Allen and Johnston. J. Am. Chcm. Soc, 82 (1910), 500; Johiision m d Adnma, Ibid., 83 (1911), 029. sulfatc tends to low results, while the nitrate has the opposite effect. Only by chance will t h caiicei one another.' A few determinalions made xith doniile the usual amounts of reagents seein to show that t!ie net error due to occlusion is small. tile flasii is thoroughly shaken every haif hour for several hours, and aliowed to settle. The petrolcum ethcr is decanted through a filter i n t o a IVilcy tube, a n d a sccoiid smaller quantity of the solvent is added to the flask, sh:iken, settled, and filtered, as before. llie washing is continued in this way until rhe sulfur and the filter paper are free of oil, and the IViIey tube contxins about 175 cc. of solvent, or enough to fill tlic tube above the top of the coppcr roll or coil. ,~ The present paper is a preliminary one, and it is intended to puhlish a more iietailed discussion of the n-liok subject later on. NOTE ON THE SOLUBILITY OF BENZIDINE SULFATE IN WATER A 100 cc. volumetric flask; a thin glass beaker, tall form, withdut lip, 300 cc. capacity; and a Wiley continuous extractor, without crucible or thimble, complete the required apparatus. By C. S. Bisson and A. W. Christie U>IYSKSITY "11 CILIYURNIA, UCKltEI.LY, CILiFOnnlA Received September 9, 1919 By C. S. Bisson and A. W. Christie U>IYSKSITY "11 CILIYURNIA, UCKltEI.LY, CILiFOnnlA Received September 9, 1919 A L OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY Vol. 12, NO. 5 J O U R N A L OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY Vol. 12, NO S T R I A L A N D ENGINEERING CHEMISTRY Vol. 12, NO. 5 failed to reveal any definite data on its solubility except a statement by Muller' which gives it as from 0.01 to 0.03 per cent at 2 j o C. We have determined its solubility at different temperatures by agitating an excess of benzidine sulfate with pure water in a constant temperature thermostat. The solubility of benzidine sulfate in water as de- termined by the above methods is given in the accom- panying table. SOLUBILITY OF BENZIDINE SULFATE IN WATER AT VARIOUS TEMPERATUR D t i ti SOLUBILITY OF BENZIDINE SULFATE IN WATER AT VARIOUS TEMPERATUR Determination--- B y Weight B y Titration Temwure G. per Liter G. per Liter 0 25 50 80 0.049 0.098 0.141 0.290 BENZIDINE SULFATE IN WATER AT VARIOUS TEMPERATU D t i ti p The benzidine sulfate was prepared by adding an excess of dilute sulfuric acid to an aqueous solution of Kahlbaum's benzidine. The precipitated sulfate was filtered and washed with distilled water till free of acid. From 3 to 5 g. were placed in a 500 cc. Pyrex flask containing pure water and the flask im- mersed in a thermostat which was regulated to 0.5' C. The contents of the flask were continually agitated by a revolving glass stirrer. After a specified number of days the solution was removed and immediately filtered, the first 2 0 cc. of filtrate being discarded. Solubility determinations were made at oo, z j O , soo, and 80" C. The two methods gave concordant results a t all the temperatures except 80' C. The solutions for the determination of solubility a t oo and 2 5 O C. were stirred in the thermostat for four days. Those at 50' and 80' C. remained in the thermostat only 2 4 hrs., since it was found that after several days the solu- tions became badly discolored, indicating that the benzidine sulfate had undergone decomposition. The solution obtained at 80' C. was slightly discolored even after 24 hrs., as was also the residue obtained on evaporation. A L OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY Vol. 12, NO. 5 This is probably due to the partial oxidation of the benzidine at the higher temperature, as evidenced by the low figure for solubility obtained by titration. The residue obtained from the soo solution showed only a very slight discoloration and the results may be considered to give the ap- proximate solubility at this temperature. Two methods for determining the solubility of the benzidine sulfate were used. One method consisted in evaporating a measured volume of IOO or 2 0 0 cc. to dryness in a platinum dish on the steam bath. The residue was dried at rooo C., cooled in a desiccator, and weighed. The other method consisted in titrating a measured volume of the solution with 0.0; N potas- sium permanganate after the addition of 5 per cent of sulfuric acid. The latter method is described in detail elsewhere.2 From a consideration of these results it is evident that in washing the benzidine sulfate in a quantitative determination of sulfate, the minimum amount of cold water should be used. 1 Ber., 55 (1902), 1587. * A. W. Christie and C. S. Bisson, THIS JOURNAL, 12 (1920), 171. 1 A common procedure is to place a measured volume of oil in a suitable graduated tube, dilute with naphtha, and spin in a centrifuge. The water is driven to the bottom of the graduated tube and its volume can be read directly. 1 Published by the permission of the Director of the U. S. Bureau of 2 See Bibliography, p. 490. Mines. 1 Published by the permission of the Director of the U. S. Bureau of 2 See Bibliography p 490 Mines. A CONVENIENT METHOD FOR THE DETERMINATION OF WATER 1N PETROLEUM AND OTHER ORGANIC EMULSIONS' paper reports the work of the Bureau in modifying this latter method to obviate certain of its admitted disadvantages. paper reports the work of the Bureau in modifying this latter method to obviate certain of its admitted disadvantages. DISTILLATION I N THE PRESENCE OF A N IMMISCIBLE SOLVENT DISTILLATION I N THE PRESENCE OF A N IMMISCIBLE SOLVENT There are various conditions under which it is desir- able to determine water in petroleum emulsions and no one method is best in all cases. The method of gravity separation' is a convenient one and is satis- factorily reliable for emulsions that are not too viscous or that contain water in not too fine a state of sub- division. For the estimation of extremely small per- centages of water, in such products as transformer oils, special methods are necessary which are too delicate for ordinary needs. The distillation method is without doubt most generally applicable, highly reliable, and sufficiently accurate for all usual requirements. The use of a solvent not miscible with water offers several advantages, the most important of which is prevention of frothing. Its principle disadvantages concern details of operation, and the modified procedure described in the present paper has been found to obviate most of these difficulties. 1 Ber., 55 (1902), 1587. * A. W. Christie and C. S. Bisson, THIS JOURNAL, 12 (1920), 171. 2 See Bibliography, p. 490. LABORATORY AND PLANT A CONVENIENT METHOD FOR THE DETERMINATION OF WATER 1N PETROLEUM AND OTHER ORGANIC EMULSIONS' By E. W. Dean and D. D. Stark CHEMICAL SECTION, PETROLBUM DIVISION, u. s. BUREAU O F MINES, PITTSBURGH, PA. Received January 15, 1920 INTRODUCTION A CONVENIENT METHOD FOR THE DETERMINATION OF WATER 1N PETROLEUM AND OTHER ORGANIC EMULSIONS' By E. W. Dean and D. D. Stark CHEMICAL SECTION, PETROLBUM DIVISION, u. s. BUREAU O F MINES, PITTSBURGH, PA. Received January 15, 1920 INTRODUCTION By C. S. Bisson and A. W. Christie U>IYSKSITY "11 CILIYURNIA, UCKltEI.LY, CILiFOnnlA Received September 9, 1919 One of the methods for the determination of sulfates which has lound considerable application consists in precipitation as benzidine sulfate, which may be quantitatively titrated with standard alkali or potas- sium permanganate. In most of the articles de- scribing such methods, the benzidine sulfate is said to be insoluble in water. .% review of the literature PR0CEI)URE Fifty grams of the pulverized sulfur (or a smaller quantity, if the oil content is known to be high) are ~eiglrcd and transferred to the IOO cc. flask. On this are poured about 50 cc. of redistilled petrolcum ether; PR0CEI)URE Fifty grams of the pulverized sulfur (or a smaller quantity, if the oil content is known to be high) are ~eiglrcd and transferred to the IOO cc. flask. On this are poured about 50 cc. of redistilled petrolcum ether; 4.86 T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY Vol. 12, NO. 5 INTRODUCTION One of the problems frequently confronting the petroleum chemist is the determination of emulsified water. Numerous methods have been devised, several of which have been described in a previous publica- tion2 issued by the Bureau of Mines. Allen and Jacobs discuss in that paper the advantages and limitations of various types of procedure and recom- mend as most desirable the scheme of distilling in a special electrically heated still. This type of still involves heating the entire outer surface of a 250 cc. distilling flask and its use permits the "breaking" of troublesome froth that is usually formed when a vis- cous petroleum emulsion is heated. Certain difficul- ties in the construction and operation of this still have led to discontinuing its use in favor of the more com- mon method of diluting the emulsion before distillation with a solvent immiscible with water. The present This method of determining water has been used in a
https://openalex.org/W2060892147	https://europepmc.org/articles/pmc4044153?pdf=render	English	null	Possible involvement of altered expression of BDNF exon II gene and specific dopamine receptors in simvastatin induced beneficial effects in depression	Molecular cytogenetics	2,014	cc-by	651	Background The up regulation of central BDNF gene expression has been suggested in the treatment of major depression. Chronic administration of dopaminergic agents activates the function of CREB which results into the up regulation of the BDNF gene expression. Statin therapy is associated with a reduced risk of depression and could be of thera- peutic potential for major depression. Conclusion The results of the above study suggests linkage between the function of dopamine or dopamine D2 like receptor and differential expression patterns for the expression of BDNF exon II gene in brain of mouse which further strengthen the emerging hypothesis, suggesting the ability of neuronal systems to exhibit the appropriate adaptive plasticity could contribute to the treatment of depression. Further, the dopaminergic agents in accordance with the cholesterol lowering drug as adjuncts may reduce the depressive like behavior more significantly and facilitation of antidepres- sant action of dopaminergic agents may be correlated with HMGR or cholesterol or mevalonate pathway. Possible involvement of altered expression of BDNF exon II gene and specific dopamine receptors in simvastatin induced beneficial effects in depression Digvijay G Rana1, Amrut Patel2, CG Joshi2, Ramesh K Goyal3 compared to simvastatin alone whereas the mice treated with haloperidol in combination with simvastatin for 14 days could abolish the for the expression of up regulation of specific BDNF exon II transcript compared to simvasta- tin alone. POSTER PRESENTATION Open Access Materials and methods We have examined a possible link amongst simvastatin, bromocriptine, haloperidol and levodopa in accordance with BDNF exon II gene expression using RT-PCR method in mice treated with standard paradigm of chronic mild stress procedure for 14 days. We specifically determined if the oral administration of simvastatin would affect the efficacy of bromocriptine, haloperidol or levodopa in med- iating the regulation of the BDNF exon II gene expression. Correspondence: dgrana3755@yahoo.com 1Department of Pharmacology, Sigma Institute of Pharmacy, Vadodara, India Full list of author information is available at the end of the article Results The results of RT-PCR method revealed the differential expression patterns for the expression of BDNF exon II gene in brain of mouse by indicating the three different bands, as evidenced previously to be the three different exon II transcript variants in mouse namely BDNF IIA, BDNF IIB and BDNF IIC. Mice treated with bromocrip- tine or levodopa in combination with simvastatin for 14 days could synergize the up regulation of for the expression of specific BDNF exon II transcript as Rana et al. Molecular Cytogenetics 2014, 7(Suppl 1):P67 http://www.molecularcytogenetics.org/content/7/S1/P67 © 2014 Rana 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. 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. Authors’ details 1 Authors details 1Department of Pharmacology, Sigma Institute of Pharmacy, Vadodara, India. 2Department of Biotechnology, Anand Veterinary College, Anand, India. 3Institute of Life Sciences, Ahmedabad University, Ahmedabad, Gujarat, India. Published: 21 January 2014 Published: 21 January 2014 doi:10.1186/1755-8166-7-S1-P67 Cite this article as: Rana et al.: Possible involvement of altered expression of BDNF exon II gene and specific dopamine receptors in simvastatin induced beneficial effects in depression. Molecular Cytogenetics 2014 7(Suppl 1):P67. doi:10.1186/1755-8166-7-S1-P67 Cite this article as: Rana et al.: Possible involvement of altered expression of BDNF exon II gene and specific dopamine receptors in simvastatin induced beneficial effects in depression. Molecular Cytogenetics 2014 7(Suppl 1):P67. © 2014 Rana 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. 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.
https://openalex.org/W4395464847	https://link.springer.com/content/pdf/10.1007/s13402-024-00945-7.pdf	English	null	Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing leukemia cells to CAR-T-mediated cytotoxicity and reducing CAR-T exhaustion	Cellular oncology	2,024	cc-by	7,671	Abstract Purpose Despite chimeric antigen receptor (CAR) T-cell therapy has achieved great advances in recent year, approxi- mately 50% of relapsed/refractory B cell acute lymphoblastic leukemia (r/r B-ALL) patients treated with CAR-T experience relapse 6 months post CAR-T treatment. CD20 express on 30 to 50% of B-ALL, which makes CD20 Monoclonal Antibody as one of the potential therapy strategies to decrease the tumor burden and improve the efficacy of CAR-T therapy. Adding Rituximab to chemotherapy protocol had been demonstrated to improve the outcome for CD20-positive ALL. However, rare study explored the influence of Rituximab combined with CAR-T therapy. Purpose Despite chimeric antigen receptor (CAR) T-cell therapy has achieved great advances in recent year, approxi- mately 50% of relapsed/refractory B cell acute lymphoblastic leukemia (r/r B-ALL) patients treated with CAR-T experience relapse 6 months post CAR-T treatment. CD20 express on 30 to 50% of B-ALL, which makes CD20 Monoclonal Antibody as one of the potential therapy strategies to decrease the tumor burden and improve the efficacy of CAR-T therapy. Adding Rituximab to chemotherapy protocol had been demonstrated to improve the outcome for CD20-positive ALL. However, rare study explored the influence of Rituximab combined with CAR-T therapy. Methods We retrospectively analyzed 20 r/r B-ALL patients who received CAR-T therapy, all of whom had failed multiple lines of therapy. Before CAR-T infusion, we administered Rituximab to 10 patients with high CD20 expression at a dose of 375 mg/m2 for 1 day. Meanwhile, we selected 10 patients with the comparable features who underwent CAR-T treatment without Rituximab in the same period as the control group. In vitro, the surface molecule expression and killing of CAR-T post Rituximab-treated B-ALL cells co-incubated with CAR-T cells were detected by flow cytometry. Results The median follow-up of Rituximab and Control groups were 29.27 and 9.83 months. We found that adding Rituximab may confer a favorable prognosis compared with Control group. The 2-year overall survival (OS) and leukemia-free survival (LFS) rates both were longer in the Rituximab group (90% vs. 26.7%, p = 0.0342; 41.7% vs. 25%, p = 0.308). In vitro, we observed that Rituximab-treated tumour cells are more sensitive to CAR-T killing and a broad range of cytokines and chemokines were produced when Rituximab-treated Nalm-6 cells co-cultured with 19- 22CAR-T cells, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2). Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing leukemia cells to CAR-T-mediated cytotoxicity and reducing CAR-T exhaustion Yangzi Li1,2 · Qingya Cui1,2 · Sining Liu1,2 · Lingling Liu1,2 · Megyn Li1,2 · Jun Gao1,2 · Zheng Li1,2 · Wei Cui1,2 · Xiaming Zhu1,2 · Liqing Kang3,4 · Lei Yu3,4 · Depei Wu1,2 · Xiaowen Tang1,2 Accepted: 29 March 2024 / Published online: 25 April 2024 © The Author(s) 2024 Accepted: 29 March 2024 / Published online: 25 April 2024 © The Author(s) 2024 Cellular Oncology (2024) 47:1649–1661 https://doi.org/10.1007/s13402-024-00945-7 Cellular Oncology (2024) 47:1649–1661 https://doi.org/10.1007/s13402-024-00945-7 RESEARCH RESEARCH 1 National Clinical Research Center for Hematologic Diseases, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China Abstract To investigate whether Rituximab has an effect on CAR-T persistence, we stimulated CAR-T cells repeatedly in vitro with Rituximab- treated Nalm-6 to evaluate the changes in CAR-T surface exhaustion molecules at different times. We found that the expression of exhaustion molecules (LAG-3, PD-1, TIM-3) on CAR-T cells were significantly lower in the Rituximab group than in the Control group. Results The median follow-up of Rituximab and Control groups were 29.27 and 9.83 months. We found that adding Rituximab may confer a favorable prognosis compared with Control group. The 2-year overall survival (OS) and leukemia-free survival (LFS) rates both were longer in the Rituximab group (90% vs. 26.7%, p = 0.0342; 41.7% vs. 25%, p = 0.308). In vitro, we observed that Rituximab-treated tumour cells are more sensitive to CAR-T killing and a broad range of cytokines and chemokines were produced when Rituximab-treated Nalm-6 cells co-cultured with 19- 22CAR-T cells, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2). To investigate whether Rituximab has an effect on CAR-T persistence, we stimulated CAR-T cells repeatedly in vitro with Rituximab- treated Nalm-6 to evaluate the changes in CAR-T surface exhaustion molecules at different times. We found that the expression of exhaustion molecules (LAG-3, PD-1, TIM-3) on CAR-T cells were significantly lower in the Rituximab group than in the Control group. 1 National Clinical Research Center for Hematologic Diseases, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China 2 Institute of Blood and Marrow Transplantation, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China 3 East China Normal University, Shanghai, China 4 Shanghai Unicar-Therapy Bio-medicine Technology Co., Ltd, Shanghai, China 1 Introduction group with the Rituximab group at a ratio of 1:1 based on: (1) age; (2) sex; (3) molecular mutations; (4) cytogenetic abnorm- alities; (5) CAR-T type; (6) conditioning regimen; (7) donor type. The study regimen was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University, and all patients provided written informed consent. Despite chimeric antigen receptor (CAR) T-cell therapy has achieved great advances in recent year, approximately 50% of relapsed/refractory B cell acute lymphoblastic leukemia (r/r B-ALL) patients treated with CAR-T experience relapse 6 months post CAR-T treatment. Clinical factors such as high disease burden and CAR-T cell exhaustion have been reported to increase the risk of recurrence. Additionally, high disease burden was also accompanied by a markedly higher incidence of cytokine release syndrome [1]. Hence, mitigating pre-CAR disease burden and CAR-T cell exhaus- tion may represent a crucial and viable strategy for aug- menting the effectiveness and safety of CAR-T therapy. 2.4 Clinical response assessment All disease assessments were conducted per protocol or institutional practice. Response assessment was per- formed 28 days after CAR-T cell infusion. Complete remission (CR) was defined as less than 5% bone mar- row (BM) blasts, the absence of circulating blasts, and no extramedullary sites of disease, regardless of cell count recovery. Complete response with incomplete count recovery was a complete response with cytopenia. MRD negativity was defined as less than 0.01% bone marrow blasts by flow cytometry. Relapsed disease was defined as the reappearance of blasts in the blood or bone marrow or in an extramedullary site after a complete remission. 1650 Y. Li et al. Conclusion Rituximab combined with CAR-T therapy is effective for improving the long-term prognosis of B-ALL patients who have failed multiple lines of therapy. In vitro, we observed that rituximab potentially improves CAR-T efficacy by sensitizing ALL to CART-mediated cytotoxicity and reducing CAR-T exhaustion. Keywords Chimeric antigen receptor T cell Rituximab r/r B-ALL 2.2 Manufacture of tandem CD19/CD22 CAR-T cells CAR-T cells were provided by Shanghai Unicar-Therapy Bio-medicine Technology Co. Ltd. The manufacture of CD19/CD22 CAR-T cells mainly consisted of retroviral vector (mCD19-mCD22 scFv.CD28/4-1BB/z) production, T cell transduction, and CAR-T cell expansion (Fig. 3A). CAR constructs (mCD19-mCD22) were synthesized and cloned into a third-generation lentiviral plasmid backbone. The CAR constructs contained a CD8 transmembrane domain in tandem with CD28, OX40, and CD3ζ transmem- brane domain. CAR-T cells were successfully manufactured, and qualified tests were conducted before infusion. CD20 express on 30–50% of B-ALL [2–5], making CD20 monoclonal antibody a potential therapeutic strategy for reducing tumor burden and enhancing the efficacy of CAR-T therapy. Intravenously administered rituximab was the first therapeutic mAb (Monoclonal Antibody) to be used in the field of oncology, establishing a new class of anti- tumor drugs. Adding rituximab to chemotherapy protocols has been shown to improve outcomes in CD20-positive ALL. In preclinical studies, rituximab augmented CD19 chimeric antigen receptor (CAR) T-cell function and increased tumor reduction and survival in murine models through synergistic targeting with CAR T-cells. However, few studies have explored how rituximab improves CAR-T efficacy. Therefore, we evaluated the efficacy and safety of rituximab administration before CAR T therapy in relapsed or refractory B-ALL and investigated the influence of ritux- imab on CAR-T cells as well as B-ALL cells in vitro. 2.3 Lymphodepletion chemotherapy Patients received lymphodepletion regimens: FC (Fludarabine 30 mg/m2/day and Cyclophosphamide 300 mg/m2/day) on days −5 to −3, with or without Decitabine (DAC) (total dose 100 mg/m2 in 3 days from day −6 to −4). The median dose of CAR-T cells was 1 × 107 cells/kg (1–4 × 107 cells/kg) during dose escalation. 2.5 Toxicity assessment For the sequential killing assay, the numbers of residual CAR-T cells and Nalm-6 were quantified every 3 days using flow cytometry with counting beads, as previously described. Tumor cells were then added to the wells with CAR T cells to restore the original effector:target (E:T) ratio of 1:1. CCK8 is measured every 3 days to assess the ability of CAR-T to kill tumor cells. Adverse events that occurred within one month after CAR- T therapy were recorded. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to ASTCT Consensus Grading [6]. The diagnostic criteria for macro- phage activation syndrome (MAS) were based on the MD Anderson diagnosis for patients post CAR-T cell therapy [7]. Other toxicities were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. The TUNEL assay was performed using the BrightRed Apoptosis Detection Kit (Vazyme, China), according to the manufacturer’s instructions. Images were obtained under a microscope (Nikon, Japan). 2.7 In vitro functional assays OS was defined as the time from CAR-T cell infusion to death or the end of follow-up. LFS was defined as the time from CAR-T cell infusion to first relapse or death, the end of follow-up. OS and LFS were censored at the last follow- up. The probabilities of OS and LFS were estimated by the Kaplan-Meier (KM) method and assessed with the use of the log-rank test. Median follow-up time was estimated by using the reverse KM method. Categorical variables were performed with Fisher’s exact tests. Group comparisons were determined by two-tailed parametric t-tests for unpaired or paired data. All reported P values were two- sided, and statistical significance was defined as P < 0.05. All analyses were conducted using GraphPad Prism 8. The statistical test used for each figure is described in the corresponding figure legend. The proliferation of T cells was determined by CSFE staining in vitro. Individual groups of CAR T cells (1 × 106 cells/tube) were labeled with CSFE (Beyotime, China) at 37 °C for 30 min. After being washed, individual groups of cells were stimulated with the same number of target cells that had previously been treated by mitomycin. CAR- T cells were collected after 5 days, and fluorescence was detected by flow cytometry. To detect the optimized E:T ratio, CAR T cells were co- cultured with target cells at different E:T ratios (0.5:1, 1:1, 5:1) in 96-well plate. CCK8 was added to each well, and the optical density (OD) was measured after 4 h using Epoch (BioTek). The killing rate was calculated by the following equation: 1-([OD of sample]-[OD of negative control])/ ([OD of positive control]-[OD of negative control]). 3 Result For the apoptosis assay, CAR-T cells were incubated with target cells at E/T ratios (1:1) for 24 h, and the target cells stained by CSFE and PI were tested with Flow cytometry to evaluate the killing rate of effect cells. Effector cells and target cells were cultured at an E:T ratio of 1:1 in RPMI 1640 media for 24 h. Supernatant of culture was analyzed by Cytometric bead array according to the manufacturer’s instructions (BD Biosciences). 2.1 Study design and patient population We retrospectively analyzed 10 r/r B-ALL patients who received CAR-T therapy at the First Affiliated Hospital of Soochow University between May 2017 and September 2022, all of whom had failed multiple lines of therapy and had high CD20 expression. Before CAR-T infusion, we admi- nistered Rituximab to all patient at a dose of 375 mg/m2 for 1 day. Meanwhile, we selected the patients with the compar- able features who did not receive Rituximab treatment during the same period as the control group. We matched the control Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing… 1651 2.6 Cell lines CAR-T cells were co-cultured with Nalm-6 cells in the medium at the ratio of 1:1 E:T for 24, 48, and 72 h. The following anti-human antibodies were also used in this study: CD3 (Percp-cy5.5, 1:100 dilution), CD4 (FITC, 1:100 dilution), CD8 (APC, 1:100 dilution), CD25 (BB700, 1:100 dilution), CD69 (PE, 1:100 dilution), CD62L (PE, 1:100 dilution), PD-1 (PE, 1:100 dilution), TIM-3 (BB515, 1:100 dilution), LAG-3 (AF674, 1:100 dilution) were purchased from BD Biosciences. The result- ing datas were analyzed by FlowJo software. The B lymphocyte leukemia cell line Nalm-6 was pur- chased from ATCC (Manassas, VA). Primary human ALL specimens were acquired from National Clinical Research Center for Hematologic Diseases. All cell lines were cul- tured in RPMI-1640 medium (Pricella, China)) supplemen- ted with 10% foetal bovine serum (Sigma-Aldrich, USA), 2 mM L-glutamine (Pricella) and 100 U/mL peni-cillin /streptomycin (Pricella, China). This study design was approved by the First Affiliated Hospital of Soochow University Ethics Board. Written informed consent for publication of their clinical details was obtained from the patient/relative of the patient. 3.1 Patient characteristics We retrospectively analyzed 20 r/r B-ALL patients who received CAR-T treatment between June 2017 and September 2021 at the First Affiliated Hospital of Soochow University. T Ten patients received Rituximab Y. Li et al. 1652 Y. Li et al. Table 1 Patient characteristics Patient characteristics Rituximab group (n = 10) Control group (n = 10) P value Median age (range) years 33 (7–58) 26 (7–56) 0.5681 Sex no(%) >0.99 Female sex 7 (70%) 5 (50%) Male sex 3 (30%) 5 (50%) Disease B-ALL 10 (100%) 10 (100%) >0.99 Refractory/relapsed 10 (100%) 10 (100%) >0.99 Number of recurrences before >0.99 CAR-T, no.(%) ≤5 4 (40%) 3 (30%) >5 6 (60%) 7 (70%) Extramedullary disease, no(%) 0.6285 NO 6 (60%) 8 (80%) YES 4 (40%) 2 (20%) Complex karyotype aberrations, no(%) >0.99 NO 7 (70%) 7 (70%) YES 3 (30%) 3 (30%) PH+, no(%) >0.99 NO 8 (80%) 7 (70%) YES 2 (20%) 3 (30%) Lymphodepletion regimens, no(%) >0.99 FC 8 (80%) 8 (80%) FC+DAC 2 (20%) 2 (20%) CAR-T cell dose (×106/kg), median (range) 10 (4–20) 10 (1–20) 0.9016 CAR-T cells origin >0.99 Patient 10 (100%) 9 (90%) Donor 0 1 (10%) CRS grade 0.814 CRS grade 0 1 (10%) 2 (20%) CRS grade 1–2 7 (70%) 6 (60%) CRS grade 3 (hematological toxicity) 2 (20%) 2 (20%) CAR-T response 0.6285 MRD-CR 2 (20%) 4 (40%) MRD+CR 8 (80%) 6 (60%) Bridging therapy (HSCT) >0.99 No 3 (30%) 3 (30%) Yes 7 (70%) 7 (70%) Table 1 Patient characteristics treatment before CAR-T cell infusion (Rituximab group), while the remaining ten patients did not receive rituximab treatment (Control group). The demographic and clinical characteristics of the r/r B-ALL patients are shown in Table 1, and there were no significant differences in baseline characteristics between the two groups. Both groups received treatment during the same period, ensuring no significant differences in supportive care (Table 1). Before treatment, the expression levels of CD19, CD20, and CD22 on the surface of leukemia cells were measured by flow cytometry in both groups (Rituximab group: CD19: 65.65%, CD20: 49.47%, CD22: 74.12%; Control group: CD19: 83.1%, CD20: 6.84%, CD22: 67.08%) (Fig. 1A). 3.2 Clinical response 28 days after CAR-Tcells infusion, 8 out of 10 patients (80%) in the Rituximab group achieved minimal residual disease negative complete remission (MRD-negative CR), maintain- ing CR for a median time of 11.15 months (0.46–46.93) post- CAR-T therapy. In comparison, 6 out of 10 patients (60%) in the Control group achieved MRD-negative CR, maintaining CR for a median time of 5.03 months (0.13–28) post-CAR-T therapy (p = 0.158) (Table 1, Fig. 1B). At the time of analysis, 8 out of 10 patients (80%) remained alive in the Rituximab group, while 3 out of 10 patients (30%) remained alive in the Control group (Fig. 1C). Among the patients in the Rituximab group, 6 (60%) proceeded to allo-HSCT, with 5 out of 6 (83.3%) patients achieving CR and survival. In contrast, 8 patients (80%) in the Control group underwent allo-HSCT, but only 3 out of 8 (37.5%) patients were alive with CR (Fig. 1D). Immune-modulating cytokines, including TNF-α, IFN-γ, IL-2, IL-6, IL-4, and IL-10, were induced in patients following 19–22 CAR-T and rituximab infusion, showing more significant elevation in the Rituximab Group compared to the Control Group (Fig. 1E). 3.3 Long-term survival The median follow-up periods for the Rituximab and Control groups were 29.27 and 9.83 months, respec- tively. Our findings suggest that adding Rituximab may lead to a more favorable prognosis compared to the Control group. The 2-year overall survival (OS) and leukemia-free survival (LFS) rates were both higher in the Rituximab group compared to the Control group (90% vs 26.7%, p = 0.0342; 41.7% vs 25%, p = 0.308) (Fig. 1G). Subgroup analysis revealed that Rituximab significantly improved OS and LFS for relapsed patients (88.9% vs 25%, p = 0.0203; 87.5% vs 15%, p = 0.0112) (Fig. 2A, B). Furthermore, the combination of CAR-T and Rituximab improved long-term prognosis in patients who had failed multiple lines of therapy (>5) (OS 100% vs 28.5%, p = 0.0205; LFS 50% vs 0%, p = 0.0119) (Fig. 2C, D). Following CAR-T treatment, subgroups of patients who underwent bridging allo-HSCT or not were analyzed. The long-term prognosis of the Rituximab group was found to be superior to that of the Control group. For patients who received allo-HSCT after CAR-T therapy, OS in the vs 28.5%, p = 0.0205; LFS 50% vs 0%, p = 0.0119) (Fig. 2C, D). Following CAR-T treatment, subgroups of patients who underwent bridging allo-HSCT or not were analyzed. The long-term prognosis of the Rituximab group was found to be superior to that of the Control group. For patients who received allo-HSCT after CAR-T therapy, OS in the vs 28.5%, p = 0.0205; LFS 50% vs 0%, p = 0.0119) (Fig. 2C, D). Following CAR-T treatment, subgroups of patients who underwent bridging allo-HSCT or not were analyzed. The long-term prognosis of the Rituximab group was found to be superior to that of the Control group. For patients who received allo-HSCT after CAR-T therapy, OS in the Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing… 1653 1 Clinical outcomes in the Rituximab combined with CAR-T control groups. A CD19, CD20, and CD22 expression on the ace of leukemia cells in both groups of patients before receiving ment. B Clinical outcomes, treatment response of each patient Rituximab combined with CAR-T therapy and the duration of onse. Patient number is shown to the left. C 6 of the 10 patient %) remained alive in the Rituximab group. 3 of the 10 patients %) remained alive in the Control group. 3.3 Long-term survival D In the Rituximab group, 6 patients proceeded to allo-HSCT, of whom 5 (5/6, 8 patients were alived with CR. In the Control group, 8 patients u went allo-HSCT, of whom 3 (3/8, 37.5%) patients were alive CR. E The peak levels of plasma cytokine release after C infusion. F, G Survival analysis. OS and LFS from the d CAR-T cell infusion is shown for patients who received C combined with Rituximab (Rituximab, n = 10) compared with who received CAR-T alone (CON, n = 10) ximab potentially improves clinical outcomes of CAR T therapy for r/r B ALL via sensitizing… group, 6 patients proceeded to allo-HSCT, of whom 5 (5/6, 83.3%) patients were alived with CR. In the Control group, 8 patients under- went allo-HSCT, of whom 3 (3/8, 37.5%) patients were alive with CR. E The peak levels of plasma cytokine release after CAR-T infusion. F, G Survival analysis. OS and LFS from the day of CAR-T cell infusion is shown for patients who received CAR-T combined with Rituximab (Rituximab, n = 10) compared with those who received CAR-T alone (CON, n = 10) Fig. 1 Clinical outcomes in the Rituximab combined with CAR-T and control groups. A CD19, CD20, and CD22 expression on the surface of leukemia cells in both groups of patients before receiving treatment. B Clinical outcomes, treatment response of each patient after Rituximab combined with CAR-T therapy and the duration of response. Patient number is shown to the left. C 6 of the 10 patient (60%) remained alive in the Rituximab group. 3 of the 10 patients (30%) remained alive in the Control group. D In the Rituximab Fig. 2 Effect of Rituximab combined with CAR-T in subgroups of patients. A, B OS and LFS in patients infused with CD19/CD22 CAR-T. (OS 66.7% vs 26.7%, p = 0.0927, LFS 66.7% vs 25.2%, p = 0.0314). C, D OS and LFS in relapsed patients. (OS 88.9% vs 25%, p = 0.0203, LFS 87.5% vs 15%, p = 0.0112). E, F The long-term prognosis in patients who had failed multiple lines of therapy (OS 100% vs 28.5%, p = 0.041; LFS 50% vs 0%, p = 0.004). G–J The long-term prognosis of patients whether or not they were bridged to transplantation after CAR-T treatment 1654 Y. Li et al. Y. Li et al. 3.5 Expansion and killing of CD19/CD22 CAR-T cells was improved by pre-treatment of tumor cells with Rituximab Our clinical data indicated that Rituximab combined with CD19/CD22 CAR-T treatment effectively improved the long- term prognosis of r/r B-ALL patients (Fig. 3A). Subsequently, we evaluated the killing ability of Rituximab combined with CAR-Ton tumor cells and the proliferation of CAR-T in vitro.f To examine the effector function of rituximab combined with CAR-T, a panel of cytokines were measured during the in vitro cytotoxicity assay. Compared with the control group, a broad range of cytokines was produced by Rituximab combined with CD19-CD22 CAR-T when co- cultured with NALM-6. Increased secretion of effector cytokines and chemokines such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-2 (IL-2) was observed (Fig. 4A). Firstly, we evaluated the tumor killing effect by selecting CD19, CD20, and CD22 simultaneously antigen-expressing B-ALL cells (NALM-6) as well as primary cells (Fig. 3B). Subsequently, we identified the concentration of Rituximab in vitro that was effective for killing tumor cells (NALM-6, Primary cells) without impacting CAR-T activity (Fig. 3C, Supplementary 1A). We used a concentration of 350 nM Rituximab to treat tumor cells, followed by flow cytometry to detect the expression of CD19 and CD22. The results showed that the expression of CD19 and CD22 had not been obviously altered (Supplementary 1C). We also selected an efficacy target ratio based on the killing of NALM-6 by Rituximab combined with CAR-T cells, and eventually, we chose a 1:1 efficacy target ratio for the subsequent experi- ments (Fig. 3D). 3.4 Safety There were no significant differences between the two groups in terms of hematological and non-hematological toxicities. All patients experienced grade 1–3 cytokine release syndrome (CRS) symptoms, such as fever and hypo- tension, which were effectively managed through sympto- matic treatment. Two patients in the Rituximab group developed headaches, while patients in the Control group did not exhibit any neurotoxicity syndromes. Moreover, all patients in our study experienced hematological toxicities. The median time to platelet and neutrophil count recovery was similar in both groups. Additionally, 50% (5/10) of patients in the Rituximab group experienced infections, compared to 30% (3/10) in the control group. All adverse events were reversible and manageable (Table 1). We utilized a sequential killing assay in which CAR-T cells were plated with Nalm-6 cells at a 1:1 effector-to- target ratio and replated every 3 days with fresh tumor cells to restore the initial ratio. Unlike the control groups, the combination of Rituximab with CAR-T repeatedly elimi- nated tumor cells for at least three rounds (Supplementary 2A). Collectively, the Rituximab combined with CAR-T demonstrated robust and sustained cytotoxicity against ALL cell lines in vitro. 3.3 Long-term survival 1654 prognosis in patients who had failed multiple lines of therapy (OS 100% vs 28.5%, p = 0.041; LFS 50% vs 0%, p = 0.004). G–J The long-term prognosis of patients whether or not they were bridged to transplantation after CAR-T treatment prognosis in patients who had failed multiple lines of therapy (OS 100% vs 28.5%, p = 0.041; LFS 50% vs 0%, p = 0.004). G–J The long-term prognosis of patients whether or not they were bridged to transplantation after CAR-T treatment Fig. 2 Effect of Rituximab combined with CAR-T in subgroups of patients. A, B OS and LFS in patients infused with CD19/CD22 CAR-T. (OS 66.7% vs 26.7%, p = 0.0927, LFS 66.7% vs 25.2%, p = 0.0314). C, D OS and LFS in relapsed patients. (OS 88.9% vs 25%, p = 0.0203, LFS 87.5% vs 15%, p = 0.0112). E, F The long-term 1655 Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing… Rituximab group was significantly longer than in the Control group (83.3% vs 33.3%, p = 0.0469) (Fig. 2E, F). For patients who did not undergo allo-HSCT after CAR-T therapy, LFS in the Rituximab group was significantly better than in the Control group (33.3% vs 0%, p = 0.0389) (Fig. 2G, H). Several mechanisms could be working through the ther- apeutic efficacies of anti-CD20 antibodies, including com- plement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and the induction of apoptosis [8]. Previous studies have indicated that direct effects mediated through binding of CD20 to the cell surface include inhibition of proliferation, induction of apoptosis, and sensitization of cancer cells to chemotherapy [9]. We divided mitomycin-treated NALM-6 and primary cells into two groups based on whether they were pretreated with Rituximab or not, and then incubated them with CD19/ CD22 CAR-T cells. The other group was treated with Rituximab alone for tumor cells. FCM analysis showed that Rituximab combined with CAR-T significantly pro- moted tumor cell apoptosis compared to the control groups (p < 0.001). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays demonstrated that the number of apoptotic cells in the Rituximab combined with CAR-T group was significantly higher than that in the control groups (Fig. 3F, Supplementary 1D). 3.6 Pre-treatment of tumor cells with Rituximab affects the surface molecule expression of CD19- CD22 CAR-T To investigate the effects of Rituximab on the expression of surface marker molecules on CD19/CD22 CAR-T cells, we co-cultured Rituximab-treated tumor cells with CAR-T cells for 24 h. Subsequently, we assessed the expression of acti- vation molecules and memory molecules on CAR-T cells using flow cytometry. The analysis revealed that CD25+, CD8+CD69+, and CD4+CD62L+ molecules on the surface of CD19/CD22 CAR-T cells were significantly upregulated in the Rituximab pre-treatment group compared to the group without Rituximab treatment (p < 0.001) (Fig. 4B–D). In proliferation experiments, we found CD19-CD22 CAR-T cells rapidly proliferated upon Nalm-6 challenge, reaching a peak level of expansion on day 5 (Fig. 3E). Rituximab presensitized tumor cells facilitated the prolif- eration of CAR-T cells. Y. Li et al. 1656 Fig. 3 Expansion and Killing of CD19/CD22 CAR-T Cells was improved by Pre-treatment of Tumor Cells With Rituximab. A Schematic diagram of anti-CD19/CD22 CAR. B Expression of CD19, CD20 and CD22 antigens on NALM-6 as well as on primary cells. C The inhibition of NALM-6 by rituximab at different drug concentrations as measured by CCK8. Curves were plotted to calcu- late IC50 (335.2 nM). D Nalm-6 killing by rituximab combined with CAR-T was detected at different E/T ratio (0.5:1, 1:1, 5:1). E Upper panel:CAR T cells were labeled with CSFE and incubated with NALM-6 for 3–5 days. The proliferation of CD19/CD22 CAR-T cells was analyzed by flow cytometry. Lower panel: Histogram plots of the proliferation efficiency of CD19/CD22 CAR-T cells (n = 3 samples examined over three independent experiments). F Uppe panel: CD19/CD22 CAR-T were co-incubated target cells (NALM-6 primary cells) at E/T ratios (1:1) for 24 h, and the target cells stained by PI were tested with Flow cytometry. Lower panel: Histogram plot of the apoptotic efficiency of CAR-T on target cells (NALM-6 primary cells) (n = 3 samples examined over three independen experiments) 1656 Y. Li et a Fig. 3 Expansion and Killing of CD19/CD22 CAR-T Cells was improved by Pre-treatment of Tumor Cells With Rituximab. A Schematic diagram of anti-CD19/CD22 CAR. B Expression of CD19, CD20 and CD22 antigens on NALM-6 as well as on primary cells. C The inhibition of NALM-6 by rituximab at different drug concentrations as measured by CCK8. Curves were plotted to calcu- late IC50 (335.2 nM). D Nalm-6 killing by rituximab combined with CAR-T was detected at different E/T ratio (0.5:1, 1:1, 5:1). 3.6 Pre-treatment of tumor cells with Rituximab affects the surface molecule expression of CD19- CD22 CAR-T E Upper panel:CAR T cells were labeled with CSFE and incubated with NALM-6 for 3–5 days. The proliferation of CD19/CD22 CAR-T Fig. 3 Expansion and Killing of CD19/CD22 CAR-T Cells was improved by Pre-treatment of Tumor Cells With Rituximab. A Schematic diagram of anti-CD19/CD22 CAR. B Expression of CD19, CD20 and CD22 antigens on NALM-6 as well as on primary cells. C The inhibition of NALM-6 by rituximab at different drug concentrations as measured by CCK8. Curves were plotted to calcu- late IC50 (335.2 nM). D Nalm-6 killing by rituximab combined with CAR-T was detected at different E/T ratio (0.5:1, 1:1, 5:1). E Upper panel:CAR T cells were labeled with CSFE and incubated with NALM-6 for 3–5 days. The proliferation of CD19/CD22 CAR-T cells was analyzed by flow cytometry. Lower panel: Histogram plots of the proliferation efficiency of CD19/CD22 CAR-T cells (n = 3 samples examined over three independent experiments). F Upper panel: CD19/CD22 CAR-T were co-incubated target cells (NALM-6, primary cells) at E/T ratios (1:1) for 24 h, and the target cells stained by PI were tested with Flow cytometry. Lower panel: Histogram plots of the apoptotic efficiency of CAR-T on target cells (NALM-6, primary cells) (n = 3 samples examined over three independent experiments) Fig. 4 CD19/CD22 CAR-T cells undergo activation, antigen-induced differentiation. A CD19/CD22 CAR-T cells produced higher levels of cytokines. CAR-T cells and T cells were incubated with tumor cells for 12 h; the levels of cytokines in the culture supernatants were determined by ELISA assay. B–D Left panel: CD19/CD22 CAR-T were incubated with NALM-6 for 48 h, CD25, CD8+CD69, CD4 +CD62L were analyzed by flow cytometry. Quantitative analysis of the frequency of CD25, CD8+CD69, CD4+CD62L; Right panel: Histogram plots of CD25, CD8+CD69, and CD4+CD62L expression on CD19/CD22 CAR T cells (n = 3 samples examined over three independent experiments) Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing… 1657 Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing… 1657 Fig. 4 CD19/CD22 CAR-T cells undergo activation, antigen-induced differentiation. A CD19/CD22 CAR-T cells produced higher levels of cytokines. CAR-T cells and T cells were incubated with tumor cells for 12 h; the levels of cytokines in the culture supernatants were determined by ELISA assay. B–D Left panel: CD19/CD22 CAR-T were incubated with NALM-6 for 48 h, CD25, CD8+CD69, CD4 +CD62L were analyzed by flow cytometry. 3.6 Pre-treatment of tumor cells with Rituximab affects the surface molecule expression of CD19- CD22 CAR-T Quantitative analysis of the frequency of CD25, CD8+CD69, CD4+CD62L; Right panel: Histogram plots of CD25, CD8+CD69, and CD4+CD62L expression on CD19/CD22 CAR T cells (n = 3 samples examined over three independent experiments) Y. Li et al. 1658 patients infused with CD19/CD22 CAR-T cells, Rituximab combined with CAR-T effectively improved the leukemia-free survival (LFS) of r/r B-ALL patients. We observed that pretreatment of tumor cells with Rituximab enhanced the expansion of CD19/CD22 CAR-T cells in vitro. Concurrently, flow cytometry (FCM) analysis showed significant upregulation of CD25+, CD8+CD69, and CD4+CD62L+ on the surface of CD19/CD22 CAR-T cells (p < 0.001). To investigate the influence of Rituximab on the persis- tence of CD19/CD22 CAR-T cells, the expression of inhi- bitory molecules PD-1, LAG-3 and Tim-3 on CD19/CD22 CAR-T cells were monitored. Flow cytometry (FCM) ana- lysis at 24 h showed that the expression of CD19/CD22 CAR-T cell surface exhaustion molecules (LAG-3, PD-1, TIM-3) was significantly lower in the Rituximab group than in the Control group (p < 0.001). Interestingly, com- pared to the control group, FCM analysis at 96 h showed that pretreatment of tumor cells with rituximab did not significantly upregulate CD19/CD22 CAR-T surface exhaustion molecules (PD-1, TIM-3) (Fig. 5A–C). Subsequently, in Cytotoxicity Assays, we demonstrated that Rituximab pretreatment of tumor cells increased the killing activity of CAR-T cells. Our data in vitro demon- strated that Rituximab efficiently reduced tumor burden, and the combination of CAR-T with Rituximab enhanced the killing efficiency of CAR-T in vitro. Accordingly, we conclude that Rituximab might enhance the efficacy of CAR-T cells by reducing tumor burden. To establish whether Rituximab combined with CAR-T was resistant to functional exhaustion, we used a sequential killing assay in which CAR-T cells were plated with Nalm- 6 cells at a 1:1 effector-to-target ratio and replated every 3 days with fresh tumor cells to restore the initial ratio. After three rounds of repeated challenges, Rituximab combined with CAR-T effectively reduced CAR-T exhaustion (Supplementary 2B–D). y g In a subgroup analysis, we found that Rituximab com- bined with CAR-T effectively improved the long-term prognosis of relapsed patients. Furthermore, Rituximab combined with CAR-T significantly improved leukemia- free survival (LFS) in patients who had failed multiple lines of therapy, suggesting that Rituximab may be effec- tive in relapsed patients, particularly those who have failed multiple lines of therapy. 3.6 Pre-treatment of tumor cells with Rituximab affects the surface molecule expression of CD19- CD22 CAR-T In relapsed patients, CAR-T cells are depleted due to repetitive antigen stimulation, as evi- denced by the upregulation of CAR-T exhaustion mole- cules and dysregulation of activation molecules [18, 19]. Nevertheless, there was limited evidence on whether Rituximab was an effective approach to reduce CAR-T exhaustion. T To investigate whether Rituximab has an effect on CAR-T persistence, we stimulated CAR-T cells in vitro with Rituximab-treated Nalm-6 cells to evaluate the changes in CAR-T surface exhaustion molecules at different times. We found that the expression of CAR-T cell surface exhaustion molecules (LAG-3, PD-1, TIM-3) was significantly lower in the Rituximab group than in the control group. Additionally, the upregulation of CAR-T cell surface exhaustion molecules was significantly slower in the Rituximab group compared to the control group. 4 Discussion CAR-T cells have emerged as a potent therapeutic weapon against B cell hematologic malignancies, leading to an expo- nential increase in clinical trials [10]. Despite the high success rate in treating ALL, there is still a 40% recurrence rate. The strong activation during clinical therapy drives CAR-T cell exhaustion and even apoptosis, which limits their antitumor efficacy [11, 12]. A Long-Term Follow-up study of CD19 CAR-T Therapy revealed that patients with a low disease burden had a significantly longer event-free survival (EFS) and overall survival (OS) compared to those with a high disease burden [1]. Therefore, reducing tumor burden prior to CAR-T therapy could be a strategy with beneficial effects on the long-term survival of relapsed/refractory B-ALL. g p y Rituximab has revolutionized the treatment of B-cell malignancies, being part of the standard of care for FL [13], DLBCL [14], and CLL [15]. Recent clinical studies have shown that Rituximab also exhibits good antitumor effects in other B lineage hematologic tumors. Clinical trials have demonstrated that Rituximab combined with chemotherapy can increase event-free survival (EFS) compared to chemotherapy alone in Philadelphia chro- mosome-negative acute lymphoblastic leukemia [16– 18]. Thus, we successfully treated relapsed/refractory (r/r) B-ALL patients who had failed multiple lines of therapy with Rituximab combined with CAR-T. Our clinical data showed that Rituximab combined with CAR-T improved the long-term prognosis of r/r B-ALL patients, regardless of whether they were bridged to transplantation after CAR-T treatment. Additionally, for In the sequential killing assay, we observed that Rituximab combined with CAR-T demonstrated robust and sustained cytotoxicity against ALL cell lines in vitro. After three rounds of repeated challenges, Rituximab combined with CAR-T effectively reduced CAR-T exhaustion. Therefore, we speculated that Rituximab combined with CAR-T treatment could potentially enhance CAR-T efficacy by reducing its exhaustion. In terms of safety, the combina- tion of rituximab with CAR-T therapy did not induce higher hematological and non-hematological toxicities.f We hypothesize that these effects may be attributed to changes in apoptotic products of tumor cells induced by rituximab pretreatment. These apoptotic products stimulate 5 Rituximab reduced the expression of exhaustion molecules 1, LAG-3, Tim-3) on CD19/CD22 CAR-T. A–C Left panel: 9/CD22 CAR-T were incubated with NALM-6 for 24–96 h, , LAG-3, and Tim-3 were analyzed by flow cytometry. Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s13402-024-00945-7. 2. G. Pavlasova, M. Borsky, V. Svobodova, Rituximab primarily targets an intra-clonal BCR signaling proficient CLL subpopula- tion characterized by high CD20 levels. Leukemia 32(9), 2028– 2031 (2018) Acknowledgements The authors would like to thank all members of the study team, the patient and their family, and Shanghai Unicar- Therapy Bio-medicine Technology Co., Ltd. 3. G.J. Weiner, Rituximab: mechanism of action. Semin. Hematol. 47(2), 115–123 (2010) 4. P. Boross, J.H. Leusen, Mechanisms of action of CD20 antibo- dies. Am. J. Cancer Res. 2(6), 676–690 (2012) Author contributions YL collected, analyzed research data, designed the research, performed statistical analyses, wrote and edited the manuscript; SL, LL and JG collected research data; XT, QC, ML, ZL, XZ, and WC treated the patients; LK and LY designed the clinical CAR vector, supervised the production of CAR T-cell Product; DW and XT conceived of the study and revised the paper. All authors have read and approved the final manuscript. 5. G. Salles, M. Barrett, R. Foà et al., Rituximab in B-Cell hema- tologic malignancies: a review of 20 years of clinical experi- ence. Adv. Ther. 34(10), 2232–2273 (2017) 6. D.W. Lee, B.D. Santomasso, F.L. Locke, A. Ghobadi, C.J. Turtle, S.S. Neelapu et al., ASTCT consensus grading for cyto- kine release syndrome and neurologic toxicity associated with immune effector cells. Biol. Blood Marrow Transplant. 25(4), 625–638 (2019) Funding This work was supported by research grants from National Natural Science Foundation of China (82070162), Frontier Clinical Technical Project of Suzhou Science and Technology plan (SKY2022001), Bethune Charitable Foundation (BCF-IBW-XY -20220930-13), Suzhou diagnosis and treatment project of Clinical Key Diseases (LCZX202201), China International Medical Foundation (Z-2018-31-2102-4), Boxi clinical research project of The First Affiliated Hospital of Soochow University (BXLC005), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). 7. S.S. Neelapu, S. Tummala, P. Kebriaei, W. Wierda, C. Gutierrez, F.L. Locke, E.J. Shpall et al., Chimeric antigen receptor T-cell therapy-assessment and management of toxicities. Nat. Rev. Clin. Oncol. 15(1), 47–62 (2018) 8. T. Cerny, B. Borisch, M. Introna, P. Johnson, A.L. Rose, Mechanism of action of rituximab. Anticancer Drugs 13 (Suppl 2), S3–S10 (2002) 9. S.L. Maude, N. Frey, P.A. Shaw, R. Aplenc, D.M. Barrett, N.J. Bunin, A. Chew et al., Chimeric antigen receptor T cells for sustained remissions in leukemia. N. Engl. J. Med. 4 Discussion Quantitative analysis of the frequency of PD-1, LAG-3, and Ti Right panel: Histogram plots of PD-1, LAG-3, and Tim-3 expres on CD19/CD22 CAR T cells (n = 3 samples examined over t independent experiments) imab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing… Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing… 1659 Fig. 5 Rituximab reduced the expression of exhaustion molecules (PD 1 LAG 3 Tim 3) on CD19/CD22 CAR T A C Left panel: Quantitative analysis of the frequency of PD-1, LAG-3, and Ti Right panel: Histogram plots of PD 1 LAG 3 and Tim 3 expre Quantitative analysis of the frequency of PD-1, LAG-3, and Tim-3; Right panel: Histogram plots of PD-1, LAG-3, and Tim-3 expression on CD19/CD22 CAR T cells (n = 3 samples examined over three independent experiments) Fig. 5 Rituximab reduced the expression of exhaustion molecules (PD-1, LAG-3, Tim-3) on CD19/CD22 CAR-T. A–C Left panel: CD19/CD22 CAR-T were incubated with NALM-6 for 24–96 h, PD-1, LAG-3, and Tim-3 were analyzed by flow cytometry. 1660 Y. Li et al. article. Meanwhile, the datasets involved in the current study are available from the corresponding author on reasonable request. CAR-T cells to enhance their tumor-killing efficacy, result- ing in a significant upregulation of exhaustion molecules and memory phenotypes in CAR-T cells. Subsequently, we are prepared to conduct metabolomics analysis to further explore the mechanism by which rituximab enhances the functionality of CAR-T cells.i article. Meanwhile, the datasets involved in the current study are available from the corresponding author on reasonable request. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University (2022445) and was conducted in accordance with the Declaration of Helsinki principles. All patients provided written informed consent. To the best of our knowledge, this is the first report assessing the efficacy and safety of rituximab combined with CAR-T therapy in patients with relapsed or refractory B-ALL. This study represented the successful application of rituximab combined with CAR-T therapy in patients with r/r B-ALL who have failed multiple lines of therapy. In our study, rituximab combined with CAR-T therapy showed an excellent safety record with manageable adverse effects in patients. Additionally, we conducted in vitro assays to inves- tigate how rituximab enhances CAR-T capabilities. Despite the limitations of the data, our study pioneers the observa- tion of the clinical benefits associated with rituximab com- bined with CAR-T therapy and further investigates the effect of rituximab on CAR-T cells in vitro, partially illus- trating the effectiveness of this combination. Competing interests The authors declare there’s no competing interests and all authors consent to publish the data. 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/. 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https://openalex.org/W1970099049	https://europepmc.org/articles/pmc3355134?pdf=render	English	null	Notch2 and Notch3 Function Together to Regulate Vascular Smooth Muscle Development	PloS one	2,012	cc-by	7,466	Qingqing Wang1, Ning Zhao1,2, Simone Kennard3, Brenda Lilly1,2* Qingqing Wang1, Ning Zhao1,2, Simone Kennard3, Brenda Lilly1,2* 1 Center for Cardiovascular and Pulmonary Research, Nationwide Children’s Hospital, Columbus, Ohio, United States of America, 2 Department of Pediatrics, The Ohio State University, Columbus, Ohio, United States of America, 3 Vascular Biology Center, Medical College of Georgia, Augusta, Georgia, United States of America 1 Center for Cardiovascular and Pulmonary Research, Nationwide Children’s Hospital, Columbus, Ohio, United States of America, 2 State University, Columbus, Ohio, United States of America, 3 Vascular Biology Center, Medical College of Georgia, Augusta, Georg Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: brenda.lilly@nationwidechildrens.org mice, while viable, display vascular smooth muscle defects associated with postnatal maturation and arterial specification [12,13]. We previously demonstrated that differentiation of vascular smooth muscle cells by endothelial cells was dependent upon NOTCH3 [14]. Notch2 is more widely expressed than Notch3, but is also present in vascular smooth muscle cells [11,15,16]. Two studies in particular have hinted at a role in smooth muscle cell regulation. McCright et al., reported that Notch2 hypomorphic knockout mice exhibit widespread hemor- rhaging mid to late gestation [17]. Whereas, a neural crest-specific deletion of Notch2 causes an underdeveloped outflow tract due to decreased smooth muscle [15]. Thus, given the collective evidence for a role of the Notch receptors in regulating smooth muscle differentiation, we hypothesized that multiple Notch receptors, particularly Notch2 and Notch3 act together to regulate vascular smooth muscle differentiation. Here, we perform a phenotypic analysis of mice deficient in the Notch2 and Notch3 genes. Our results show that together these genes regulate vascular smooth muscle development. Mice deficient in both genes die during mid- gestation from severe vascular defects associated with an absence of differentiated smooth muscle cells. These data are the first to demonstrate a critical role for these Notch receptors in vascular development, and highlight the combined role they play in regulating smooth muscle differentiation. Abstract Notch signaling has been implicated in the regulation of smooth muscle differentiation, but the precise role of Notch receptors is ill defined. Although Notch3 receptor expression is high in smooth muscle, Notch3 mutant mice are viable and display only mild defects in vascular patterning and smooth muscle differentiation. Notch2 is also expressed in smooth muscle and Notch2 mutant mice show cardiovascular abnormalities indicative of smooth muscle defects. Together, these findings infer that Notch2 and Notch3 act together to govern vascular development and smooth muscle differentiation. To address this hypothesis, we characterized the phenotype of mice with a combined deficiency in Notch2 and Notch3. Our results show that when Notch2 and Notch3 genes are simultaneously disrupted, mice die in utero at mid-gestation due to severe vascular abnormalities. Assembly of the vascular network occurs normally as assessed by Pecam1 expression, however smooth muscle cells surrounding the vessels are grossly deficient leading to vascular collapse. In vitro analysis show that both Notch2 and Notch3 robustly activate smooth muscle differentiation genes, and Notch3, but not Notch2 is a target of Notch signaling. These data highlight the combined actions of the Notch receptors in the regulation of vascular development, and suggest that while these receptors exhibit compensatory roles in smooth muscle, their functions are not entirely overlapping. Citation: Wang Q, Zhao N, Kennard S, Lilly B (2012) Notch2 and Notch3 Function Together to Regulate Vascular Smooth Muscle Development. PLoS ONE 7(5): e37365. doi:10.1371/journal.pone.0037365 Editor: Qingzhong Xiao, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, United Kingdom Editor: Qingzhong Xiao, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary U Kingdom Received February 13, 2012; Accepted April 20, 2012; Published May 17, 2012 Copyright: 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) grant R01 HL076428 to BL. 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 National Institutes of Health (NIH) grant R01 HL076428 to BL. The funders had no role in study analysis, decision to publish, or preparation of the manuscript. Combined mutations in Notch2 and Notch3 genes cause embryonic lethality y y Prior analysis showed that Notch3 null mice are viable and fertile with minor, yet significant postnatal defects in smooth muscle structure and vascular function [12,13,18]. Because Notch2 is also expressed in smooth muscle and a neural crest-specific Notch2 mutation causes vascular patterning defects [11,15,16], we set out to determine the phenotype of mice with a combined deficiency in the Notch2 and Notch3 genes. We crossed previously generated Notch2 hypomorph [17] and Notch3 null [19] mice to create double heterozygotes (Notch22/+;Notch32/+), which we intercrossed to generate the genotypes for comparison. From a double heterozy- gous cross, nine different genotypes were produced. Initial analysis indicated that the Notch22/2;Notch32/2 embryos did not survive to embryonic day (E)12.5 of gestation (Table S1). Double mutant embryos were recovered at expected Mendelian ratios at this time point, however all Notch22/2;Notch32/2 embryos were small, pale (bloodless) and were being resorbed. At E11.5 the Notch22/2; Notch32/2 embryos were easily distinguishable from wildtype and heterozygous littermates with obvious signs of hemorrhaging and yolk sac defects. This primary analysis indicated that the combined loss of Notch2 and Notch3 resulted in embryonic lethality around E11.5; thus we focused on this time point and those just prior to determine the cause of lethality. Figure 1. Combined mutations in Notch2 and Notch3 cause defects in vascular development. Yolk sacs (A) and embryos (B) at E10.5 and E11.5 were dissected and photographed with a stereo microscope. At E10.5, Notch22/2;Notch32/2 (N22/2;N32/2) embryos exhibit a decrease in yolk sac blood vessels, while the embryo proper is relatively normal in appearance. At E11.5, Notch22/2;Notch32/2 mice show severe vascular defects in both yolk sac and embryo. Yolk sac blood vessels are not visible and extensive hemorrhaging is seen in the embryo (arrowhead). Blood vessels are grossly normal in Notch22/2 (N22/2;N3+/+) and Notch32/2 (N2+/+;N32/2) embryos. Scale bar = 1 mm. doi:10.1371/journal.pone.0037365.g001 y For simplicity, we chose to perform a comparative examination of wildtype embryos with the three genotypes harboring homo- zygous mutant alleles alone and in combination (Notch22/2; Notch3+/+, Notch2+/+;Notch32/2, Notch22/2;Notch32/2). Gross anal- ysis of the yolk sacs by light microscopy revealed normal vascular patterning in wildtype and single mutant embryos at E10.5, however 45% of the double mutant yolk sacs had a decrease in visible blood vessels (Figure 1A and Table 1). Combined mutations in Notch2 and Notch3 genes cause embryonic lethality At E11.5, 92% of double mutant embryos had reduced blood vessels in the yolk sacs (Figure 1A, and Table 1), while only a small percentage of the Notch2 and Notch3 mice had observable vascular defects within their yolk sacs (Table 1). Examination of the embryo proper at E10.5 showed that all genotypes were phenotypically normal and appeared similar in size with no apparent structural defects (Figure 1B). At E11.5, double mutant embryos had a combination of abdominal and cerebral hemorrhaging, pericardial effusion, and in some cases were smaller in size (Figure 1B). The Notch32/2 mice were indistinguishable from the wildtype embryos and a small percentage of Notch22/2 mice exhibited visible hemorrhaging, consistent with previous findings [17]. These data suggested that the primary defect of the mutant embryos was vascular insufficiency leading to the demise of the embryos. To examine potential structural defects of blood vessels, embryos were cross- sectioned and stained with hematoxylin and eosin (H&E). At E10.5, the overall structure of the blood vessels from the four different genotypes were indistinguishable, with all having intact vessel walls (Figure S1). At E11.5, embryos deficient in both Notch2 and Notch3 had severely compromised blood vessel structure (Figure 2). Vessels were enlarged and filled with blood, suggesting improper flow, while the vessel walls were thin and in certain cases appeared to incompletely enclose the lumen. The defect was observed in the dorsal aorta and caudal aorta (Figure 2), and was also apparent in smaller arteries like the carotid arteries (not shown). In contrast, the overall structure of the blood vessels in the Notch22/2 and Notch32/2 embryos at E11.5 was similar to wildtype. Introduction The Notch family of receptors is evolutionarily conserved and critical for cell fate determination and differentiation [1,2]. Each of the four Notch receptors present in mammals (Notch 1–4) is activated by a membrane-bound ligand (Jagged-1,2/Delta-like- 1,3,4), which promotes receptor cleavage releasing a Notch intracellular domain (NICD) that translocates to the nucleus. In the nucleus, the NICD binds to the transcription factor CSL (CBF- 1/RBP-Jk, Su(H), and Lag-1) and regulates downstream gene expression such as Hes (hairy/enhancer of split) and Hey (Hairy- related, also referred to as Hrt, CHF, HESR) family members [3]. In the vasculature, Notch activation regulates the expression of angiogenic factors, including members of the vascular endothelial growth factor (VEGF) pathway [4], and platelet-derived growth factor receptor-ß [5]. Not surprisingly, functional studies have demonstrated a role for Notch signaling in angiogenic remodeling, arterial/venous specification, and in endothelial tip cell differen- tiation [6,7]. While, many of these studies focused on the actions of Notch signaling in the endothelium, others have identified a role for Notch activation in vascular smooth muscle development [8]. One report showed that expression of the Notch ligand Jagged1 (Jag1) on endothelial cells is essential for neighboring vascular smooth muscle differentiation [9], indicating a requirement for Notch receptors on smooth muscle cells. The Notch3 receptor is highly enriched in smooth muscle [10,11] and Notch3 knockout May 2012 \| Volume 7 \| Issue 5 \| e37365 1 PLoS ONE \| www.plosone.org Notch Receptors Regulate Smooth Muscle Development Figure 1. Combined mutations in Notch2 and Notch3 cause defects in vascular development. Yolk sacs (A) and embryos (B) at E10.5 and E11.5 were dissected and photographed with a stereo microscope. At E10.5, Notch22/2;Notch32/2 (N22/2;N32/2) embryos exhibit a decrease in yolk sac blood vessels, while the embryo proper is relatively normal in appearance. At E11.5, Notch22/2;Notch32/2 mice show severe vascular defects in both yolk sac and embryo. Yolk sac blood vessels are not visible and extensive hemorrhaging is seen in the embryo (arrowhead). Blood vessels are grossly normal in Notch22/2 (N22/2;N3+/+) and Notch32/2 (N2+/+;N32/2) embryos. Scale bar = 1 mm. doi:10.1371/journal.pone.0037365.g001 Results Combined mutations in Notch2 and Notch3 genes cause embryonic lethality Notch2 and Notch3 double mutants show deficiencies in smooth muscle cells We stained whole embryos with the endothelial cell marker Pecam1 and the smooth muscle marker smooth muscle a-actin (SMA) to assess the overall structure of the blood vessels in mutant embryos. At E10.5, Pecam1 staining in the double mutant embryos appeared similar to wildtype and the single mutant embryos, indicating that vascular patterning was grossly normal and endothe- lial cells were being properly positioned (Figure 3A). Staining with SMA showed similar expression levels in the heart of all genotypes, but the Notch22/2;Notch32/2 embryos and Notch22/2 embryos exhibited a decrease in the SMA staining within the paired dorsal aorta (Figure 3B). These data indicated that the loss of Notch2 and the combined loss of Notch2 and Notch3 result in a decrease in smooth muscle cells surrounding the blood vessel walls. Section staining through blood vessels at E10.5 and E11.5 showed a consistent result with the whole mount analysis. At E10.5, Pecam1 expression showed a similar patterning in all genotypes, while SMA expression was greatly reduced in both the Notch22/2 and Notch22/2; Notch32/2 embryos (Figure 4A, Figure S2). At this stage SMA expression appeared mottled, with many vessels exhibiting SMA staining that was asymmetrically localized. At E11.5, the expression pattern of Pecam1 was decreased in the double mutant embryos, with SMA expression barely detectable (Figure 4B, Figure S2). Notch2 mutant mice had reduced Pecam1 staining compared to wildype and Notch32/2 embryos, and showed SMA staining that was moderately reduced from wildtype (Figure 4B, Figure S2). Collectively, our results show that at E10.5, vessels from Notch22/2 and Notch22/2;Notch32/2 embryos are similar, however at E11.5, the loss of both genes causes a dramatic reduction in SMA staining and compromised vessel structure. These defects were exclusive to smooth muscle of P May 2012 \| Volume 7 \| Issue 5 \| e37365 PLoS ONE \| www.plosone.org 2 Notch Receptors Regulate Smooth Muscle Development Notch2/Notch3 mutant embryos, as expression of SMA in the myocardium was comparable to wildtype (Figure S3). Table 1. Notch2 (N2) and Notch3 (N3) mutant embryos with yolk sac defects. Genotypes Age N2+/+;N3+/+ N22/2;N3+/+ N2+/+;N32/2 N22/2;N32/2 E10.5 % 0% 0% 0% 45% # 0 of 14 0 of 17 0 of 18 10 of 22 E11.5 % 0% 11% 8% 92% # 0 of 16 2 of 19 1 of 13 12 of 13 doi:10.1371/journal.pone.0037365.t001 Figure 3. Notch22/2 and Notch22/2;Notch32/2 embryos devel- op a normal vascular plexus but have disrupted vascular smooth muscle cells. Combined Notch2 and Notch3 mutations cause yolk sac defects Consistent with our immunohistochemistry results, Pecam1 expression was normal at E10.5, and showed a reduction at E11.5 in all mutant genotypes. Examination of Notch target genes indicated no significant difference in Hes1 expression and a small decrease in Hey2. Heyl expression was dramatically affected by the loss of Notch2 and the combined Notch2/Notch3 mutant, but only slightly affected by the loss of Notch3. Notch2 and Notch3 double mutants show deficiencies in smooth muscle cells Whole-mount embryos at E10.5 were stained for Pecam1 (A) or SMA (B). The vascular plexus is well formed in all mutant embryos with normal vessel patterning seen in large vessels (A, upper panels) and smaller intersomitic vessels (A, lower panels). Whole- mount immunostaining for SMA demonstrates less SMA-positive cells in the dorsal aorta of Notch22/2 (N22/2;N3+/+) and Notch22/2;Notch32/2 (N22/2;N32/2) (B, lower panels) embryos compared to wildtype and Notch32/2 (N2+/+;N32/2) embryos. Arrowheads point to paired dorsal aorta. Scale bar = 1 mm. (H) heart. doi:10.1371/journal.pone.0037365.g003 Genotypes Age N2+/+;N3+/+ N22/2;N3+/+ N2+/+;N32/2 N22/2;N32/2 E10.5 % 0% 0% 0% 45% # 0 of 14 0 of 17 0 of 18 10 of 22 E11.5 % 0% 11% 8% 92% # 0 of 16 2 of 19 1 of 13 12 of 13 doi:10.1371/journal.pone.0037365.t001 Notch2/Notch3 mutant embryos, as expression of SMA in the myocardium was comparable to wildtype (Figure S3). Notch2/Notch3 mutant embryos, as expression of SMA in the myocardium was comparable to wildtype (Figure S3). Notch2/Notch3 mutant embryos, as expression of SMA in the myocardium was comparable to wildtype (Figure S3). Combined Notch2 and Notch3 mutations cause yolk sac defects Our initial analysis showed that the yolk sac vasculature is disrupted in double mutant embryos, so we additionally analyzed these blood vessels to determine if similar defects existed in this vascularized tissue. Yolk sac blood vessels at E11.5 had slightly reduced amounts of Pecam1 staining in both the Notch22/2 and Notch22/2;Notch32/2 genotypes and reduced SMA expression (Figure 5). Similar to the embryos, the yolk sac vessels of the double mutant mice appeared structurally fragile, and in many Figure 3. Notch22/2 and Notch22/2;Notch32/2 embryos devel- op a normal vascular plexus but have disrupted vascular smooth muscle cells. Whole-mount embryos at E10.5 were stained for Pecam1 (A) or SMA (B). The vascular plexus is well formed in all mutant embryos with normal vessel patterning seen in large vessels (A, upper panels) and smaller intersomitic vessels (A, lower panels). Whole- mount immunostaining for SMA demonstrates less SMA-positive cells in the dorsal aorta of Notch22/2 (N22/2;N3+/+) and Notch22/2;Notch32/2 (N22/2;N32/2) (B, lower panels) embryos compared to wildtype and Notch32/2 (N2+/+;N32/2) embryos. Arrowheads point to paired dorsal aorta. Scale bar = 1 mm. (H) heart. doi:10.1371/journal.pone.0037365.g003 Figure 2. Embryos lacking both Notch2 and Notch3 have disrupted blood vessels. Hematoxylin and eosin staining of transverse sections of E11.5 embryos through the heart and midsection (A–D), descending aorta (E–H), and caudal aorta (I–L). In the Notch22/2; Notch32/2 (N22/2;N32/2) embryos, the paired dorsal aorta is expanded in size and filled with blood (D, arrowheads). Higher magnification of blood vessels in double mutant embryos show a lack of cells surrounding the lumen (H, L). The overall structure of blood vessels appears relatively normal in the single Notch22/2 (N22/2;N3+/+) and Notch32/2 (N2+/+;N32/2) mice. Scale bar = 100 mm. (H) heart, (nt) neural tube. doi:10.1371/journal.pone.0037365.g002 instances were collapsed. Because yolk sacs are a highly vascularized tissue, we used this tissue to assess gene expression of smooth muscle markers and Notch signaling targets by qPCR (Figure 6). SMA RNA levels showed a decrease consistent with immunofluorescence staining, and another early marker of smooth muscle cells, SM22a exhibited a decrease at E10.5 but not at E11.5. The pronounced decrease observed at E10.5 compared to E11.5 likely reflects continued expression of these markers in nonvascular cells within the whole yolk sac. A later smooth muscle marker gene, Calponin-h1 (Cnn1) exhibited decreased transcripts in all mutant genotypes at E11.5, but not E10.5. Notch2 and Notch3 activate downstream targets with similar efficiency Previously we showed that NOTCH3 was induced in smooth muscle by Notch signaling and could activate its own transcription through autoregulation [14]. Consistent with this, NICD3 activated transcription of the endogenous NOTCH3 gene, but was not able to induce the expression of NOTCH2 in aortic smooth muscle cells. NICD2 could also promote the expression of NOTCH3, but could not induce the expression of endogenous NOTCH2 transcripts (Figure 7C). Overall, our data demonstrate a critical role for Notch signaling in the development of vascular smooth muscle cell, and indicate that Notch2 and Notch3 have overlapping yet distinct roles in governing smooth muscle differentiation. Similarly, overexpression of NICD2 and NICD3 activated known Notch target genes (Figure 7B). Previously we showed that NOTCH3 was induced in smooth muscle by Notch signaling and could activate its own transcription through autoregulation [14]. Consistent with this, NICD3 activated transcription of the endogenous NOTCH3 gene, but was not able to induce the expression of NOTCH2 in aortic smooth muscle cells. NICD2 could also promote the expression of NOTCH3, but could not induce the expression of endogenous NOTCH2 transcripts (Figure 7C). Overall, our data demonstrate a critical role for Notch signaling in the development of vascular smooth muscle cell, and indicate that Notch2 and Notch3 have overlapping yet distinct roles in governing smooth muscle differentiation. While previous data showed that other Notch receptors do not compensate in the absence of Notch3 through increased expres- sion [11], their presence in smooth muscle cells may contribute to smooth muscle differentiation. Indeed, Notch2 has been shown to be strongly expressed in smooth muscle and data from knockout mice show cardiac outflow tract anomalies consistent with smooth muscle defects [15,17]. Because Notch2 null mice die very early due to massive cell death [16], in this study we utilized a hypomorphic allele, which had been previously characterized to have cardiovascular defects [17]. However the smooth muscle differentiation profile of these hypomorphic mice had not been reported. In our analysis we show for the first time, that Notch2 hyopmorphic mice have a loss of smooth muscle markers as early as E10.5, which likely leads to later defects associated with hemorrhage and outflow tract defects. In contrast, Notch3 null mice at E10.5 exhibit normal vascular structure, with no apparent signs of defects. Loss of function of the two Notch genes gives rise to a complete breakdown of the vascular wall at E11.5. Notch2 and Notch3 activate downstream targets with similar efficiency Figure 2. Embryos lacking both Notch2 and Notch3 have disrupted blood vessels. Hematoxylin and eosin staining of transverse sections of E11.5 embryos through the heart and midsection (A–D), descending aorta (E–H), and caudal aorta (I–L). In the Notch22/2; Notch32/2 (N22/2;N32/2) embryos, the paired dorsal aorta is expanded in size and filled with blood (D, arrowheads). Higher magnification of blood vessels in double mutant embryos show a lack of cells surrounding the lumen (H, L). The overall structure of blood vessels appears relatively normal in the single Notch22/2 (N22/2;N3+/+) and Notch32/2 (N2+/+;N32/2) mice. Scale bar = 100 mm. (H) heart, (nt) neural tube. Because our results suggested that both Notch2 and Notch3 contribute to smooth muscle differentiation we wanted to determine if they could directly activate smooth muscle-specific gene expression. We overexpressed the intracellular domains of the Notch2 (NICD2) and Notch3 (NICD3) by lentiviral infection of human aortic smooth muscle cells and examined gene expression by qPCR. Both NICD2 and NICD3 activated the expression of smooth muscle differentiation genes SMA, CNN1 and smooth muscle myosin heavy chain (SM-MHC) (Figure 7A). PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37365 3 Notch Receptors Regulate Smooth Muscle Development Figure 5. SMA expression is decreased in yolk sacs of Notch2 and Notch2/Notch3 double mutant embryos. Yolk sacs collected at E11.5 were stained for SMA (red) and Pecam1 (green). SMA is prominently expressed around the blood vessels in yolk sacs of wildtype and Notch32/2 (N2+/+;N32/2) embryos, whereas its expression is significantly decreased in Notch22/2 (N22/2;N3+/+) and Notch22/2; Notch32/2 (N22/2;N32/2) vessels. Yolk sacs of the Notch22/2;Notch32/2 embryos have blood vessels that appear structurally deficient compared to the other genotypes. Scale bar = 100 mm. doi:10.1371/journal.pone.0037365.g005 Figure 4. Notch2 and Notch2/Notch3 double mutant embryos exhibit diminished smooth muscle cell marker expression. Transverse sections of embryos at E10.5 (A) and E11.5 (B) were stained for SMA (red) and Pecam1 (green). Notch22/2 (N22/2;N3+/+) and Notch22/2;Notch32/2 (N22/2;N32/2) embryos exhibit less SMA-positive cells in the dorsal aorta at E10.5 compared to wild-type and Notch32/2 (N2+/+;N32/2) embryos, while Pecam1 levels are similar in all genotypes. At E11.5, Notch22/2;Notch32/2 embryos show an even greater loss of SMA expression, with increased vessel diameter. Merged images also show DAPI stain (blue) to highlight nuclei. Scale bar = 100 mm. doi:10.1371/journal.pone.0037365.g004 Figure 4. Notch2 and Notch2/Notch3 double mutant embryos exhibit diminished smooth muscle cell marker expression. Notch2 and Notch3 activate downstream targets with similar efficiency At E10.5, the expression of SMA looks comparable in the Notch2 and double mutant embryos, suggesting that Notch2 activity predom- inates at this early stage, however at E11.5, the double mutant vessels appear to lose most of their smooth muscle cells, while the Notch2 embryos retain the expression of smooth muscle marker, Notch2 and Notch3 activate downstream targets with similar efficiency Transverse sections of embryos at E10.5 (A) and E11.5 (B) were stained for SMA (red) and Pecam1 (green). Notch22/2 (N22/2;N3+/+) and Notch22/2;Notch32/2 (N22/2;N32/2) embryos exhibit less SMA-positive cells in the dorsal aorta at E10.5 compared to wild-type and Notch32/2 (N2+/+;N32/2) embryos, while Pecam1 levels are similar in all genotypes. At E11.5, Notch22/2;Notch32/2 embryos show an even greater loss of SMA expression, with increased vessel diameter. Merged images also show DAPI stain (blue) to highlight nuclei. Scale bar = 100 mm. doi:10.1371/journal.pone.0037365.g004 Figure 5. SMA expression is decreased in yolk sacs of Notch2 and Notch2/Notch3 double mutant embryos. Yolk sacs collected at E11.5 were stained for SMA (red) and Pecam1 (green). SMA is prominently expressed around the blood vessels in yolk sacs of wildtype and Notch32/2 (N2+/+;N32/2) embryos, whereas its expression is significantly decreased in Notch22/2 (N22/2;N3+/+) and Notch22/2; Notch32/2 (N22/2;N32/2) vessels. Yolk sacs of the Notch22/2;Notch32/2 embryos have blood vessels that appear structurally deficient compared to the other genotypes. Scale bar = 100 mm. doi:10.1371/journal.pone.0037365.g005 Figure 4. Notch2 and Notch2/Notch3 double mutant embryos exhibit diminished smooth muscle cell marker expression. Transverse sections of embryos at E10.5 (A) and E11.5 (B) were stained for SMA (red) and Pecam1 (green). Notch22/2 (N22/2;N3+/+) and Notch22/2;Notch32/2 (N22/2;N32/2) embryos exhibit less SMA-positive cells in the dorsal aorta at E10.5 compared to wild-type and Notch32/2 (N2+/+;N32/2) embryos, while Pecam1 levels are similar in all genotypes. At E11.5, Notch22/2;Notch32/2 embryos show an even greater loss of SMA expression, with increased vessel diameter. Merged images also show DAPI stain (blue) to highlight nuclei. Scale bar = 100 mm. doi:10.1371/journal.pone.0037365.g004 athy with subcortical infarcts and leukoencephalopathy (CADA- SIL) [10,11,20]. Data from knockout mice indicated that loss of Notch3 results in smooth muscle maturation defects and causes deficiencies in arterial specification [12]. Further studies with postnatal mice revealed a role for Notch3 in the regulation of proliferation and vascular tone [18,21]; however, Notch3 null mice undergo a normal restenosis response following vascular injury [22]. These data clearly indicate that Notch3 regulates aspects of smooth muscle function, but is non-essential for smooth muscle differentiation. In vitro data from our lab indicated that Notch3 drives expression of smooth muscle genes, and in a model of endothelial cell-induced smooth muscle differentiation, Notch3 is critical for smooth muscle gene expression [14]. Similarly, overexpression of NICD2 and NICD3 activated known Notch target genes (Figure 7B). PLoS ONE \| www.plosone.org Discussion Although several lines of evidence have implicated Notch signaling in vascular smooth muscle development, the data supporting this notion has been less conclusive. Here we show that combined mutations of Notch2 and Notch3 genes in mice results in severe cardiovascular defects with the underlying cause due to a lack of smooth muscle differentiation. Previous studies on Notch receptor function in vascular smooth muscle cells have largely focused on Notch3, due to its relatively specific expression within this cell type and the known association with the human cerebral vascular disease, cerebral autosomal dominant arteriop- PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37365 May 2012 \| Volume 7 \| Issue 5 \| e37365 4 Notch Receptors Regulate Smooth Muscle Development Figure 6. Smooth muscle differentiation and Notch target genes are down regulated in Notch2 and Notch2/Notch3 double mutants. Gene expression analysis (qPCR) using yolk sac RNA demonstrates a reduction of smooth muscle-specific genes SMA, SM22a, and Cnn1 in Notch22/2 (N22/2;N3+/+) and Notch22/2;Notch32/2 (N22/2;N32/2). Notch signaling target gene Heyl shows a robust decrease at E10.5 and E11.5 in the Notch22/2 (N22/2;N3+/+) and Notch22/2;Notch32/2 (N22/2;N32/2) yolk sacs, while Hey2 shows a slight decrease and Hes1 expression is unaffected in the mutant genotypes. Data represent relative mRNA expression levels normalized to 18S rRNA. * P,0.05, ** P,0.01 compared to wildtype. doi:10 1371/journal pone 0037365 g006 p g p Figure 6. Smooth muscle differentiation and Notch target genes are down regulated in Notch2 and Notch2/Notch3 double mutants. Gene expression analysis (qPCR) using yolk sac RNA demonstrates a reduction of smooth muscle-specific genes SMA, SM22a, and Cnn1 in Notch22/2 (N22/2;N3+/+) and Notch22/2;Notch32/2 (N22/2;N32/2). Notch signaling target gene Heyl shows a robust decrease at E10.5 and E11.5 in the Notch22/2 (N22/2;N3+/+) and Notch22/2;Notch32/2 (N22/2;N32/2) yolk sacs, while Hey2 shows a slight decrease and Hes1 expression is unaffected in the mutant genotypes. Data represent relative mRNA expression levels normalized to 18S rRNA. * P,0.05, ** P,0.01 compared to wildtype. doi 10 1371/journal pone 0037365 g006 Notch3 expression and together they drive the differentiation program. In the absence of Notch2, smooth muscle differentiation is delayed slightly, however the presence of Notch3 can maintain vascular integrity. In the absence of Notch3, Notch2 expression is sufficient for the initiation of smooth muscle differentiation, but is not capable of the fine-tuning required for the later steps of maturation. PLoS ONE \| www.plosone.org Discussion In the absence of both genes, smooth muscle cells cannot differentiate properly, the vessels carrying blood through the embryos fail, resulting in hemorrhaging and eventual collapse of the vessel. Overall, our data show for the first time a critical role for Notch receptors in vascular smooth muscle development, and suggest that propagation of Notch signaling in this cell type requires the combined efforts of Notch2 and Notch3. The actual mechanisms by which Notch2 and Notch3 coordinate to regulate vascular development are not known. Differences in how they are SMA. Importantly, the double mutant embryos still express some SMA and SM22a, implying that these Notch receptors are not completely necessary for the expression of these smooth muscle marker genes during differentiation. Additional signaling pathways are likely involved in maintaining expression during differentia- tion, such as those driven by TGFß or serum response factor (SRF) [23]. Notch3 expression and together they drive the differentiation program. In the absence of Notch2, smooth muscle differentiation is delayed slightly, however the presence of Notch3 can maintain vascular integrity. In the absence of Notch3, Notch2 expression is sufficient for the initiation of smooth muscle differentiation, but is not capable of the fine-tuning required for the later steps of maturation. In the absence of both genes, smooth muscle cells cannot differentiate properly, the vessels carrying blood through the embryos fail, resulting in hemorrhaging and eventual collapse of the vessel. Overall, our data show for the first time a critical role for Notch receptors in vascular smooth muscle development, and suggest that propagation of Notch signaling in this cell type requires the combined efforts of Notch2 and Notch3. The actual mechanisms by which Notch2 and Notch3 coordinate to regulate vascular development are not known. Differences in how they are Previously it was shown that endothelial cell-expressed Notch ligand Jagged1 is critical for smooth muscle differentiation [9]. Our previous data and findings shown here, indicate that Notch3 expression, but not Notch2 is induced by Notch signaling [14]. Possibly, within smooth muscle cells Notch2 is activated by the Jagged1 ligand on neighboring endothelial cells to establish the first wave of Notch activation and differentiation. Mouse lines, genotyping and crosses All strains were maintained in C57Bl/6 background. Notch2del1 [17] and Notch3dl [19] single mutant mice were generated and generously provided by Dr. Thomas Gridley. Notch2del1/+, referred to here as Notch22/+ (N22/+) mice were crossed with Notch3dl/dl, referred to here as Notch32/2 (N32/2) mice to generate Notch22/+; Notch32/+ double heterozygous mice. To produce embryos for analysis, Notch22/+;Notch32/+ mice were intercrossed. Nine differ- ent genotypes of embryos were generated and collected as shown Discussion During this phase, Notch2 activates not only smooth muscle genes, but also May 2012 \| Volume 7 \| Issue 5 \| e37365 May 2012 \| Volume 7 \| Issue 5 \| e37365 5 Notch Receptors Regulate Smooth Muscle Development regulated by upstream transcription factors might contribute to Hospital. The Nationwide Children’s Hospital Research Institute Figure 7. NICD2 and NICD3 can directly activate Notch targets and smooth muscle marker genes. Activated forms of Notch2 (NICD2) and Notch3 (NICD3) were introduced into human aortic smooth muscle cells by lentiviral transduction, followed by qPCR to analyze gene expression. Both NICD2 and NICD3 robustly activate expression of smooth muscle genes, SMA, CNN1, and SM-MHC (A), and also activate Notch targets HEYL, HEY2, and HES1 (B), compared to a GFP-expressing control. Both NICD2 and NICD3 activate endogenous NOTCH3 expression, but not NOTCH2 expression (C). Western blot demonstrates expression of the NICD2 and NICD3 constructs with a FLAG antibody (D). Data represent relative mRNA expression levels normalized to 18S rRNA. * P,0.05, ** P,0.01 compared to GFP control. doi:10.1371/journal.pone.0037365.g007 Notch Receptors Regulate Smooth Muscle Development Figure 7. NICD2 and NICD3 can directly activate Notch targets and smooth muscle marker genes. Activated forms of Notch2 (NICD2) and Notch3 (NICD3) were introduced into human aortic smooth muscle cells by lentiviral transduction, followed by qPCR to analyze gene expression. Both NICD2 and NICD3 robustly activate expression of smooth muscle genes, SMA, CNN1, and SM-MHC (A), and also activate Notch targets HEYL, HEY2, and HES1 (B), compared to a GFP-expressing control. Both NICD2 and NICD3 activate endogenous NOTCH3 expression, but not NOTCH2 expression (C). Western blot demonstrates expression of the NICD2 and NICD3 constructs with a FLAG antibody (D). Data represent relative mRNA expression levels normalized to 18S rRNA. * P,0.05, ** P,0.01 compared to GFP control. doi:10.1371/journal.pone.0037365.g007 Hospital. The Nationwide Children’s Hospital Research Institute IACUC specifically approved this study. regulated by upstream transcription factors might contribute to individual temporal functions that converge within blood vessels. Alternatively, unique structural features of each Notch family member could contribute to distinct downstream interactions and responses that when acting together shape the vascular landscape. Cell culture and lentiviral expression of NICD2 and NICD3 Cell culture and lentiviral expression of NICD2 and NICD3 Human aortic smooth muscle cells (HAoSMC) were purchased from Lonza and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Mediatech, Inc.) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 2 mM glutamine, 1 mM sodium pyruvate and 100 U/ml penicillin-streptomycin. Cells between passages 6– 9 were used for all experiments. For virus production, TN293 cells were purchased from Stratagene and cultured in 10% DMEM as above. All cultures were maintained in humidified 5% CO2 at 37uC. Human NOTCH2 intracellular domain (NICD2) cDNA (a gift from Dr. Igor Prudovsky) was cloned with a 36-FLAG-tag attached to the 59 end into pCDF1-MCS2-EF1-copGFP (System Biosciences). NOTCH3 intracellular domain (NICD3) with a 36- FLAG-tag was made as described previously [14]. The lentivirus plasmids were transfected into TN-293 cells using Lipofectamine 2000 (Invitrogen), and the viral particles were amplified and purified as described [14]. Equal volumes of viral particles were diluted in 10% FBS in DMEM and were incubated with cells for 24 hours. The efficiency of infection was evaluated using GFP expression and qPCR. Viral particles were titrated to achieve 90% to 100% infection. Expression of cDNAs were confirmed using qPCR (not shown) and Western blot analysis using a FLAG antibody (SIGMA, F1804) (Figure 7). Quantitative RT-PCR (qPCR) Total RNA was extracted using RNeasy Mini Kit (QIAGEN, Cat: 74104) from a minimum of five yolk sacs corresponding to each genotype. RNA from cultured cells was isolated by TRIzol (Invitrogen). Reverse transcription was performed using M-MLV reverse transcriptase (Invitrogen, Cat: 28025-013). SYBR green detection of PCR amplicons was performed using an ABI qPCR machine. Corresponding gene expression level was normalized to 18S rRNA or Gapdh from the same sample. Primer sequences are listed in Table S2. Statistical analysis Data shown are representatives of at least three independent experiments and are presented as mean 6 standard error of the mean (SEM). Data analyses were conducted using GraphPad Prism and comparisons of the data among 4 different groups were made using One-way ANOVA, followed by Newman-Keuls test. Differences were considered significant if P,0.05. Whole-mount immunohistochemistry Embryos or yolk sacs were harvested in cold phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde overnight at 4uC or 1 hour at room temperature. For embryo staining, the embryos were dehydrated in a graded methanol series, and bleached with Methanol: DMSO: 3% H2O2 (4:1:1) or PBS: 3% H2O2 (4:1) to block endogenous peroxidase. Embryos were then blocked and permeabilized with 5% instant nonfat milk or goat serum and 0.3% Triton-X-100. Primary antibodies, Smooth muscle a-actin (SMA) (1:500, SIGMA, Cat: A2547) or Pecam1 (1:50, BD Pharmingen, Cat: 550274) were incubated overnight at 4uC, followed by incubation with appropriate HRP-conjugated second- ary antibody (1:500) overnight at 4uC. The color reaction was done in PBT (PBS+0.1% tween-20) containing 0.5 mg/ml 3,39- diaminobenzidine (DAB, SIGMA, Cat: D-5673) and 0.01% H2O2. Embryos were dehydrated and cleared in benzyl alcohol: benzyl benzoate (1:2) (SIGMA). Pictures were captured with a dissecting microscope (Leica, M156C). Immunostaining of whole yolk sacs was performed as previously described with modifications [13]. In brief, after fixation, yolk sacs were placed in cold methanol for 10 minutes, blocked in 5% goat serum and 0.3% Triton-X-100 for 1 hour at room temperature, followed by Pecam1 and SMA antibody incubation for 2 hours at 37uC. Yolk sacs were then incubated with AlexaFluor-conjugated second antibody overnight at 4uC. After washing in PBT, yolk sacs were flattened and mounted in aqueous mounting medium (LERNER LABORA- TORIES, Cat: 13800). Images were captured using a confocal microscope (Zeiss LSM 710). Supporting Information Figure S1 Hematoxylin and eosin staining of transverse sections of E10.5 embryos through the descending aorta. The overall structure of blood vessels appears relatively normal in the single mutant Notch22/2 (N22/2;N3+/+), Notch32/2 (N2+/+; N32/2) and double mutant, Notch22/2;Notch32/2 (N22/2;N32/2) embryos. 406 magnification. (PDF) Figure S2 Quantification of Pecam1 and SMA staining of sectioned aortas. Staining of Pecam1 and SMA was quantified by measuring the number of pixels with a set intensity and normalizing to vessel circumference. (A) At E10.5, the Notch22/2 (N22/2;N3+/+) and Notch22/2;Notch32/2 (N22/2;N32/2) embryos exhibit less SMA-positive staining intensity. (B) At E11.5, both Pecam1 and SMA expression is significantly reduced in the Notch2 and double mutant aortas. Notch22/2;Notch32/2 embryos show an even greater loss of SMA expression compared to the Notch22/2 mice. P,0.05, * compared to wildtype control, # compared to Notch22/2. (PDF) Ethics statement All mouse studies were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Research Institute at Nationwide Children’s PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37365 6 Notch Receptors Regulate Smooth Muscle Development with primary antibody, SMA (1:1000, SIGMA, Cat: A2547), and Pecam1 (1:250, Santa Cruz, sc-1506-R) overnight at 4uC. After washing in PBT, sections were incubated with appropriate AlexaFluor–conjugated secondary antibody (1:500 Invitrogen) for 1 hour at room temperature, counterstained with DAPI, and mounted in Vectashield mounting medium (Vector Laboratories, H-1400). Pictures were taken using a fluorescence microscope (OLYMPUS, 1X51). in Table 1. Their numbers were tested for goodness of fit to expected Mendelian segregation. Embryos were considered embryonic (E) day 0.5 (E0.5) at the day when vaginal plug was observed. Genotyping of mice and embryos was carried out by PCR with Notch2wtsp3: 59-CCA GTG TGC CAC AGG TAA GTG-39, Notch2wtsp4: 59-TCT CCA TAT TGA TGA GCC ATG C-39, Notch2dlsp6: 59 –TTC CTG ACT AGG GGA GGA GTA G-39. Notch3wt1: 59-CCA TGA GGA TGC TAT CTG TGA C-39, Notch3wt2: 59-CAC ATT GGC ACA AGA ATG AGC C-39, Notch3dl1: 59-GGT ACT GAG AAC CAA ACT CAG C-39, Notch3dl2: 59-TCG CCT TCT ATC GCC TTC TTG A-39. in Table 1. Their numbers were tested for goodness of fit to expected Mendelian segregation. Embryos were considered embryonic (E) day 0.5 (E0.5) at the day when vaginal plug was observed. Genotyping of mice and embryos was carried out by PCR with Notch2wtsp3: 59-CCA GTG TGC CAC AGG TAA GTG-39, Notch2wtsp4: 59-TCT CCA TAT TGA TGA GCC ATG C-39, Notch2dlsp6: 59 –TTC CTG ACT AGG GGA GGA GTA G-39. Notch3wt1: 59-CCA TGA GGA TGC TAT CTG TGA C-39, Notch3wt2: 59-CAC ATT GGC ACA AGA ATG AGC C-39, Notch3dl1: 59-GGT ACT GAG AAC CAA ACT CAG C-39, Notch3dl2: 59-TCG CCT TCT ATC GCC TTC TTG A-39. Acknowledgments The authors wish to thank Vidu Garg and Mary Cismowski for critical reading of the manuscript. We wish to extend a special thanks to Hua Liu for assistance with the initial phase of embryo collections. Histological analysis of embryos After fixation, embryos were processed, embedded in paraffin, and sectioned at 8 mm. Hematoxylin and eosin staining was performed by standard staining protocol. For immunohistochem- istry, sections were baked at 60uC for 1 hour, cleared in xylene, rehydrated through a descending concentration of ethanol for 2 minutes each ending in distilled water. Antigen retrieval was done in citrate buffer (0.01 M, PH = 6.0) using a pressure cooker for 30 minutes. Sections were cooled to room temperature and blocked with 5% goat serum diluted in PBS with 0.5% Triton-X- 100 for 1 hour at room temperature. Sections were then incubated PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37365 7 Notch Receptors Regulate Smooth Muscle Development Table S1 Recovery of Notch2 (N2) and Notch3 (N3) mutant embryos at various gestational ages. (PDF) Author Contributions Conceived and designed the experiments: QW NZ BL. Performed the experiments: QW NZ SK BL. Analyzed the data: QW NZ BL. Contributed reagents/materials/analysis tools: QW NZ SK BL. Wrote the paper: QW NZ BL. Table S1 Recovery of Notch2 (N2) and Notch3 (N3) mutant embryos at various gestational ages. (PDF) Figure S3 Notch2 and Notch3 double mutant embryos have structurally normal hearts. Transverse sections from wildtype and mutant embryos at E10.5 were stained for SMA (red) and Pecam1 (green) (A). All mutant embryos have normal SMA expression in the cardiomyocytes and Pecam1 staining of the endocardial cells. H&E staining of transverse sections through the outflow tract of wildtype and mutant embryos at E10.5 and E11.5 (B). At E10.5, hearts of the double mutant embryos are comparable to wildtype and single mutant embryos. A day later the Notch2/ Notch3 double mutant embryo’s outflow tract show signs of cellular atrophy, whereas the other three genotypes appear structurally normal. Notch22/2 (N22/2;N3+/+), Notch32/2 (N2+/+;N32/2) and double mutant, Notch22/2;Notch32/2 (N22/2;N32/2) embryos. 106magnification. (PDF) Figure S3 Notch2 and Notch3 double mutant embryos have structurally normal hearts. Transverse sections from wildtype and mutant embryos at E10.5 were stained for SMA (red) and Pecam1 (green) (A). All mutant embryos have normal SMA expression in the cardiomyocytes and Pecam1 staining of the endocardial cells. H&E staining of transverse sections through the outflow tract of wildtype and mutant embryos at E10.5 and E11.5 (B). At E10.5, hearts of the double mutant embryos are comparable to wildtype and single mutant embryos. A day later the Notch2/ Notch3 double mutant embryo’s outflow tract show signs of cellular atrophy, whereas the other three genotypes appear structurally normal. Notch22/2 (N22/2;N3+/+), Notch32/2 (N2+/+;N32/2) and double mutant, Notch22/2;Notch32/2 (N22/2;N32/2) embryos. 106magnification. (PDF) References 13. Liu H, Zhang W, Kennard S, Caldwell RB, Lilly B (2010) Notch3 is critical for proper angiogenesis and mural cell investment. Circ Res 107: 860–870. 1. Baron M (2003) An overview of the Notch signalling pathway. Semin Cell Dev Biol 14: 113–119. 14. Liu H, Kennard S, Lilly B (2009) NOTCH3 expression is induced in mural cells through an autoregulatory loop that requires endothelial-expressed JAGGED1. Circ Res 104: 466–475. 2. Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7: 678–689. 3. Fischer A, Gessler M (2007) Delta-Notch–and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors. Nucleic Acids Res 35: 4583–4596. 15. Varadkar P, Kraman M, Despres D, Ma G, Lozier J, et al. (2008) Notch2 is required for the proliferation of cardiac neural crest-derived smooth muscle cells. Dev Dyn 237: 1144–1152. 4. Siekmann AF, Covassin L, Lawson ND (2008) Modulation of VEGF signalling output by the Notch pathway. BioEssays : news and reviews in molecular, cellular and developmental biology 30: 303–313. 16. Hamada Y, Kadokawa Y, Okabe M, Ikawa M, Coleman JR, et al. (1999) Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality. Development 126: 3415–3424. 5. Gaengel K, Genove G, Armulik A, Betsholtz C (2009) Endothelial-Mural Cell Signaling in Vascular Development and Angiogenesis. Arterioscler Thromb Vasc Biol. 17. McCright B, Gao X, Shen L, Lozier J, Lan Y, et al. (2001) Defects in development of the kidney, heart and eye vasculature in mice homozygous for a hypomorphic Notch2 mutation. Development 128: 491–502. 6. Gridley T (2007) Notch signaling in vascular development and physiology. Development 134: 2709–2718. 18. de Chantemele EJ, Retailleau K, Pinaud F, Vessieres E, Bocquet A, et al. (2008) Notch3 is a major regulator of vascular tone in cerebral and tail resistance arteries. Arterioscler Thromb Vasc Biol 28: 2216–2224. p 7. Gridley T (2010) Notch signaling in the vasculature. Current topics in developmental biology 92: 277–309. 19. Krebs LT, Xue Y, Norton CR, Sundberg JP, Beatus P, et al. (2003) Characterization of Notch3-deficient mice: normal embryonic development and absence of genetic interactions with a Notch1 mutation. Genesis 37: 139–143. p gy 8. Morrow D, Guha S, Sweeney C, Birney Y, Walshe T, et al. (2008) Notch and vascular smooth muscle cell phenotype. Circ Res 103: 1370–1382. p gy 8. References Morrow D, Guha S, Sweeney C, Birney Y, Walshe T, et al. (200 vascular smooth muscle cell phenotype. Circ Res 103: 1370–1 9. High FA, Lu MM, Pear WS, Loomes KM, Kaestner KH, et al. (2008) Endothelial expression of the Notch ligand Jagged1 is required for vascular smooth muscle development. Proc Natl Acad Sci U S A 105: 1955–1959. 20. Joutel A, Corpechot C, Ducros A, Vahedi K, Chabriat H, et al. (1996) Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature 383: 707–710. 10. Joutel A, Andreux F, Gaulis S, Domenga V, Cecillon M, et al. (2000) The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients. J Clin Invest 105: 597–605. 21. Li X, Zhang X, Leathers R, Makino A, Huang C, et al. (2009) Notch3 signaling promotes the development of pulmonary arterial hypertension. Nat Med 15: 1289–1297. 11. Kitamoto T, Takahashi K, Takimoto H, Tomizuka K, Hayasaka M, et al. (2005) Functional redundancy of the Notch gene family during mouse embryogenesis: analysis of Notch gene expression in Notch3-deficient mice. Biochem Biophys Res Commun 331: 1154–1162. 22. Li Y, Takeshita K, Liu PY, Satoh M, Oyama N, et al. (2009) Smooth muscle Notch1 mediates neointimal formation after vascular injury. Circulation 119: 2686–2692. y g y g p Biochem Biophys Res Commun 331: 1154–1162. 12. Domenga V, Fardoux P, Lacombe P, Monet M, Maciazek J, et al. (2004) Notch3 is required for arterial identity and maturation of vascular smooth muscle cells. Genes Dev 18: 2730–2735. 23. Owens GK, Kumar MS, Wamhoff BR (2004) Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84: 767–801. PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37365 8
https://openalex.org/W2584306935	https://koedoe.co.za/index.php/koedoe/article/download/1375/2097	English	null	Using genetics to prioritise headwater stream fish populations of the Marico barb, <i>Enteromius motebensis</i> Steindachner 1894, for conservation action	Koedoe	2,017	cc-by	667	KOEDOE - African Protected Area Conservation and Science ISSN: (Online) 2071-0771, (Print) 0075-6458 Page 1 of 1 Corrigendum KOEDOE - African Protected Area Conservation and Science ISSN: (Online) 2071-0771, (Print) 0075-6458 Page 1 of 1 Corrigendum KOEDOE - African Protected Area Conservation and Science ISSN: (Online) 2071-0771, (Print) 0075-6458 KOEDOE - African Protected Area Conservation and Science ISSN: (Online) 2071-0771, (Print) 0075-6458 Page 1 of 1 Page 1 of 1 Corrigendum: Using genetics to prioritise headwater stream fish populations of the Marico barb, Enteromius motebensis Steindachner 1894, for conservation action In the version of this article initially published, Kerry-Ann van der Walt’s and Darragh J. Woodford’s second affiliation, and Olaf L.F. Weyl’s first affiliation was omitted. The initials for Darragh J. Woodford and Olaf L.F. Weyl was also omitted. Read online: Scan this QR code with your smart phone or mobile device Authors: Kerry-Ann van der Walt1,2 Ernst R. Swartz3 Darragh J. Woodford3,4 Olaf L.F. Weyl1,2,3 Affiliations: 1Department of Ichthyology and Fisheries Science, Rhodes University, South Africa 2Centre for Invasion Biology, South African Institute for Aquatic Biodiversity, South Africa 3South African Institute for Aquatic Biodiversity, Grahamstown, South Africa 4School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, South Africa Corresponding author: Kerry-Ann van der Walt, k.vanderwalt@saiab.ac.za Dates: Published: 30 Nov. 2017 How to cite this article: Van der Walt, K-A., Swartz, E.R., Woodford, D.J. & Weyl, O.L.F., 2017, ‘Corrigendum: Using genetics to prioritise headwater stream fish populations of the Marico barb, Enteromius motebensis Steindachner 1894, for conservation action’, Koedoe 59(1), a1506. https://doi. org/10.4102/koedoe. v59i1.1506 Copyright: © 2017. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Scan this QR code with your smart phone or mobile device Authors: Kerry-Ann van der Walt1,2 Ernst R. Swartz3 Darragh J. Woodford3,4 Olaf L.F. Weyl1,2,3 Affiliations: 1Department of Ichthyology and Fisheries Science, Rhodes University, South Africa 2Centre for Invasion Biology, South African Institute for Aquatic Biodiversity, South Africa 3South African Institute for Aquatic Biodiversity, Grahamstown, South Africa 4School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, South Africa Corresponding author: Kerry-Ann van der Walt, k.vanderwalt@saiab.ac.za Dates: Published: 30 Nov. 2017 How to cite this article: Van der Walt, K-A., Swartz, E.R., Woodford, D.J. & Weyl, Authors: Kerry-Ann van der Walt1,2 Ernst R. Swartz3 Darragh J. Woodford3,4 Olaf L.F. Weyl1,2,3 The author list and affiliations are hereby corrected to: Authors: Kerry-Ann van der Walt1,2 Ernst R. Swartz3 Darragh J. Woodford3,4 Olaf L.F. Weyl1,2,3 Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Affiliations: i 1Department of Ichthyology and Fisheries Science, Rhodes University, South Africa 1Department of Ichthyology and Fisheries Science, Rhodes University, South Africa 2Centre for Invasion Biology, South African Institute for Aquatic Biodiversity, South Africa p y gy y 2Centre for Invasion Biology, South African Institute for Aquatic Biodiversity, South Africa 3South African Institute for Aquatic Biodiversity, Grahamstown, South Africa 3South African Institute for Aquatic Biodiversity, Grahamstown, South Africa 4School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, South Africa 4School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, South Africa The errors have been corrected in the PDF version of the article. The corresponding author apologises for any inconvenience that this omission may have caused. How to cite this article: Van der Walt, K-A., Swartz, E.R., Woodford, D.J. & Weyl, O.L.F., 2017, ‘Corrigendum: Using genetics to prioritise headwater stream fish populations of the Marico barb, Enteromius motebensis Steindachner 1894, for conservation action’, Koedoe 59(1), a1506. https://doi. org/10.4102/koedoe. v59i1.1506 Copyright: © 2017. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Note: Doi of original article: https://doi.org/10.4102/koedoe.v59i1.1375 Note: Doi of original article: https://doi.org/10.4102/koedoe.v59i1.1375 Open Access http://www.koedoe.co.za
https://openalex.org/W4226104279	https://acp.copernicus.org/articles/21/18333/2021/acp-21-18333-2021.pdf	English	null	Changes in satellite retrievals of atmospheric composition over eastern China during the 2020 COVID-19 lockdowns	Atmospheric chemistry and physics	2,021	cc-by	14,165	Research article Research article Atmos. Chem. Phys., 21, 18333–18350, 2021 https://doi.org/10.5194/acp-21-18333-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. Robert D. Field1,2, Jonathan E. Hickman1, Igor V. Geogdzhayev1,2, Kostas Tsigaridis1,3, and Susanne E. Bauer1 1NASA Goddard Institute for Space Studies, 2880 Broadway, New York, NY, 10025, USA 2Department of Applied Physics and Applied Mathematics, Columbia University, 2880 Broadway, New York, NY, 10025, USA 1NASA Goddard Institute for Space Studies, 2880 Broadway, New York, NY, 10025, USA 2Department of Applied Physics and Applied Mathematics, Columbia University, 2880 Broadway, New York, NY, 10025, USA 3Center for Climate Systems Research, Columbia University, 2880 Broadway, New York, NY, 10025, USA 3Center for Climate Systems Research, Columbia University, 2880 Broadway, New York, NY, 10025, USA Correspondence: Robert D. Field (robert.ﬁeld@columbia.edu) Correspondence: Robert D. Field (robert.ﬁeld@columbia.edu) 1 Introduction In an effort to control the spread of COVID-19, the Chinese government implemented a range of restrictions on move- ment. These led to reductions in industrial and other work- related and personal activities starting 23 January 2020 in Wuhan, Hubei province, and then extending to other cities and regions in the days that followed. On 8 April 2020, Wuhan was the last city to reopen after a complete lockdown that prevented most people from leaving their homes. These measures have been linked to changes in air quality. A net- work of surface monitoring stations in northern China ob- served 35 % decreases in PM2.5 and 60 % decreases in NO2 concentrations during 29 January through 29 February, as compared to the preceding 3 weeks; CO and SO2 also de- clined (Shi and Brasseur, 2020). In and around Wuhan, de- creases in NO2 and PM2.5 were similar to regional changes, but there was a slight increase in SO2 concentrations (Shi and Brasseur, 2020). Observations by the Tropospheric Monitor- ing Instrument (TROPOMI) showed large decreases in tro- pospheric NO2 column densities over Chinese cities, on the order of 40 % for 11 February to 24 March 2020 compared to the same period in 2019, ranging from roughly 25 % for cities not affected by lockdown to 60 % for Wuhan and Xi’an (Bauwens et al., 2020). Prospective simulations suggested that meteorology may limit the effect of reduced emissions on PM2.5 concentrations, with Chinese cities experiencing less than 20 % reductions (Wang et al., 2020). Bottom-up estimates suggest that SO2 emissions peaked earlier, with declines starting around 2005, primarily as a result of power and industrial pollution control measures as well as the elimination of small industrial boilers (Sun et al., 2018; Zheng et al., 2018b). An earlier peak in SO2 emis- sions is consistent with observations by multiple satellite in- struments, which revealed declines in SO2 column densities since 2005 (Fioletov et al., 2016; Krotkov et al., 2016; Wang and Wang, 2020; Zhang et al., 2017; Si et al., 2019). AOD retrievals from the Along Track Scanning Radiome- ter instruments show a steady increase over southeastern China from 1995 to 2005 (Sogacheva et al., 2020) and a de- cline since 2005 in the MODIS AOD (He et al., 2019). 1 Introduction The AOD peak has been argued to match the ∼2011 peak in NO2 (Zheng et al., 2018b; Xie et al., 2019), to match the ∼2005 peak of SO2, or to have occurred at some point in between (Ma et al., 2016), with more rapid decreases in AOD after 2011 (Lin et al., 2018). The recent decrease in AOD is also seen in Visible Infrared Imaging Radiometer Suite (VIIRS) retrievals (Sogacheva et al., 2020). Most mitigation of direct PM2.5 emissions since 2010 was by industry, with residential emissions also decreasing substantially (Zheng et al., 2018b). The decline in SO2 emissions also exerted an important in- ﬂuence, with the sulfate concentration of PM2.5 decreasing substantially between 2013 and 2017 (Shao et al., 2018), re- ﬂecting the negative trend in SO2 emissions. g The goal of our study was to consider these changes against pollution trends in China using NASA Earth Ob- serving System data by combining several products to give a holistic view covering several emission sectors that are responsible for the observed changes. Over the last 2 to 3 decades, air pollution in China appears to have followed the pattern described by the environmental Kuznets curve (Selden and Song, 1994). This framework describes a re- lationship in which economic growth is initially accompa- nied by an increase in air pollution, when poverty remains widespread. But as growth continues, air pollution is ex- pected to level off and decline as a consequence of changes in social awareness of environmental degradation and the economic, political, and technological capacity to limit it (Sarkodie and Strezov, 2019; Selden and Song, 1994). The peak in concentrations of CO, which has an atmo- spheric lifetime ranging from weeks to months, is less eas- ily identiﬁed. Some studies suggest that trends have been negative potentially throughout the 21st century (Han et al., 2018; Strode et al., 2016; Wang et al., 2018; Yumimoto et al., 2014; Zheng et al., 2018a), but others suggest that emissions and/or column densities were increasing or ﬂat during at least the ﬁrst decade of the century (Sun et al., 2018; Zhao et al., 2013, 2012). The negative trend has been attributed largely to reductions in emissions from industrial activity, as well as from residential and transportation sectors (Zheng et al., 2018a, b). Received: 7 June 2020 – Discussion started: 16 July 2020 Revised: 30 September 2021 – Accepted: 15 November 2021 – Published: 17 December 2021 Field et al.: Atmospheric composition over eastern China during COVID-19 but this trend is reversed sometime between 2010 and 2014 (Georgoulias et al., 2019; Krotkov et al., 2016; Lin et al., 2019; Xu et al., 2020; Si et al., 2019; Shah et al., 2020). The trend reversal has been attributed to a combination of emis- sion control measures (Zheng et al., 2018a) and variations in economic growth (Krotkov et al., 2016). Received: 7 June 2020 – Discussion started: 16 July 2020 Revised: 30 September 2021 – Accepted: 15 November 2021 – Published: 17 December 2021 Received: 7 June 2020 – Discussion started: 16 July 2020 Revised: 30 September 2021 – Accepted: 15 November 2021 – Published: 17 December 2021 Abstract. We examined daily level-3 satellite retrievals of Atmospheric Infrared Sounder (AIRS) CO, Ozone Monitoring Instrument (OMI) SO2 and NO2, and Moderate Resolution Imaging Spectroradiometer (MODIS) aerosol optical depth (AOD) over eastern China to understand how COVID-19 lockdowns affected atmospheric composition. Changes in 2020 were strongly dependent on the choice of background period since 2005 and whether trends in atmospheric composition were accounted for. Over central east China during the 23 January– 8 April lockdown window, CO in 2020 was between 3 % and 12 % lower than average depending on the back- ground period. The 2020 CO was not consistently less than expected from trends beginning between 2005 and 2016 and ending in 2019 but was 3 %–4 % lower than the background mean during the 2017–2019 period when CO changes had ﬂattened. Similarly for AOD, 2020 was between 14 % and 30 % lower than averages beginning in 2005 and 14 %–17 % lower compared to different background means beginning in 2016. NO2 in 2020 was be- tween 30 % and 43 % lower than the mean over different background periods and between 17 % and 33 % lower than what would be expected for trends beginning later than 2011. Relative to the 2016–2019 period when NO2 had ﬂattened, 2020 was 30 %–33 % lower. Over southern China, 2020 NO2 was between 23 % and 27 % lower than different background means beginning in 2013, the beginning of a period of persistently lower NO2. CO over southern China was signiﬁcantly higher in 2020 than what would be expected, which we suggest was partly because of an active ﬁre season in neighboring countries. Over central east and southern China, 2020 SO2 was higher than expected, but this depended strongly on how daily regional values were calculated from individual retrievals and reﬂects background values approaching the retrieval detection limit. Future work over China, or other regions, needs to take into account the sensitivity of differences in 2020 to different background periods and trends in order to separate the effects of COVID-19 on air quality from previously occurring changes or from variability in other sources. Published by Copernicus Publications on behalf of the European Geosciences Union. 18334 18334 R. D. R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18335 ter in the UV–visible range (Krotkov et al., 2017). We used NASA’s L3 tropospheric NO2 column density standard product v3 (OMNO2d_003) and the OMI principal com- ponent analysis planetary boundary layer (PBL) SO2 prod- uct (OMSO2e_003), which grid retrievals to 0.25◦resolu- tion (Krotkov et al., 2017; Li et al., 2013). Both products are cloud-screened; only pixels that are at least 70 % cloud-free are included in the NO2 product, and those that are at least 80 % cloud-free are included in the SO2 product. The NO2 product relies on air mass factors (AMFs) calculated with the assistance of an atmospheric chemical transport model and are sensitive to model representations of emission, chem- istry, and transport data. Instead of AMFs, the SO2 product uses spectrally dependent SO2 Jacobians but can be inter- preted as having a ﬁxed AMF that is representative of sum- mertime conditions. We applied basic transient SO2 plume ﬁltering, excluding retrievals with SO2 > 15 DU (Wang and Wang, 2020). Changes in pollution over China have also come from short-term interventions. To improve air quality for the 2008 summer Olympics – a time when emissions in China were high and still increasing – the Chinese government im- posed a series of strict emission control measures from July through 21 September 2008, which were qualitatively similar to the emission reductions expected to have accompanied the COVID-19 lockdown (UNEP, 2009). As a result, NO2 con- centrations over Beijing were estimated to have declined by between 40 % and 60 % based on satellite observations, with substantial but smaller reductions in surrounding cities of- ten on the order of 20 % to 30 % compared to previous years (Mijling et al., 2009; Witte et al., 2009). Regional reductions of SO2 and CO during the months of the games were esti- mated to be 13 % and 19 %, respectively (Witte et al., 2009). These results are broadly consistent with on-road observa- tions (Wang et al., 2009) but larger than some surface obser- vations comparing concentrations before and after the emis- sion control measures were implemented (Wang et al., 2010). Because our trend analysis uses a seasonal mean as the response variable, we assume that random errors cancel out, leaving only systematic errors, which do not contribute to un- certainty in the trend analysis. R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 Systematic errors in the OMI NO2 product have an uncertainty of 20 % (McLinden et al., 2014) and are associated with AMFs and tropospheric ver- tical column contents. The OMI NO2 products use an im- plicit aerosol correction to account for the optical effects of aerosols, but retrievals can be biased when aerosol loading is extreme (Castellanos et al., 2015). Under these conditions, the OMI NO2 retrieval is biased low by roughly 20 % to 40 % (Chimot et al., 2016). Note that any aerosol-related error would have the potential effect of underestimating the magni- tude of decreases in NO2 column densities when comparing 2020 to previous years. Additional bias in the NO2 product may be introduced due to the reliance on nearly cloud-free pixels, in which greater sunlight may induce higher photo- chemical rates. For example, the current NO2 product is bi- ased roughly 30 % low over the Canadian oil sands (McLin- den et al., 2014). The level-2 OMI-NO2 product has been val- idated against in situ and surface-based observations showing good agreement (Lamsal et al., 2014). The use of ﬁxed Jaco- bians in the SO2 product introduces systematic errors of 50 % to 100 % for cloud-free observations (Krotkov et al., 2016). The COVID-related lockdowns provide a similar natural experiment to the 2008 Beijing Olympics but on the other side of the Kuznets curve. The fact that the lockdowns oc- curred during years of decreasing air pollution needs to be taken into account in attributing changes in atmospheric composition to COVID-19 lockdowns, independent of the long-term trend. Following Chen et al.’s (2020) analysis of air quality improvements on mortality which controlled for changes in air quality since 2016, in this study we determine whether changes in 2020 in satellite retrievals of CO, SO2, NO2, and AOD departed signiﬁcantly from the expected de- clines associated with the long-term decreases in concen- trations resulting from pollution controls and technological change. 1 Introduction Bottom-up and top-down assessments of air pollutant emissions and concentrations suggest that China has fol- lowed this pattern during the era of satellite monitoring of atmospheric composition, with concentrations of SO2, NO2, CO, and aerosol optical depth (AOD) mostly exhibiting marked and steady declines over the last decade. In the case of NO2, multi-instrument analyses, which extend the obser- vational record beyond the lifetime of a single instrument, depict a consistent regional picture of NO2 trends in China since 1996 (Geddes et al., 2016; Georgoulias et al., 2019; Wang and Wang, 2020; Xu et al., 2020). Column totals show an increasing trend during the ﬁrst part of the satellite record, In addition to these long-term trends, a number of air pol- lutants also exhibit strong seasonal variation in China. An- thropogenic emissions of CO, SO2, and PM2.5 are highest in winter, reﬂecting large variation in emissions from the resi- dential sector and, in the case of CO, increased emissions as- sociated with cold-start processes in the transportation sector (Li et al., 2017). Outﬂow of CO and AOD has a spring max- imum, resulting from transport of pollution, dust, and boreal biomass burning emissions (Han et al., 2018; Luan and Jae- gle, 2013). https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 https://doi.org/10.5194/acp-21-18333-2021 2 Data and methods We used daily level-3 (L3) retrievals from four different in- struments on three different NASA Earth Observing System satellites. The Atmospheric Infrared Sounder (AIRS) instru- ment aboard NASA’s Aqua satellite is a 2300-channel in- frared grating spectrometer in a sun-synchronous orbit with northward Equator crossing time of 13:30. AIRS carbon monoxide (CO) proﬁles are retrieved with horizontal resolu- tion of 45 km at nadir, in a swath width of 30 ﬁelds of view or about 1600 km. The retrieval uses a cloud-clearing methodol- ogy providing CO with sensitivity that peaks around 500 hPa, with ∼0.8–1.2 degrees of freedom of signal for 50 %–70 % of scenes. More sampling and higher information content is obtained in clear scenes (Warner et al., 2013). We used the daily version 6 (AIRS3STD.006) product. Starting in 2007, the quality of level 1B radiance data for some OMI viewing directions has been affected, known as the row anomaly. The L3 products used here exclude all pix- els affected by the row anomaly from each observation, but the locations of the row anomaly pixels were dynamic be- tween 2007 and 2011, which could affect any comparisons including those years. Since 2011, the pixels affected by the row anomaly problem are the same, so comparisons for data only since 2011 are not affected by changes in the row anomaly. The Ozone Monitoring Instrument (OMI) aboard NASA’s Aura satellite was launched in July 2004 and has a lo- cal Equator crossing time of roughly 13:45. OMI is a nadir-viewing spectrometer, which measures solar backscat- Moderate Resolution Imaging Spectroradiometer (MODIS) sensors observe the Earth from polar orbit, from the Terra satellite since 2000 and from Aqua since https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18336 Figure 1. Groupings of provinces for central east China and south- ern China. grid cell with higher-quality retrievals, identiﬁed as those have less than 30 % cloud fraction and not affected by the row anomaly problem. We also calculated the daily value from the median of all retrievals to understand whether in- dividual high values (mainly SO2) had any effect on the sig- niﬁcance of trends or differences between 2020 and different background periods. 2 Data and methods In this study we use MODIS-derived AOD at 550 nm obtained by merging Dark Target and Deep Blue retrievals (Sayer et al., 2014). Speciﬁcally, we use the Deep_Blue_Aerosol_Optical_Depth_550_Land_Mean ﬁeld over land and the over ocean AOD_550_Dark_Target_Deep_Blue_Combined_Mean the from Collection 6.1 L3 Gridded products MYD08 and MOD08 (Hubanks et al., 2019), though very few retrievals over ocean are included in our analysis. L3 values are computed on 1◦× 1◦spatial grid from L2 AOD products with resolution of 10 km × 10 km. Over land 66 % of MODIS-retrieved Dark Target AOD values were shown to be ± 0.05 ± 0.15AOD AErosol RObotic NETwork (AERONET)-observed values, with high corre- lation (R = 0.9) (Levy et al., 2010). Around 78 % of the Deep Blue retrievals are within the expected error range of ± 0.05± 0.20AOD (Sayer et al., 2013). MODIS AOD data have been extensively used by the modeling and remote sensing scientiﬁc communities and inter-compared with a wide range of satellite AOD products (see Schutgens et al., 2020, and references therein). g, ) p g We also considered how the analysis depended on how the lockdown period was deﬁned. Emissions and pollution can decrease during the Chinese New Year holidays (Chen et al., 2020), which started as early as 23 January in 2012 and as late as 19 February in 2015, complicating COVID- 19-related analyses of atmospheric composition over China (Bauwens et al., 2020; Chen et al., 2020). The timing and extent of lockdowns also varied between provinces and we assume that “slowdowns” could have happened before or af- ter stricter, ofﬁcial lockdowns – for example, ground and air transportation remaining below lockdown levels nation- ally at least through 14 April 2020 (International Energy Agency, 2020). Excluding the holiday period from all years is a straightforward approach to excluding any New Year holiday effects but will exclude simultaneous lockdown ef- fects during the initial, and presumably most strict, stages of the lockdown. Rather than specifying different combinations of New Year holiday period and provincial-level lockdown timing, we used 23 January–8 April as our baseline period (which will include all holiday periods since 2005) but exam- ined the sensitivity of the statistics to the length of the lock- down period, namely a longer lockdown period beginning 1 week earlier and 1 week later, and a shorter lockdown pe- riod for February only. 2 Data and methods Monthly averages were calculated from the daily regional averages, with each day weighted in the monthly average by the number of valid retrievals so as to not overrepresent days with little satellite coverage or signif- icant cloud cover. The monthly data were used to visually identify COVID-19-related changes against background sea- sonality and trends since 2005. We examined the difference in the distribution of daily data during the 23 January to 8 April 2020 lockdown pe- riod to the same period during previous years since 2005. We compared 2020 to 2019 to different background peri- ods and to the expected value for 2020 estimated from trends over different background periods. Given the uneven nature of changes in atmospheric composition over different parts of China identiﬁed in previous studies, background periods were deﬁned for each possible starting year between 2005 and 2018, with each ending in 2019. Retrieved quantities in 2020 were compared to the background means over each period and to the value expected for 2020 estimated from the linear trend over each period. We tested the signiﬁcance of these differences using bootstrap resampling (Efron and Gong, 1983) with a resampling size of 2000. Figure 1. Groupings of provinces for central east China and south- ern China. mid-2002. In this study we use MODIS-derived AOD at 550 nm obtained by merging Dark Target and Deep Blue retrievals (Sayer et al., 2014). Speciﬁcally, we use the Deep_Blue_Aerosol_Optical_Depth_550_Land_Mean ﬁeld over land and the over ocean AOD_550_Dark_Target_Deep_Blue_Combined_Mean the from Collection 6.1 L3 Gridded products MYD08 and MOD08 (Hubanks et al., 2019), though very few retrievals over ocean are included in our analysis. L3 values are computed on 1◦× 1◦spatial grid from L2 AOD products with resolution of 10 km × 10 km. Over land 66 % of MODIS-retrieved Dark Target AOD values were shown to be ± 0.05 ± 0.15AOD AErosol RObotic NETwork (AERONET)-observed values, with high corre- lation (R = 0.9) (Levy et al., 2010). Around 78 % of the Deep Blue retrievals are within the expected error range of ± 0.05± 0.20AOD (Sayer et al., 2013). MODIS AOD data have been extensively used by the modeling and remote sensing scientiﬁc communities and inter-compared with a wide range of satellite AOD products (see Schutgens et al., 2020, and references therein). mid-2002. 3 Results 3c), with a July–August minimum and December– January peak, which has been attributed to increased heating needs (Yu et al., 2017; Si et al., 2019) and longer chemi- cal lifetime owing to lower OH and RO2 (Shah et al., 2020). NO2 has also decreased since 2011, and during most years, there is a departure from a smooth seasonal cycle in January and February associated with the Chinese New Year holi- day period. January and February 2020 NO2 was consider- ably lower than previous years, increased during March, and had recovered to typical, recent levels by April. AOD has consistent seasonal peaks in summer, which have been at- tributed to hygroscopic growth and agricultural residue burn- ing (Filonchyk et al., 2019), but had less regular seasonal- ity otherwise and has decreased since 2011. AODs during February and particularly March of 2020 were lower than recent years, but during which time there was considerable variability in the monthly data. R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18337 18337 Figure 2. The 2020–2019 differences during 23 January to 8 April over China in (a) AIRS carbon monoxide (CO) at 500 hPa, (b) OMI PBL sulfur dioxide (SO2), (c) OMI tropospheric nitrogen dioxide (NO2), and (d) Aqua MODIS aerosol optical depth (AOD). Figure 2. The 2020–2019 differences during 23 January to 8 April over China in (a) AIRS carbon monoxide (CO) at 500 hPa, (b) OMI PBL sulfur dioxide (SO2), (c) OMI tropospheric nitrogen dioxide (NO2), and (d) Aqua MODIS aerosol optical depth (AOD). https://doi.org/10.5194/acp-21-18333-2021 3 Results 3 Results ber, with the minima usually in November and December (Fig. 3a). There has been a decrease since 2005 in CO. The seasonal decrease from January to February in 2020 is sim- ilar to that which has occurred occasionally before, but the CO during February and March 2020 was the lowest for that time of the year since 2005. By April, CO had returned to levels typical of 2015–2019. The main characteristics of the monthly SO2 over the region are that it has decreased since 2005 (Fig. 3b) and that early 2020 SO2 was within the range of recent levels. There is a strong seasonal NO2 cy- cle (Fig. 3c), with a July–August minimum and December– January peak, which has been attributed to increased heating needs (Yu et al., 2017; Si et al., 2019) and longer chemi- cal lifetime owing to lower OH and RO2 (Shah et al., 2020). NO2 has also decreased since 2011, and during most years, there is a departure from a smooth seasonal cycle in January and February associated with the Chinese New Year holi- day period. January and February 2020 NO2 was consider- ably lower than previous years, increased during March, and had recovered to typical, recent levels by April. AOD has consistent seasonal peaks in summer, which have been at- tributed to hygroscopic growth and agricultural residue burn- ing (Filonchyk et al., 2019), but had less regular seasonal- ity otherwise and has decreased since 2011. AODs during February and particularly March of 2020 were lower than recent years, but during which time there was considerable variability in the monthly data. ber, with the minima usually in November and December (Fig. 3a). There has been a decrease since 2005 in CO. The seasonal decrease from January to February in 2020 is sim- ilar to that which has occurred occasionally before, but the CO during February and March 2020 was the lowest for that time of the year since 2005. By April, CO had returned to levels typical of 2015–2019. The main characteristics of the monthly SO2 over the region are that it has decreased since 2005 (Fig. 3b) and that early 2020 SO2 was within the range of recent levels. There is a strong seasonal NO2 cy- cle (Fig. 2 Data and methods In interpreting the data, we put more conﬁdence in 2020 differences that were insensitive to these choices. We analyzed these retrievals over two large regions (Fig. 1). Central east China was comprised of Shaanxi, Hubei, Anhui, Jiangsu, Shanxi, Henan, Hebei, Shandong, Beijing, and Tianjin provinces. Southern China was com- prised of Guizhou, Guangxi, Hunan, Jiangxi, Guangdong, Fujian, and Zhejiang provinces. Daily mean quantities were calculated across all valid retrievals falling within the provinces comprising the regions. For the OMI NO2 columns, individual retrievals were weighted by the L3 “weight” ﬁeld, which is proportional to the fraction of the https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18337 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18338 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 Figure 3. Monthly mean (a) AIRS CO, (b) OMI PBL SO2, (c) OMI tropospheric NO2, and (d) MODIS AOD over central east China since 2005. As in Bauwens et al. (2020), each year starts in August to show any departure from the seasonal cycle during the 23 January to 8 April lockdown period, shown by the thin gray vertical lines. Figure 3. Monthly mean (a) AIRS CO, (b) OMI PBL SO2, (c) OMI tropospheric NO2, and (d) MODIS AOD over central east China since 2005. As in Bauwens et al. (2020), each year starts in August to show any departure from the seasonal cycle during the 23 January to 8 April lockdown period, shown by the thin gray vertical lines. Figure 3. Monthly mean (a) AIRS CO, (b) OMI PBL SO2, (c) OMI tropospheric NO2, and (d) MODIS AOD over central east China since 2005. As in Bauwens et al. (2020), each year starts in August to show any departure from the seasonal cycle during the 23 January to 8 April lockdown period, shown by the thin gray vertical lines. Figure 4 shows the four retrieved quantities over southern China. There is a springtime maximum in CO (Fig. 4a), a less regular maximum during September–January, and an annual minimum in July. The range of CO is similar to central east China. CO over the last 5 years is lower than earlier in the record, and early 2020 CO was higher than recent years. SO2 (Fig. 4b) is lower than central east China, and any seasonal cycle is also hard to identify. The high June 2011 values are due to the Nabro eruption in Ethiopia (Fromm et al., 2014) which is still apparent in the time series despite excluding individual SO2 retrievals that are greater than 15 DU and are due to a combination of higher overall background values and individual retrievals with very high (>10 DU) SO2. NO2 (Fig. 4c) is lower than over central east China, but both re- gions share a similar seasonality. NO2 during January–April 2020 was slightly lower than in 2019. AOD (Fig. 4d) has weak seasonal peaks in October, March, and June; has de- creased since 2011; and fell within the range of 2015–2019 in 2020. 3.1 Regional patterns and seasonality Figure 2 shows the 2020–2019 differences over China dur- ing the 23 January–8 April lockdown period for the four satellite-retrieved quantities. There were decreases of 5– 10 ppbv in AIRS CO over central east China (Fig. 2a) and increases of 20–25 ppbv over southern China in 2020 com- pared to 2019. The increase in southern China is adjacent to a stronger positive CO anomaly over the upper Mekong re- gions of Myanmar, Thailand, and Laos. There were no co- herent regional changes in OMI SO2 (Fig. 2b) but rather smaller localized differences of either sign. There were de- creases in NO2 (Fig. 2c) across central east China exceeding 8 × 1015 moleccm−2 coincident with the weaker decrease in CO. Over southern China, there were comparable dif- ferences over Guangdong province, with smaller differences elsewhere. There was a decrease in MODIS AOD (Fig. 2d) in central east China coincident with the decreases in CO and NO2 but smaller in magnitude. There was a region of higher AOD in and northeast of the upper Mekong region coincident with the CO increase, both presumably because of biomass burning. To put the 2020/2019 difference maps in a longer-term and seasonal context, Fig. 3 shows monthly averages of the four retrieved quantities over central east China since 2005. There are seasonal CO peaks in March–April, June, and Septem- Atmos. Chem. Phys., 21, 18333–18350, 2021 https://doi.org/10.5194/acp-21-18333-2021 18338 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18339 18339 R. D. Field et al.: Atmospheric composition over eastern China during COVID 19 18339 Figure 4. Same as Fig. 3 but for southern China. tal lines and the mean shown by the black dot. The asso- ciated statistics comparing 2020 and 2019 are provided in Table 1, and comparing 2020 to longer background periods with and without trends accounted for is shown in Tables S1– S4 in the Supplement. The AIRS CO is shown in Fig. 5a. The variation during 23 January–8 April of each year is due to weather-related factors and observational error. The mean CO of 133.5 ppbv in 2020 was 3.2 % less than the 2019 mean of 137.9 ppbv, which was only marginally signiﬁcant, hav- ing a 95 % conﬁdence interval (−6.3 %–0.1 %) spanning 0. CO to the background average and to that which might be expected given any trends over the background period. Be- cause there was no obvious starting year for the background period, we considered different periods starting in each year between 2005 and 2018 and ending in 2019 (Fig. 6a, Ta- ble S1). The difference between 2020 and the background depended strongly on the starting year of the background pe- riod, ranging from −11.5 % lower than the 2005–2019 mean to −3.1 % lower than over 2018–2019, but all were statisti- cally signiﬁcant. Signiﬁcant trends over years beginning be- Figure 4. Same as Fig. 3 but for southern China. Figure 4. Same as Fig. 3 but for southern China. Figure 4. Same as Fig. 3 but for southern China. CO to the background average and to that which might be expected given any trends over the background period. Be- cause there was no obvious starting year for the background period, we considered different periods starting in each year between 2005 and 2018 and ending in 2019 (Fig. 6a, Ta- ble S1). The difference between 2020 and the background depended strongly on the starting year of the background pe- riod, ranging from −11.5 % lower than the 2005–2019 mean to −3.1 % lower than over 2018–2019, but all were statisti- cally signiﬁcant. Signiﬁcant trends over years beginning be- tween 2005 and 2016 (shown in Fig. 6a by the red line and shading) ranged between −1.5 ppbvyr−1 when starting in 2013 to −3.6 ppbvyr−1 if starting in 2016. 3.2 Central east China Figure 5 shows the CO, SO2, NO2, and AOD for 23 January– 8 April of each year over central east China as box-and- whisker plots with the median, interquartile range, and 2.5th and 97.5th percentiles over all daily mean data as horizon- Atmos. Chem. Phys., 21, 18333–18350, 2021 https://doi.org/10.5194/acp-21-18333-2021 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 The uncertainty in the trends increased for trends over shorter periods and were, tal lines and the mean shown by the black dot. The asso- ciated statistics comparing 2020 and 2019 are provided in Table 1, and comparing 2020 to longer background periods with and without trends accounted for is shown in Tables S1– S4 in the Supplement. The AIRS CO is shown in Fig. 5a. The variation during 23 January–8 April of each year is due to weather-related factors and observational error. The mean CO of 133.5 ppbv in 2020 was 3.2 % less than the 2019 mean of 137.9 ppbv, which was only marginally signiﬁcant, hav- ing a 95 % conﬁdence interval (−6.3 %–0.1 %) spanning 0. During years prior, there were increases and decreases in CO from year to year, but an overall decreasing trend since 2005. To quantify if the 2020 departure was signiﬁcant against this background, we compared the distribution of observed 2020 tal lines and the mean shown by the black dot. The asso- ciated statistics comparing 2020 and 2019 are provided in Table 1, and comparing 2020 to longer background periods with and without trends accounted for is shown in Tables S1– S4 in the Supplement. The AIRS CO is shown in Fig. 5a. The variation during 23 January–8 April of each year is due to weather-related factors and observational error. The mean CO of 133.5 ppbv in 2020 was 3.2 % less than the 2019 mean of 137.9 ppbv, which was only marginally signiﬁcant, hav- ing a 95 % conﬁdence interval (−6.3 %–0.1 %) spanning 0. During years prior, there were increases and decreases in CO from year to year, but an overall decreasing trend since 2005. To quantify if the 2020 departure was signiﬁcant against this background, we compared the distribution of observed 2020 tal lines and the mean shown by the black dot. The asso- ciated statistics comparing 2020 and 2019 are provided in Table 1, and comparing 2020 to longer background periods with and without trends accounted for is shown in Tables S1– S4 in the Supplement. The AIRS CO is shown in Fig. 5a. The variation during 23 January–8 April of each year is due to weather-related factors and observational error. R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 The mean CO of 133.5 ppbv in 2020 was 3.2 % less than the 2019 mean of 137.9 ppbv, which was only marginally signiﬁcant, hav- ing a 95 % conﬁdence interval (−6.3 %–0.1 %) spanning 0. During years prior, there were increases and decreases in CO from year to year, but an overall decreasing trend since 2005. To quantify if the 2020 departure was signiﬁcant against this background, we compared the distribution of observed 2020 https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18340 Table 1. Summary statistics for central east China comparing 2020 and 2019 during 23 January–8 April. Variable 2020 mean 2019 mean 2020 % difference from 2019 CO 133.5 137.9 −3.2 (ppbv) (130.3, 136.8) (134.7,141.3) (−6.3, 0.1) SO2 0.057 0.031 95 (DU) (0.045, 0.070) (0.018, 0.046) (14.8, 249.6) NO2 6.5 9.6 −32.1 (1015 moleccm−2) (5.8, 7.2) (8.7, 10.5) (−42.1, −21.7) AOD 0.41 0.48 −14.3 (0.36, 0.46) (0.41, 0.55) (−29.4, 3.1) Table 1. Summary statistics for central east China comparing 2020 and 2019 during 23 January–8 April. mary statistics for central east China comparing 2020 and 2019 during 23 January–8 April. Figure 5. The 23 January–8 April box plots over central east China for (a) AIRS CO, (b) OMI PBL SO2, (c) OMI tropospheric NO2, and (d) Aqua and Terra MODIS AOD from 2005 to 2020. The black box plots show the median, interquartile range, and 2.5th and 97.5th percentiles over all daily data, with the mean shown by the black dot. Figure 5. The 23 January–8 April box plots over central east China for (a) AIRS CO, (b) OMI PBL SO2, (c) OMI tropospheric NO2, and (d) Aqua and Terra MODIS AOD from 2005 to 2020. The black box plots show the median, interquartile range, and 2.5th and 97.5th percentiles over all daily data, with the mean shown by the black dot. https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18341 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18341 Figure 6. Dependence of trends (red) and difference between 2020 observations and predicted value (magenta) on detrending start year over central east China for (a) AIRS CO, (b) OMI PBL SO2, (c) OMI tropospheric NO2, and (d) MODIS AOD. The solid line shows the mean of the estimate for each year and the shading shows the 95 % conﬁdence interval. 3.3 Southern China 6c), with the strongest trend of −0.7 5 × 1015 moleccm−2 yr−1 for the period beginning in 2011. The 2020 NO2 was signiﬁcantly less than the predicted value for all background periods but varied from 16.8 % less than predicted from the 2011–2019 trend to 27.1 % less than predicted from the 2015–2019 trend, the last period when there was a signiﬁcant, although weak, decrease. OMI NO2 (Fig. 7c) increased toward 2011 and 2012, declining after to 2005–2010 levels. The 2020 mean of 3.3 × 1015 moleccm−2 was 22 % less than the 2019 mean of 4.3 × 1015 moleccm−2. For longer background periods, 2020 was between 22.9 % and 30.6 % less than the mean (Table S7 in the Supplement), all of which were signiﬁcant. NO2 trends were signiﬁcantly negative when the start of the trend was calculated using years between 2007 and 2012 but not otherwise (Fig. 8c). The 2020 NO2 mean was signiﬁ- cantly lower than predicted, except for when the trend was estimated beginning in 2011 or 2018. A 2-year trend cannot be interpreted meaningfully, especially without considering meteorological differences. Visually, however, it is hard to tell if the 2020 NO2 distribution represents a COVID-related departure or a decrease comparable to changes during recent previous years, unlike over central east China. OMI NO2 (Fig. 7c) increased toward 2011 and 2012, declining after to 2005–2010 levels. The 2020 mean of 3.3 × 1015 moleccm−2 was 22 % less than the 2019 mean of 4.3 × 1015 moleccm−2. For longer background periods, 2020 was between 22.9 % and 30.6 % less than the mean (Table S7 in the Supplement), all of which were signiﬁcant. NO2 trends were signiﬁcantly negative when the start of the trend was calculated using years between 2007 and 2012 but not otherwise (Fig. 8c). The 2020 NO2 mean was signiﬁ- cantly lower than predicted, except for when the trend was estimated beginning in 2011 or 2018. A 2-year trend cannot be interpreted meaningfully, especially without considering meteorological differences. Visually, however, it is hard to tell if the 2020 NO2 distribution represents a COVID-related departure or a decrease comparable to changes during recent previous years, unlike over central east China. MODIS AOD (Fig. 5d) was ﬂat or slightly increasing from 2005 to 2011, decreasing thereafter and with a ﬂattening since 2016 similar to SO2 and NO2. 3.3 Southern China Figure 7 shows the distribution of daily CO, SO2, NO2, and AOD for 23 January–8 April of each year over southern China. The associated statistics comparing 2020 and 2019 are provided in Table 2. AIRS CO (Fig. 7a) in 2020 was 144.7 ppbv, 13 % higher than the 2019 mean of 128.5 ppbv, which can be seen in an upward shift in the distribution of the box plot. The 2020 CO was between 4.4 % and 8.8 % greater than the background mean for periods starting af- ter 2014 (Table S5 in the Supplement) but not signiﬁcantly different otherwise. CO decreased signiﬁcantly for periods starting between 2005 and 2016 (Fig. 8a). When these trends are taken into account, 2020 CO was between 11.2 % and 18.7 % greater than predicted, and in all cases these differ- ences were signiﬁcant. OMI SO2 (Fig. 7b) ﬂuctuated from 2005 until 2013 and ﬂattened afterwards, driven by fewer high individual SO2 values in later years, as in central east China. The 2020 mean of 0.003 DU was 116 % higher than the 2019 mean of −0.02 DU but also with a wide 95 % conﬁdence interval (24 %–223 %). Year 2020 was less than the background mean periods starting between 2005 and 2011 (Table S6 in the Sup- plement) but not signiﬁcantly different otherwise. SO2 trends were consistently negative for all periods (Fig. 8b), although not as strong as over central east China. Whether 2020 SO2 was greater than predicted from trends depended more on the background period than over central east China. Differences in 2020 were also not signiﬁcantly different from predicted when daily values were calculated from the median SO2 of individual retrievals for any background period (Fig. S2b in the Supplement). OMI NO2 (Fig. 5c) increased from 2005 to 2011 and de- creased thereafter with an apparent ﬂattening since 2016. The 2020 mean NO2 of 6.5 × 1015 moleccm−2 was 32 % less than the 2019 mean of 9.6 × 1015 moleccm−2; the pronounced regional difference between 2020 and 2019 (Figs. 2c and 5c) in part reﬂects a 2019 uptick from 2018. For different background periods (Table S3), 2020 NO2 ranged from 43.3 % less than the 2010–2019 mean to 30 % less than the 2018–2019 mean, with all differences signiﬁcant. Trends were negative and signiﬁcant for starting years be- tween 2007 and 2015 (Fig. R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 2012–2019 (Fig. 6b), during which the trend could explain a maximum of 32 % of the variation in the data. For pe- riods starting in 2007 and after, the observed 2020 mean was signiﬁcantly higher than predicted. Relative to the value predicted from the 2012–2019 trend of −0.06, the observed 2020 SO2 was 200 % higher; the large percent difference re- ﬂects a predicted value close to zero, and we note that the retrieved SO2 can be negative for individual values and aver- ages (Li et al., 2013; Wang and Wang 2020). The observed 2020 SO2 was much higher than expected from trends calcu- lated over 2016–2019 when SO2 was ﬂat and with less vari- ability, but the low SO2 approaching the detection limit over this period makes these estimates not particularly meaning- ful. Furthermore, the change in 2020 SO2 was strongly de- pendent on whether daily values were calculated from the mean or median of individual values over the region. For most background periods (Fig. S1b in the Supplement), the trends in the median values were still negative until 2015, but 2020 was only 8.4 % higher than predicted from the 2012– 2019 trend and not signiﬁcantly different from expected for trends beginning later. This likely reﬂects the greater inﬂu- ence of high individual retrieval values on the daily mean value compared to the median, even after the basic ﬁltering of transient SO2 plumes. ginning in 2008 and later, when the trends were strongest, and which approached 0 after 2014. 3.3 Southern China R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 Figure 6. Dependence of trends (red) and difference between 2020 observations and predicted value (magenta) on detrending start year over central east China for (a) AIRS CO, (b) OMI PBL SO2, (c) OMI tropospheric NO2, and (d) MODIS AOD. The solid line shows the mean of the estimate for each year and the shading shows the 95 % conﬁdence interval. Figure 6. Dependence of trends (red) and difference between 2020 observations and predicted value (magenta) on detrending start year over central east China for (a) AIRS CO, (b) OMI PBL SO2, (c) OMI tropospheric NO2, and (d) MODIS AOD. The solid line shows the mean of the estimate for each year and the shading shows the 95 % conﬁdence interval. surface at 850 hPa (not shown), but where the retrieval has less sensitivity. unsurprisingly, insigniﬁcant by 2017, with the 95 % conﬁ- dence intervals of the trends spanning 0. The differences be- tween the observed 2020 mean and the value predicted from the trend (magenta line) varied inversely with the trend and were always negative but, except for 2009, had 95 % conﬁ- dence intervals (magenta shading) spanning 0 and therefore were not considered signiﬁcant. Therefore, for CO, 2020 was signiﬁcantly lower than the background period mean but not consistently lower than predicted given the decreasing trend during the background period, no matter how this period was deﬁned. Results were similar for CO analyzed closer to the OMI SO2 (Fig. 5b) ﬂuctuated over 2005 to 2011 and de- clined steadily afterward, during which variation also de- clined, becoming narrower to a degree not seen in the CO. The 2020 mean of 0.057 was 95 % higher than the 2019 mean of 0.031 but with a wide 95 % conﬁdence interval (15 %– 250 %). For different background periods (Table S2), 2020 SO2 ranged from 83 % less than the 2005–2019 mean to 30 % less than the 2016–2019 mean, with insigniﬁcant differences compared to more recent periods. Trends varied signiﬁcantly from to −0.03 yr−1 over 2005–2019 to −0.06 DUyr−1 over https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 18342 3.3 Southern China The 2020 mean AOD of 0.41 was 14 % less than the 2019 mean of 0.48, but this was not signiﬁcant. For different background periods (Ta- ble S4), 2020 AOD ranged from 30.2 % less than the 2007– 2019 mean to 14.2 % less than the 2018–2019 mean, with conﬁdence intervals for the differences becoming closer to spanning 0 for more recent periods. Trends were negative and signiﬁcant for starting years between 2005 and 2014 (Fig. 6d), with the strongest decrease of 0.04 yr−1 over the 2012–2019 period. There was no signiﬁcant difference be- tween the observed and predicted 2020 mean for periods be- MODIS AOD (Fig. 7d) was comparable to NO2 in its in- crease toward 2012, decrease thereafter, and ﬂattening during more recent years. The 2020 mean AOD of 0.38 was 12 % higher than the 2019 mean of 0.34 but with a 95 % conﬁ- Atmos. Chem. Phys., 21, 18333–18350, 2021 https://doi.org/10.5194/acp-21-18333-2021 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18343 Table 2. Same as Table 1 but for southern China. Variable 2020 mean 2019 mean 2020 % difference from 2019 CO 144.7 128.5 12.6 (ppbv) (139.6, 150.3) (124.4, 132.8) (7.2, 18.3) SO2 0.003 −0.020 116 (DU) (−0.01, 0.020) (−0.04, −0.001) (24, 223) NO2 3.3 4.3 −22.2 (1015 moleccm−2) (3.0, 3.7) (3.9, 4.7) (−32.6, −10.4) AOD 0.38 0.34 12 (0.34, 0.43) (0.30, 0.39) (−7, 34) Figure 7. Same as Fig. 5 but for southern China. https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 Fi 7 S Fi 5 b t f th Chi Figure 7. Same as Fig. 5 but for southern China. https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 18344 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18344 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 Figure 8. Same as Fig. 6 but for southern China. dence interval (−7 %–34 %) spanning 0. Similarly, 2020 was between 14 % and 22 % lower than during background pe- riods beginning from 2005 to 2012 but not for more recent periods (Table S8 in the Supplement). The AOD trends were signiﬁcantly negative for all start years until 2015. The 2020 mean was between 32 % and 47 % higher than predicted from trends for periods starting between 2010 and 2015 but was not different from predicted for trends starting in other years. For both regions and all quantities, the differences be- tween observed and predicted values for 2020 were insensi- tive to a longer lockdown period or to whether the bootstrap resampling was weighted by the number of valid retrievals niﬁcant when starting in later years, but the differences be- tween the observed and expected values remained insignif- icant over central east China. The SO2 trends for different periods were similar. The 2020 SO2 differences from what would be expected approached 0 for later periods but were also not consistently different when the median values of in- dividual retrievals were used. Results for NO2 were unaf- fected. The AOD 2020 difference from what would be ex- pected was stronger and technically signiﬁcant but still with a very wide conﬁdence interval and therefore difﬁcult to in- terpret. We emphasize that while a February-only lockdown period is useful for comparison, it is problematic in not in- Figure 8. Same as Fig. 6 but for southern China. niﬁcant when starting in later years, but the differences be- tween the observed and expected values remained insignif- icant over central east China. The SO2 trends for different periods were similar. The 2020 SO2 differences from what would be expected approached 0 for later periods but were also not consistently different when the median values of in- dividual retrievals were used. Results for NO2 were unaf- fected. The AOD 2020 difference from what would be ex- pected was stronger and technically signiﬁcant but still with a very wide conﬁdence interval and therefore difﬁcult to in- terpret. We emphasize that while a February-only lockdown period is useful for comparison, it is problematic in not in- cluding the New Year’s holiday periods from all previous years. dence interval (−7 %–34 %) spanning 0. 4 Discussion and conclusions SO2 concentrations were relatively ﬂat from 2016– 2019; when using 2016 as the ﬁrst year of the trend, SO2 was signiﬁcantly higher than the expected value when calcu- lated from the mean of the daily SO2 retrievals but not signif- icantly different when calculated from the median. We note also that analyses of SO2 and NO2 that include years prior to 2012 may be affected by changes in observation sample size due to changes in the OMI row anomaly. The modest decreases in CO and AOD over central east China were unexpected; given its high population density and level of industrial activity, lockdowns may have been an- ticipated to lead to larger decreases. In the case of MODIS AOD, these modest decreases were possibly due to contri- butions from other sources unaffected by COVID-19-related lockdowns – limitations in the MODIS AOD retrieval un- der cloudy conditions, climatological variability from other sources such as mineral dust, and meteorology favorable to secondary aerosol formation which could have offset lower emissions (Wang et al., 2020). The 2020 increase in SO2 is more difﬁcult to interpret because of the discrepancies be- tween daily values calculated from the mean or median of individual retrievals but is broadly consistent with surface observations that ﬁnd no signiﬁcant change in in situ surface SO2 over Wuhan in the daily mean and a slight increase in daytime SO2 possibly associated with increased residential heating and cooking (Shi and Brasseur, 2020). Over southern China, retrieved 2020 SO2 was signiﬁcantly lower than the background average only for periods begin- ning between 2005 and 2011. Signiﬁcant departures from expected trends were uneven when using the mean value of daily retrievals and absent when using the median value. As with central east China, we conclude that no signiﬁcant changes could be robustly detected in 2020 SO2. NO2 in 2020 was between 23 % and 32 % less than the background average for different periods. Here, the ﬂattening in NO2 be- ginning in 2013 is easier to identify than over central east China because of the much higher NO2 during the 3 years prior; 2020 NO2 was 23 %–27 % less than different back- ground means between 2013 and 2019. The more signiﬁ- cant reductions in NO2 in central east China compared to the south is presumably due the former’s greater population and industrialization and consequently higher pollution lev- els. 4 Discussion and conclusions analysis cannot be compared directly because we include non-urban areas and deﬁne the lockdown period differently, but we do note that NO2 during the same period in 2019 ap- peared to be anomalously high relative to the previous few years, which would make the decreases in 2020 appear more signiﬁcant. The degree to which the COVID-19 lockdowns in China resulted in changes in atmospheric composition depended strongly on the background period and whether existing trends were taken into account. For AIRS CO over cen- tral east China, the 2020 mean was 3 %–12 % lower com- pared to different background periods. Relative to mean CO concentrations during periods beginning between 2005 and 2016, there were signiﬁcant decreases in CO but CO in 2020 was not consistently different from what would be expected from trends calculated over this period. These longer-term declines in CO concentrations do appear to ﬂatten out in recent years; assuming that the ﬂat CO during 2017–2019 would have persisted, we estimate a 3 %–4 % reduction in CO in 2020 relative to that period. For MODIS AOD, the 2020 mean was between 14 % and 30 % less than differ- ent background averages but not signiﬁcantly different from what would be expected for trends beginning between 2008 and 2014. As with CO trends, the negative AOD trends in the region also appear to ﬂatten in recent years. Relative to the ﬂat AOD over 2016–2019, 2020 AOD was 14 %–17 % lower than the background mean; as with CO, this range would be the more meaningful estimate of changes in 2020 if we as- sume that this ﬂattening were to persist. The 2020 SO2 was signiﬁcantly lower than background averages calculated over most periods, ranging from 83 % less than over 2005–2019 to 30 % less than over 2016–2019. Compared to the 2012–2019 period when there were no signiﬁcant SO2 increases, 2020 SO2 was 200 % greater than what would be expected based on a trend starting in 2021, only 8 % greater when the me- dian of daily retrievals was used, and not signiﬁcantly differ- ent from expected relative to the trends beginning later than 2012. R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18345 18345 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 Similarly, 2020 was between 14 % and 22 % lower than during background pe- riods beginning from 2005 to 2012 but not for more recent periods (Table S8 in the Supplement). The AOD trends were signiﬁcantly negative for all start years until 2015. The 2020 mean was between 32 % and 47 % higher than predicted from trends for periods starting between 2010 and 2015 but was not different from predicted for trends starting in other years. For both regions and all quantities, the differences be- tween observed and predicted values for 2020 were insensi- tive to a longer lockdown period or to whether the bootstrap resampling was weighted by the number of valid retrievals each day. For a February-only lockdown period (Figs. S3 and S4 in the Supplement), the CO trends were more sig- dence interval (−7 %–34 %) spanning 0. Similarly, 2020 was between 14 % and 22 % lower than during background pe- riods beginning from 2005 to 2012 but not for more recent periods (Table S8 in the Supplement). The AOD trends were signiﬁcantly negative for all start years until 2015. The 2020 mean was between 32 % and 47 % higher than predicted from trends for periods starting between 2010 and 2015 but was not different from predicted for trends starting in other years. For both regions and all quantities, the differences be- tween observed and predicted values for 2020 were insensi- tive to a longer lockdown period or to whether the bootstrap resampling was weighted by the number of valid retrievals each day. For a February-only lockdown period (Figs. S3 and S4 in the Supplement), the CO trends were more sig- https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 al.: Atmospheric composition over eastern China during COVID-19 Table 4. Bottom-up biomass Global Fire Assimilation System (Kaiser et al., 2012) burning CO emission estimates from the up- per Mekong region (17 to 24◦N, 95 to 105◦E) and AIRS CO over southern China from 23 January to 8 April, for 2005–2020. Table 4. Bottom-up biomass Global Fire Assimilation System (Kaiser et al., 2012) burning CO emission estimates from the up- per Mekong region (17 to 24◦N, 95 to 105◦E) and AIRS CO over southern China from 23 January to 8 April, for 2005–2020. Table 3. The 2014 anthropogenic emission estimates by sector (in %) over China, excluding biomass burning, from the Commu- nity Emissions Data System (CEDS) for a representative set of constituents: black carbon (BC), carbon monoxide (CO), ammo- nia (NH3), nitrogen oxides (NOx), organic carbon (OC), and sul- fur dioxide (SO2). Residential, commercial, and other sectors are combined as RCO. Year GFAS CO upper AIRS CO southern China Mekong (kt) 500 hPa (ppbv) 2005 7977 157 2006 8905 146 2007 15 734 165 2008 4542 153 2009 9990 140 2010 14 176 149 2011 3591 147 2012 11 320 153 2013 8684 145 2014 8722 142 2015 8084 143 2016 9642 149 2017 3736 131 2018 3179 139 2019 6309 128 2020 7871 145 Year GFAS CO upper AIRS CO southern China Mekong (kt) 500 hPa (ppbv) BC CO NH3 NOx OC SO2 Agriculture 0 0 61.6 1.1 0 0 Energy 32.6 8 0.4 38.5 28.3 29.4 Industrial 12.7 41.8 6.5 33 5.1 57.3 Ground transportation 8.1 7.2 0.5 17.5 1.7 0.3 RCO 38.1 36.7 5.2 4.2 38.4 12.5 Solvents 0 0 0 0 0 0 Waste 8.5 6.3 25.8 5.2 26.5 0.4 Shipping 0 0 0 0.2 0 0.1 Aircraft 0 0 0 0.2 0 0 ent emission changes could have contributed to (1) why NO2 was robustly lower in 2020 over central east China compared to CO and AOD and (2) why CO and perhaps AOD were higher over southern China compared to what would be ex- pected from recent trends. R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 To understand why NO2 differences over central east China were more signiﬁcant than other quantities, Table 3 shows the emissions by sector for a representative set of constituents from the Community Emissions Data System (CEDS) (Hoesly et al., 2018) over China for 2014, the most recent year available. Other bottom-up emission inventories will vary in absolute emission amounts and their sector con- tributions, particularly for more recent periods, but CEDS is the standard available emission dataset available globally as a baseline for the next Intergovernmental Panel on Cli- mate Change (IPCC) assessment, in anticipation of assess- ing 2020 COVID-19-related changes to atmospheric compo- sition in other regions, and for modeling studies involving a transboundary transport component. Across all species, en- ergy production, industrial activity, transportation, residen- tial/commercial/other (RCO), and waste disposal constitute the bulk of the emissions. Based on activity data for the ﬁrst quarter of 2020, energy demand across China declined by 7 % compared to 2019, and transportation sector activity de- clined by 50 % to 75 % in regions with lockdowns in place (International Energy Agency, 2020). These sectors are di- rect or indirect sources of numerous pollutants, including SO2 (the precursor of sulfate aerosol), NOx, CO, and primary anthropogenic aerosols classiﬁed broadly as organic carbon (OC) and black carbon (BC). If we apply the 7 % reduction in energy production and midpoint 62.5 % reduction to trans- portation from the International Energy Agency (IEA) and assume a 20 % reduction in industrial emissions, 5 % reduc- tion in waste emissions, and no change in RCO (with com- mercial decreases offset by residential increases), this yields a 10 % reduction in BC, 5 % reduction in OC, 14 % reduction in SO2, 14 % reduction in CO, and 21 % reduction in NO2. The larger reduction in NO2 relative to other emissions could partly explain why OMI NO2 column density changes over central east China were stronger than in the other retrievals. g Following Si et al.’s (2019) consideration of biomass burn- ing as a pollution source in China alongside anthropogenic sources, we considered transboundary smoke transport as a possible reason for the higher 2020 CO over southern China, guided by higher CO over the upper Mekong region in 2020 compared to 2019 (Fig. 2a) and the predominant westerly ﬂow during this time of year (Reid et al., 2013). 4 Discussion and conclusions This is consistent with Chen et al.’s (2020) detection of a larger 2020 decrease in surface NO2 in Wuhan compared to Shanghai. Retrieved CO in 2020 was between 4 % and 8 % greater than background averages beginning in 2014 but be- tween 11 % and 19 % higher than what would be expected given the decreasing trends over any period. AOD in 2020 was lower than background averages calculated starting with years earlier than 2012 but higher or not signiﬁcantly differ- ent from expected for trends calculated starting in years after 2012. OMI NO2 in 2020 over central east China was consistently lower than the background average and expected value from the trends. There was a 17 % decrease in 2020 relative to the value expected from a trend calculated over 2011–2019 but a 30 %–33 % decrease relative to the different background means since 2016 when the NO2 was relatively ﬂat. Again assuming that this ﬂattening were to persist, this latter range may be the more meaningful baseline for the 2020 decrease. For reference, Bauwens et al. (2020) reported a ∼40 % drop in OMI NO2 from 2019 to 2020 over cities affected by the lockdown using the QA4ECV retrieval (Boersma et al., 2018) and a ∼51 % drop in NO2 over the eight cities (Bei- jing, Jinan, Nanjing, Qingdao, Tianjin, Wuhan, Xi’an, and Zhengzhou) falling within our central east China region. Our The focus of this analysis is on whether satellite retrievals of atmospheric composition over 2020 departed signiﬁcantly from different background periods and expected values for 2020 when daily variability and trends are accounted for, but it is useful at a preliminary stage to speculate as to how differ- https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 18346 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 Table 4 compares 23 January–8 April AIRS CO over south- ern China to CO emission estimates from biomass burning from the Global Fire Assimilation System (GFAS) (Kaiser et al., 2012) over the upper Mekong region (17 to 25◦N, 95 to 105◦E) including parts of eastern Myanmar, northern Thailand, and northern Laos. From 2005 to 2020, variation in GFAS CO over this region explained a moderate (32 %) amount of variability in AIRS CO over southern China, sug- gesting it is a non-negligible contributor to variation in CO concentration and a contributor to higher CO in 2020. This illustrates that, at a minimum, sources such as biomass burn- ing smoke and dust that are less affected by COVID-19- related measures will complicate attribution studies. To that end, modeling studies following Wang et al. (2020) will be required to isolate emissions, meteorological and chemical drivers of changes in atmospheric composition, and their ef- fects at a process level. With proper instrument-equivalent comparisons, modeling studies will also help to identify the extent to which the lack of signiﬁcant changes are due to https://doi.org/10.5194/acp-21-18333-2021 Atmos. Chem. Phys., 21, 18333–18350, 2021 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 18347 Center (DAAC) at https://ladsweb.modaps.eosdis.nasa.gov/archive/ allData/61/ (last access: 29 January 2021). retrieval limitations, namely low sensitivity near the surface where differences would presumably be more pronounced, particularly given remote emission sources such as dust, biomass burning smoke, and volcanic SO2, which will arrive at higher altitudes. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/acp-21-18333-2021-supplement. g The key implication of our study is that interpreting dif- ferences in 2020 retrievals of atmospheric composition de- pends strongly on how the background period is deﬁned and whether trends over these periods are accounted for. Not taking these into account could lead to misattribution of changes in air quality to COVID-19 lockdowns. At a mini- mum, whether differences in 2020 are signiﬁcant depends on the choice of background period, which is somewhat subjec- tive. Leading up to 2020, there was an apparent ﬂattening of decreasing trends beginning earlier in the decade across the retrievals; the considerable variability in the data made iden- tifying this ﬂattening easier in some cases than in others. R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 We are more conﬁdent, for example, in our estimate of a 23 %– 27 % decrease in 2020 NO2 over southern China relative to a ﬂat background period than the 30 %–33 % decrease over central east China, where the recent variability was greater and the ﬂattening less apparent. Revisiting this type of anal- ysis in the years when regional economies have fully recov- ered post COVID-19 will help to distinguish between fur- ther decreases and ﬂat trends and will lend themselves to us- ing non-linear models in estimating the trends. We have ap- proached the issue by comparing data for 2020 to what would have been expected given recent trends and by applying a single lockdown period to two large regions, with additional analyses to gauge the sensitivity of the 2020 differences to these choices. Other studies over China or elsewhere will in- evitably use other approaches that more explicitly account for seasonality and meteorology, and which relate changes in pollution over smaller areas (e.g., single provinces or states) to region-speciﬁc lockdown measures and timing at a process level. Regardless of the approach, however, it is important to consider recent trends and variability. In places where pollu- tion has decreased, not accounting for recent context could result in over-attribution of changes in pollution to COVID- 19. In places where pollution has increased, such as parts of South Asia, this could result in under-attribution. Author contributions. All authors conceived of the study. RDF, IVG, and KT conducted the data analysis. RDF and JEH prepared the manuscript with contributions from all co-authors. Competing interests. The contact author has declared that nei- ther they nor their co-authors have any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements. The authors thank two anonymous review- ers and the editor for their constructive feedback, which improved the interpretation of the data and conclusions drawn in the paper. Financial support. This research has been supported by the NASA (grant no. 80NSSC18M0133). Financial support. This research has been supported by the NASA (grant no. 80NSSC18M0133). Review statement. This paper was edited by Michel Van Roozendael and reviewed by two anonymous referees. https://doi.org/10.5194/acp-21-18333-2021 R. D. Field et al.: Atmospheric composition over eastern China during COVID-19 19 Outbreak in China, Lancet Planetary Health, 4, E210–E212, https://doi.org/10.1016/S2542-5196(20)30107-8, 2020. 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https://openalex.org/W4312125768	https://zenodo.org/records/7479061/files/Naproxen.pdf	English	null	Formulation and Development of Naproxen loaded Polymeric Nanoparticles using the Solvent Evaporation Method	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	3,839	Abstract Oil in water (O/W) emulsion solvent evaporation was used to develop polymeric drug nanoparticles for this study. Water served as a non-solvent and the polymer's solvent was acetone. The emulsion solvent evaporation method will be used in this study to prepare nanoparticles and to analyze the effects of different processing parameters on the properties of the nanoparticles. We use two different types of acrylic polymers in this study: Eudragit E100 and Eudragit RS100. It was observed that various factors, including the polymer content in the organic solvent, the concentration of the surfactant, and the volume ratio of the oil and water phases, affect the size of the nanoparticles. By using a transmission electron microscope, the morphological structure is examined (TEM). The production of spherical nanoparticles with an average size of 90 nm was verified by TEM pictures. By using laser dynamic light scattering, the size distribution is determined. The range of the nanoparticles' size distribution, from 50 to 150 nm, was found. Fourier transform infrared spectroscopy analysis revealed no interactions between the medication and polymer. The amorphous structure was visible in the X-ray diffraction patterns of nanoparticles containing naproxen, eudragit E100, and eudragit RS 100. Keywords: Nanoparticles, Eudragit E100, Polymer There are numerous microencapsulation techniques available to produce drug nanoparticles. The drug is dissolved, dispersed, or emulsified in an organic polymer solution in the typical microencapsulation approach employing oil in water (O/W) emulsion system, which is subsequently emulsified in an external aqueous or oil phase. The drug and polymer precipitate in the droplets as the organic solvent is evaporated, generating the nanospheres or nanocapsules. A variety of biocompatible polymers, including poly (D,L-lactide-co-glycolide) 5&6, and eudragit 7, have been utilised to successfully create microspheres using the emulsion solvent evaporation technique, which was fully developed by the end of the 1970s. Kawashima et al. 8, 9 proposed an emulsion solvent diffusion method more recently. The process International Journal of Pharma Research and Technology Formulation and development of Naproxen loaded Polymeric Nanoparticles using the Solvent Evaporation Method Shinde V.R. 1, Shinde k.A. 2 Sanjivani College of Pharmaceutical Education & Research Kopargaon, 423603 KSS College of Pharmacy Shikrapur 412208 vsk.vision11@gmail.com p Shinde V.R. 1, Shinde k.A. 2 Sanjivani College of Pharmaceutical Education & Research Kopargaon, 423603 KSS College of Pharmacy Shikrapur 412208 vsk.vision11@gmail.com p Shinde V.R. 1, Shinde k.A. 2 Sanjivani College of Pharmaceutical Education & Research Kopargaon, 423603 KSS College of Pharmacy Shikrapur 412208 vsk.vision11@gmail.com Volume 1, Issue 4, 2022, Page 1-7 Volume 1, Issue 4, 2022, Page 1-7 Formulation and development of Naproxen loaded Polymeric Nanoparticles using the Solvent Evaporation Method Shinde V.R. 1, Shinde k.A. 2 Sanjivani College of Pharmaceutical Education & Research Kopargaon, 423603 KSS College of Pharmacy Shikrapur 412208 vsk.vision11@gmail.com Introduction The choice of nanoparticle material and surface modification can have an impact on these qualities, as well as targeting and controlled release. Nanoparticles are created using substances like protein, synthetic polymers, and other natural macromolecules. In the administration of therapeutic molecules, drug nanoparticles may be used for tissue targeting in cancer therapy, controlled release, carrier action for peptide transport, and an improvement in drug solubility.10 In this study, two different types of acrylic polymers, Eudragit E 100 and Eudragit RS 100, are used to t ti l i th l i l t of emulsion solvent evaporation has a number of benefits, and it is preferred to other preparation techniques like spray drying, sonication, and homogenization because it just calls for moderate conditions like room temperature and continual stirring. With the drug's activity unaffected, a stable emulsion can be created. The general emulsification solvent evaporation method, which is used to create nanoparticles, involves a number of processing and material parameters, including the amount of energy used, the power and duration used, the volume of the aqueous phase, the concentration of polymers and drugs in the organic phase, the molecular weight and end groups of the polymers, the volume of the solvent, and the concentration of surfactants. The size and/or drug content are influenced by each of these processing and material characteristics. Nanoparticles for drug delivery should be easily biocompatible and biodegradable. The choice of nanoparticle material and surface modification can have an impact on these qualities, as well as targeting and controlled release. Nanoparticles are created using substances like protein, synthetic polymers, and other natural macromolecules. In the administration of therapeutic molecules, drug nanoparticles may be used for tissue targeting in cancer therapy, controlled release, carrier action for peptide transport, and an improvement in drug solubility.10 In this study, two different types of acrylic polymers, Eudragit E 100 and Eudragit RS 100, are used to create nanoparticles using the emulsion solvent evaporation method in order to examine the impact of different processing parameters on particle size and Naproxen is a methoxynaphthalene that is 2- methoxynaphthalene substituted by a carboxy ethyl group at position 6. Naproxen is having white to creamy white, crystalline powder; soluble in water and sparingly soluble in alcohol. In this investigation, Eudragit NE 30 D and Eudragit NM 30 D polymers were used. Characterisation of Nanoparticles Characterisation of Nanoparticles We examine on the morphology, crystallinity, and particle size and size distribution of nanoparticles. Dynamic light scattering was used to determine the particle size distribution (HORIBA LB-550-Japan). A Fourier transform infrared (FT-IR) spectrometer (TEMSORTM37 - Bruner, USA) was used to record the infrared (IR) absorption spectra of raw materials We examine on the morphology, crystallinity, and particle size and size distribution of nanoparticles. Dynamic light scattering was used to determine the particle size distribution (HORIBA LB-550-Japan). A Fourier transform infrared (FT-IR) spectrometer (TEMSORTM37 - Bruner, USA) was used to record the infrared (IR) absorption spectra of raw materials Experimental Materials/Methods Preparation of the Drug Nanoparticles Preparation of the Drug Nanoparticles In this study, emulsification solvent evaporation technique with sonication is used to create nanoparticles. An organic phase made up of drug (naproxen) and polymer (Eudragit E 100 or RS 100) dissolved in acetone (10 mL). An O/W type emulsion is created by mixing this organic phase with an aqueous phase that already contains a surfactant (PVA, concentration 0.5%, 90 mL). Oil and water phases had a volume ratio of 1:9. By using outside energy through a sonicator, this emulsion is broken down into nanodroplets. When the extremely volatile organic solvent is evaporated, these nanodroplets turn into nanoparticles. During magnetic stirring at 300 rpm under atmospheric conditions for 2 hours, the organic solvent evaporates. Nanoparticles for drug delivery should be easily biocompatible and biodegradable. The choice of nanoparticle material and surface modification can have an impact on these qualities, as well as targeting and controlled release. Nanoparticles are created using substances like protein, synthetic polymers, and other natural macromolecules. In the administration of therapeutic molecules, drug nanoparticles may be used for tissue targeting in cancer therapy, controlled release, carrier action for peptide transport, and an improvement in drug solubility.10 In this study, two different types of acrylic polymers, Eudragit E 100 and Eudragit RS 100, are used to create nanoparticles using the emulsion solvent evaporation method in order to examine the impact of different processing parameters on particle size and nanoparticle properties. The concentration of polymers in the organic phase, the concentration of polyvinyl alcohol (PVA) in the aqueous phase, and the volume ratio of the oil to water phases are among the processing parameters. Non-prescription naproxen is used to treat minor discomfort from toothaches, backaches, menstrual cramps, arthritis, headaches, muscular pains, and the common cold. Naproxen belongs to the group of drugs known as NSAIDs. We evaluated at how different processing settings affected particle size. The concentration of polymers in the organic phase, the concentration of PVA in the aqueous phase, and the volume ratio of the oil and water phases are the processing parameters. Introduction They are soluble in organic solvents like acetone, ethanol, and others but insoluble in acid and water. Bi-distilled water was used as non-solvents. PVA was used as an emulsifying agent. All materials were obtained from commercial sources and used as received: naproxen (USP-30; Dr. Reddy's Laboratories) Eudragit E 100, Eudragit RS 100 (Merck, Germany), PVA, acetone (Merck, German). Introduction Nanoparticle drug delivery involves creating drug- loaded particles with 1–1000 nm in diameter. Solid, submicron-sized medication carriers known as nanoparticles may or may not be biodegradable1,2. Based on their structure, drug nanoparticles can be further divided into nanocapsules and nanospheres3. In vesicular systems called nanocapsules, the medicine is contained in a hollow with an inner liquid core and a polymeric membrane around it. Nanospheres are built like a matrix. Drugs may be contained within the particle or absorbed at the surface of the sphere. The medication is either incorporated as crystallites in the polymer matrix or solubilized in the polymer matrix to create an amorphous particle 4. 1 www.ijprt.com Volume 1, Issue 4, 2022, Page 1-7 of emulsion solvent evaporation has a number of benefits, and it is preferred to other preparation techniques like spray drying, sonication, and homogenization because it just calls for moderate conditions like room temperature and continual stirring. With the drug's activity unaffected, a stable emulsion can be created. The general emulsification solvent evaporation method, which is used to create nanoparticles, involves a number of processing and material parameters, including the amount of energy used, the power and duration used, the volume of the aqueous phase, the concentration of polymers and drugs in the organic phase, the molecular weight and end groups of the polymers, the volume of the solvent, and the concentration of surfactants. The size and/or drug content are influenced by each of these processing and material characteristics. of emulsion solvent evaporation has a number of benefits, and it is preferred to other preparation techniques like spray drying, sonication, and homogenization because it just calls for moderate conditions like room temperature and continual stirring. With the drug's activity unaffected, a stable emulsion can be created. The general emulsification solvent evaporation method, which is used to create nanoparticles, involves a number of processing and material parameters, including the amount of energy used, the power and duration used, the volume of the aqueous phase, the concentration of polymers and drugs in the organic phase, the molecular weight and end groups of the polymers, the volume of the solvent, and the concentration of surfactants. The size and/or drug content are influenced by each of these processing and material characteristics. Nanoparticles for drug delivery should be easily biocompatible and biodegradable. Experimental Materials/Methods 2 www.ijprt.com Volume 1, Issue 4, 2022, Page 1-7 concentration ranges from 3% (w/v) to 15% (w/v). The impact of polymer content on particle size in the organic phase is shown in Figure 1. The diameter of the nanoparticles gradually grows as the amount of polymer in a fixed volume of organic solvent increases. The viscosity increases with increasing polymer concentration, increasing the emulsion droplet size. The organic phase shear stresses are opposed by the viscous forces, and the ultimate particle size and size distribution rely on the net shear stress that is available to the droplet. Previous studies using different poly (lactic-co-glycolic acid)/poly lactic acid (PLGA/PLA) systems have shown the significance of polymer content in regulating the size of particles produced by the general emulsification process. and nanoparticles in the wavelength range 4000-400 cm-1. and nanoparticles in the wavelength range 4000-400 cm-1. Using a transmission electron microscope (TEM; JEM-1400, Japan) and an acceleration voltage of 100 kV, particle morphology was examined. Utilizing X-ray diffraction, the particles' crystallinity was investigated (XRD; D8 Advance – Bruker, German). Using a transmission electron microscope (TEM; JEM-1400, Japan) and an acceleration voltage of 100 kV, particle morphology was examined. 1. Effect of Polymer Concentration in the Organic Phase The polymer Eudragit E100 was used to produce the naproxen nanoparticles. The volume ratio of the oil to water phases was 1:9, and the PVA concentration in the aqueous phase was 0.5%. The polymer queous phase was 0.5%. The polymer Figure No.1: Effect of Polymer Concentration in the Organic Phase on the Particle Size Figure No.1: Effect of Polymer Concentration in the Organic Phase on the Particle Size Results and discussion Effect of Various Processing Parameter on the Size of Particles 1. Effect of Polymer Concentration in the Organic Phase 2. Effect of PVA Concentration in the Aqueous Phase content in the aqueous phase on particle size are depicted in Figure 2. Due to the high aqueous phase viscosity caused by increasing PVA concentration, particles gradually grow in size, which decreases the net shear force that may be used to break apart droplets. At high PVA concentrations, Zweers et al. 11 reported that PLGA nanoparticle size increased. The polymer Eudragit E100 was used to make the naproxen nanoparticles; the oil to water volume ratio was 1:9 and the polymer content was 5%. PVA is present in the aqueous phase at concentrations ranging from 0.5% (w/v) to 2% (w/v). The effects of PVA Figure No.2: Effect of PVA Concentration in the Aqueous Phase on the Particle Size. Figure No.2: Effect of PVA Concentration in the Aqueous Phase on the Particle Size. 3 www.ijprt.com Volume 1, Issue 4, 2022, Page 1-7 3. Effect of the Volume Ratio of oil and Water Phases ratio of the water to oil phases was different. The effects of the volume ratio of the oil and water phases on particle size are depicted in Figure 3. The size of the particles increases quickly as the volume ratio of the oil and water phases rises above 0.6. The polymer Eudragit E100 was used to create the naproxen nanoparticles; the polymer concentration was set at 5% (w/v), and the PVA concentration in the aqueous phase was set at 0.5% (w/v). The volume ueous phase was set at 0.5% (w/v). The volume Figure No.3: Effect of the Volume Ratio of oil and Water Phases on the Particle Size. Figure No.3: Effect of the Volume Ratio of oil and Water Phases on the Particle Size. Figure 4(b). Grain boundaries or crystallinity were not visible in Figure 4(a) or (b). The nanoparticles were round, amorphous, and smooth, according to TEM pictures. The particles had an average size of 90 nm. There has been evidence of the emergence of a drug- amorphous polymer structure. 4. The Characteristics of Nanoparticle A TEM was used to study the morphology of the prepared nanoparticles. Naproxen nanoparticles containing 10% (w/w) and 90% (w/w) Eudragit E100 are displayed in Fig 4(a) using TEM images. Nanoparticles with 10% (w/w) naproxen and 90% (w/w) Eudragit RS 100 are shown in TEM images in (w/w) Eudragit RS 100 are shown in TEM images in (a) (b) Figure 4: TEM images of the Nanoparticles Containing: 10% (w/w) Naproxen and 90% (w/w) Eudragit NE (a) and 10% (w/w) Naproxen and 90% (w/w) Eudragit NM (b). (b) (a) (b) (a) Figure 4: TEM images of the Nanoparticles Containing: 10% (w/w) Naproxen and 90% (w/w) Eudragit NE (a) and 10% (w/w) Naproxen and 90% (w/w) Eudragit NM (b). Figure 4: TEM images of the Nanoparticles Containing: 10% (w/w) Naproxe NE (a) and 10% (w/w) Naproxen and 90% (w/w) Eudragit wavenumber region. Naproxen possesses a carboxylic acid group that can interact with the polymer's function groups. The interactions of the drug with the polymers were studied using IR spectroscopy. Figure 5 displays the nanoparticles' IR spectra in the 4000 to 400 cm-1 4 www.ijprt.com Volume 1, Issue 4, 2022, Page 1-7 Figure No. 5: Infrared Spectra at Wavenumber of 4000 to 400 cm−1 of the Nanoparticles Containing: (a) 10% (w/w) Naproxen and 90% (w/w) Eudragit E100 and (b) 10% (w/w) Naproxen and 90% (w/w) Eudragit RS. 3. Effect of the Volume Ratio of oil and Water Phases Figure No. 5: Infrared Spectra at Wavenumber of 4000 to 400 cm−1 of the Nanoparticles Containing: (a) 10% (w/w) Naproxen and 90% (w/w) Eudragit E100 and (b) 10% (w/w) Naproxen and 90% (w/w) Eudragit RS. (Figure 6(a)), naproxen and Eudragit RS (Figure 6(b)). Size distribution were determined by dynamic light scattering in aqueous solution, the size of the particles was in the range from 50 to 150 nm and the mean diameter was 90 nm. Based on these results, we can see the sonication method is suited to produce small size nanoparticles (<300 nm diameter) with narrow size distribution. FT-IR patterns of naproxen, naproxen/eudragit® RS100/ eudragit E100 physical mixtures as well as the related nanoparticles are demonstrated in Fig. 5. Matching up to FT-IR spectrum of naproxen with the physical mixtures revealed no distinctive changes indicating that eudragit® RS100 and eudragit E100 was not involved in intermolecular interaction with naproxen in physical mixtures. Figure 6 shows the particle size distribution of nanoparticles containing: naproxen and Eudragit E100 nanoparticles containing: naproxen and Eudragit E100 Figure 6: The Particle Size Distribution of Nanoparticles Containing: (a) 10% (w/w) Naproxen and 90% (w/w) Eudragit E100 and (b) 10% (w/w) Naproxen and 90% (w/w) Eudragit RS Figure 6: The Particle Size Distribution of Nanoparticles Containing: (a) 10% (w/w) Naproxen and 90% (w/w) Eudragit E100 and (b) 10% (w/w) Naproxen and 90% (w/w) Eudragit RS 5 www.ijprt.com Volume 1, Issue 4, 2022, Page 1-7 7b). The amorphous structure and lack of crystallinity in Figure 7(a) and (b) were shown by the XRD diffraction pattern. 7b). The amorphous structure and lack of crystallinity in Figure 7(a) and (b) were shown by the XRD diffraction pattern. The nanoparticles' XRD patterns are shown in Figure 7: 10% naproxen and 90% eudragit rs (Figure 7(a)), and 10% naproxen and 90% eudragit e100 (Figure 7b). The amorphous structure and lack of crystallinity in Figure 7(a) and (b) were shown by the XRD diffraction pattern. The nanoparticles' XRD patterns are shown in Figure 7: 10% naproxen and 90% eudragit rs (Figure 7(a)), and 10% naproxen and 90% eudragit e100 (Figure 7b). The amorphous structure and lack of crystallinity in Figure 7(a) and (b) were shown by the XRD diffraction pattern. Acknowledgement 7. Bouchemal, K, Briancon, S, Perrier, E, Fessi, H, B onnet, I and Zydowicz, N. 2004. Synthesis and characterization of polyurethane and poly(etherurethane) nanocapsules using a new technique of interfacial polycondensation 7. Bouchemal, K, Briancon, S, Perrier, E, Fessi, H, B onnet, I and Zydowicz, N. 2004. Synthesis and characterization of polyurethane and poly(etherurethane) nanocapsules using a new technique of interfacial polycondensation The authors are thankful to the Sanjivani College of Pharmaceutical Education & Research Kopargaon (India) for unconditional support for the work. 3. Effect of the Volume Ratio of oil and Water Phases The nanoparticles' XRD patterns are shown in Figure 7: 10% naproxen and 90% eudragit rs (Figure 7(a)), and 10% naproxen and 90% eudragit e100 (Figure p g g p Figure 7: XRD Patterns of the Nanoparticles Containing: 10% (w/w) Naproxen and 90% (w/w) Eudragit E100 (a) and 10% (w/w) Naproxen and 90% (w/w) Eudragit RS (b). Figure 7: XRD Patterns of the Nanoparticles Containing: 10% (w/w) Naproxen and 90% (w/w) Eudragit E100 (a) and 10% (w/w) Naproxen and 90% (w/w) Eudragit RS (b). The absence of peaks corresponding to diffraction from the drug crystal lattice leads us to the conclusion that the nanoparticles generated were amorphous. 1. Couvreur, P, Dubernet, C and Puisieux, F. 1995. C ontrolled drug delivery with nanoparticles: Current possibilities and future trends. Eur. J. Pharm. Biopharm., 41: 2–13. 1. Couvreur, P, Dubernet, C and Puisieux, F. 1995. C ontrolled drug delivery with nanoparticles: Current possibilities and future trends. Eur. J. Pharm. Biopharm., 41: 2–13. Conclusion 2. Couvreur, P. 1988. Polyalkylcyanoacrylates as colloidal drug carriers. Crit. Rev. Ther. Drug Carrier Syst., 5: 1–20. Emulsion solvent evaporation was used to produce polymeric drug nanoparticles. We examined into just how different processing variables affected nanoparticle properties and particle size. Particle size increased as a result of changes in various factors, including the concentration of polymers in the organic solvent, the concentration of surfactants, and the volume ratio of the oil to water phases. The TEM observation reveals the surface morphological characteristics; the particle morphology was spherical and homogeneous. The nanoparticles were discovered to have a size distribution in the 50 to 150 nm range, with a mean diameter of 90 nm. FT-IR was used to analyze how the medication and polymer interacted. We found that the naproxen molecule's carboxylic group interacts with the eudragit. Nanoparticle XRD patterns revealed the amorphous structure. 3. Kreuter, J. 1991. Nanoparticle – based drug delivery systems. J. Controlled Release, 16: 169– 176. 4. Soppimath, KS, Aminabhavi, TM, Kulkarni, AR a nd Rudzinski, WE. 2001. Biodegradable polymeric nanoparticles as drug delivery devices. J. Controlled Release, 70: 1–20. 5. Allemann, E and Gurny, R. 1993. Drug loaded nanoparticle-preparation methods and drug targeting issues. Eur. J. Pharm. Biopharm., 39: 173–191. 6. Jung, T, Kamm, W, Breitenbach, A, Kaiserling, E, Xiao, JX and Kissel, T. 2000. Biodegradable nanoparticle for oral delivery of peptides: Is there a role for polymers to affect mucosal uptake. Eur. J. Pharm. Biopharm., 50: 147–160. 6. Jung, T, Kamm, W, Breitenbach, A, Kaiserling, E, Xiao, JX and Kissel, T. 2000. Biodegradable nanoparticle for oral delivery of peptides: Is there a role for polymers to affect mucosal uptake. Eur. J. Pharm. Biopharm., 50: 147–160. References 6 www.ijprt.com Volume 1, Issue 4, 2022, Page 1-7 Volume 1, Issue 4, 2022, Page 1-7 combined to spontaneous emulsification. Int. J. Pharm., 269: 89–100 14. Barzegar-Jalali M, Adibkia K, Valizadeh H, Shadbad MR, Nokhodchi A, Omidi Y, Mohammadi G, Nezhadi SH, Hasan M. Kinetic analysis of drug release from nanoparticles. J Pharm Pharm Sci, 2008; (1):167-177 8. Kumar, M. 2000. Nano and microparticles as controlled drug delivery devices. J. Pharm. Sci., 3: 234–258. 9. Kumar, N, Ravikumar, M and Domb, AJ. 2001. Bi odegradable block copolymers. Adv. Drug Delivery Rev., 53: 23–44. 15. Dillen K, Vandervoort J, Van den Mooter G, Ludwig A. Evaluation of ciprofloxacin-loaded Eudragit RS100 or RL100/PLGA nanoparticles. Int J Pharm, 2006;(1):72-82 10. Brigger, J, Dubernet, C and Couvreur, P. 2002. Na noparticles in the cancer therapy and diagnosis. Adv. Drug Delivery Rev., 54: 631– 651. 16. Perumal D, Dangor CM, Alcock RS, Hurbans N, Moopanar KR. Effect of formulation variables on in vitro drug release and micromeritic properties of modified release ibuprofen microspheres. J Microencapsul, 1999; (4):475-487 11. 11. Yoo, HS, Oh, JE, Lee, KH and Park, TG. 1999. Bi odegadable nanoparticles containing PLGA conjugate for sustained release. Pharm. Res., 16: 1114–1118. 17. Pignatello R, Bucolo C, Ferrara P, Maltese A, Puleo A, Puglisi G. Eudragit RS100 nanosuspensions for the ophthalmic controlled delivery of ibuprofen. Eur J Pharm Sci, 2002; (1- 2):53-61 12. Adibkia K, Javadzadeh Y, Dastmalchi S, Mohammadi G, Niri FK, Alaei-Beirami M. Naproxen-eudragit RS100 nanoparticles: preparation and physicochemical characterization. Colloids Surf B Biointerfaces. 2011 Mar;83(1):155-9. 18. Pignatello R, Bucolo C, Spedalieri G, Maltese A, Puglisi G. Flurbiprofen-loaded acrylate polymer nanosuspensions for ophthalmic application. Biomaterials, 2002; (15):3247-3255 19. Le Thi Mai Hoa , Nguyen Tai Chi , Le Huu Nguyen & Dang Mau Chien. Preparation and characterisation of nanoparticles containing ketoprofen and acrylic polymers prepared by emulsion solvent evaporation method, Journal of Experimental Nanoscience, 2012;7:2: 189-197, 13. Hornig S, Bunjes H, Heinze T. Preparation and characterization of nanoparticles based on dextran-drug conjugates. J Colloid Interface Sci, 2009; (1):56-62 7 www.ijprt.com
https://openalex.org/W2954862606	https://europepmc.org/articles/pmc4324069?pdf=render	English	null	Preface	Journal of experimental botany	2,015	cc-by	732	Preface Size control of multicellular organisms such as plants poses a long-standing biological question that has fascinated scientists from every time and generation. Currently, the question on how size is measured and fixed during the growth of an organ or an organism is far from resolved, essentially because of its complex, integrated nature of regulation at the cellular, tissue, organ, and whole organism level. g g Growth is a quantitative, dynamic, and multi-factorial trait regulated by numerous genetic and environmental factors. The study of this complex machinery therefore requires the integration of multiple approaches, or system biology approaches, at different scales (plant, organ, cell) including genetics, physiology, quantitative phenotyping, and various -omics technolo- gies in order to obtain an holistic image of the molecular regulation of organ growth. In this special issue, recent approaches developed to unravel the regulation of plant organ growth and the knowledge obtained are presented for different parts of the plant (leaf, root, flower, fruit, and seeds). p (l ) The recent advances made in quantitative biology for the identification of the genetic basis of leaf growth regulation are first described and novel approaches that could be implemented for a better understanding of leaf growth and development are discussed (Gonzalez and Inzé). In the leaf, its final size is determined by cell proliferation and cell expansion that must be tightly co-ordinated. Hisanaga et al. integrated information from recent advances in molecular and genetic studies on compen- sation, an enhanced post-mitotic cell expansion associated with a decrease in cell number during lateral organ development is suggestive of such co-ordination. gg Floral organs have been suggested, for a long time, to be leaf-like structures. As reviewed by Sablowski, links have emerged between floral homeotic genes and general regulators of lateral organ growth, such as the genes involved in organ patterning, tissue growth, and cell differentiation, which will help to understand the regulation of organ shape. Unravelling how fruit and seed sizes are regulated is important not only from a fundamental perspective but also from a more applied viewpoint. In the review from Azzi et al., current knowledge on the genes contributing to the regulation of various developmental processes governing fruit growth in tomato is presented. Li and Li have summarized current progress on the maternal control of seed size and discuss the roles of several newly identified regulators. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. j This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) Journal of Experimental Botany, Vol. 66, No. 4 p. 1043, 2015 doi:10.1093/jxb/erv037 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) doi:10.1093/jxb/erv037 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) Preface yi g The study of how the growth of the underground part of the plant is regulated also represents an important challenge and new approaches have been developed to tackle this question as presented by Satbhai et al. who provide an overview of the major mechanisms, pathways, and genes that contribute to shaping root system architecture and its responses to environmen- tal variation. Hormones play a major role as endogenous regulators of plant organ growth such as the roots. This is illustrated by the review from Pacifici et al. that summarizes recent findings on the molecular mechanisms on hormonal cross-talk and root meristem size and primary root growth control. In the review from Singh and Savaldi-Goldstein, particular attention is given to the brassinosteroid signalling pathway and its role in the regulation of root growth. Finally, Long et al. describe the SHR–SCR pathway which has a key role in regulating stem cell activities and radial patterning in the root, illustrating the importance of cell–cell communication in the regulation of growth processes. p g g p We hope that this special issue on plant organ growth will further boost world-wide research on this highly interesting but challenging biological problem. Understanding size control is not only of academic interest but also opens numerous perspec- tives to improve crop yield thorough genetic modification, gene editing, and advance breeding. Nathalie González and Dirk Inzé, Ghent, Belgium
https://openalex.org/W2886260421	https://zenodo.org/record/1341470/files/DEZ_article_27033.pdf	English	null	Revision of the Quedius fauna of Middle Asia (Coleoptera, Staphylinidae, Staphylininae)	Mitteilungen aus dem Museum für Naturkunde in Berlin. Deutsche entomologische Zeitschrift	2,018	cc-by	32,514	Dtsch. Entomol. Z. 65 (2) 2018, 117–159 \| DOI 10.3897/dez.65.27033 Dtsch. Entomol. Z. 65 (2) 2018, 117–159 \| DOI 10.3897/dez.65.27033 Abstract Twenty eight species of the genus Quedius from Middle Asia comprising Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan, are revised. Quedius altaicus Korge, 1962, Q. capitalis Eppelsheim, 1892, Q. fusicornis Luze, 1904, Q. solskyi Luze, 1904 and Q. cohaesus Eppelsheim, 1888 are redescribed. The following new synonymies are established: Q. solskyi Luze, 1904 = Q. asiaticus Bernhauer, 1918, syn. n.; Q. cohae sus Eppelsheim, 1888 = Q. turkmenicus Coiffait, 1969, syn. n., = Q. afghanicus Coiffait, 1977, syn. n.; Q. hauseri Bernhauer, 1918 = Q. peneckei Bernhauer, 1918, syn. n., = Q. ouzbekiscus Coiffait, 1969, syn. n.; Q. imitator Luze, 1904 = Q. tschinganensis Coiffait, 1969, syn. n.; Q. novus Eppelsheim, 1892 = Q. dzambulensis Coiffait, 1967, syn. n., Q. pseudonigriceps Reitter, 1909 = Q. kirklarensis Korge, 1971, syn. n. Lectotypes are designated for Q. asiaticus Bernhauer, 1918, Q. fusicornis Luze, 1904, Q. hauseri Ber nhauer, 1918, Q. imitator Luze, 1904, Q. novus Eppelsheim, 1892 and Q. solskyi Luze, 1904. For all revised species, taxonomy, distribution and bionomics are summarized. Quedius fuliginosus (Gravenhorst, 1802), Q. sundukovi Smetana, 2003 and Q. pseudon igriceps Reitter, 1909 are recorded for Middle Asia for the first time. One species from the Q. coloratus-group, found to be new to science is not described due to shortage of material. Another possibly new species is tentatively identified as Q. fulvicollis Stephens, 1833 until the taxonomy of that widespread species is revised. An identification key to all species is provided. Copyright Maria Salnitska, Alexey Solodovnikov. 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. http://zoobank.org/B1A8523C-A463-4FC4-A0C3-072C2E78BA02 Corresponding authors: Maria Salnitska (m.salnitska@gmail.com); Alexey Solodovnikov (asolodovnikov@snm.ku.dk) Revision of the Quedius fauna of Middle Asia (Coleoptera, Staphylinidae, Staphylininae) Maria Salnitska1, Alexey Solodovnikov2 1 Department of Entomology, St. Petersburg State University, Universitetskaya Embankment 7/9, Saint-Petersburg, Russia 2 Natural History Museum of Denmark, Zoological Museum, Universitetsparken 15, Copenhagen 2100 Denmark Introduction study and reclassification of this genus. Such taxonomic work is also important for an overall inventory and under standing of the Palaearctic entomofauna. Unfortunately, our knowledge of the Palaearctic Quedius is uneven and in some places very limited. For example, hardly any thing has been done on Quedius of North Africa, Middle Asia, or Near and Middle East.i The rove beetle genus Quedius Stephens, 1829 is one of the largest in the family Staphylinidae. Even according to a recent phylogenetic study (Brunke et al. 2016) which restricted Quedius to a cluster of lineages confined mostly to the Holarctic region, it remains a very speciose taxon to deal with. The greatest diversity of Quedius in this re stricted sense, ca. 700 species, is confined to the humid ar eas of the Palaearctic region (Herman 2001; Schülke and Smetana 2015). A satisfactory alpha-taxonomic knowl edge of the mega-diverse Palaearctic fauna of Quedius is crucial for implementing a badly needed phylogenetic This paper aims to fill one of these knowledge gaps and focuses on Quedius of Middle Asia in the sense of Cowan (2007), i.e. the area covering five countries: Ka zakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uz bekistan (Fig. 1). These countries are indeed dominated by arid landscapes and their faunas have much in com 118 Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... Figure 1. Middle Asia, our study region comprising five countries according to Cowan (2007). Figure 1. Middle Asia, our study region comprising five countries according to Cowan (2007). Material and methods mon (Kryzhanovsky 1965). However, one must bear in mind the poor correspondence of this large territory to biogeography. Due to certain patterns of geography, land scape mosaic and biogeographic history, various areas of Middle Asia may show stronger faunal connections with other respective neighboring regions than to each other. Nevertheless, we limit our paper by the formal political borders of the listed countries for practical reasons. As the former republics of the Soviet Union, they often were (and often still are) studied together. As a result, legacy taxonomic and faunistic literature considers Middle Asia largely within these borders. dez.pensoft.net Depositories of material Material for this paper is deposited in the public institu tions and private collections abbreviated as follows: FMNH Field Museum of Natural History, Chicago, USA (C. Mayer, M. Thayer, A. Newton) HNHM Hungarian Natural History Museum, Budapest, Hungary (G. Makranczy) MNHN National Museum of Natural History, Paris, France (A. Taghavian) NHMD Natural History Museum of Denmark (for mer ZMUC, Zoological Museum of the Uni veristy of Copenhagen) (A. Solodovnikov, S. Selvantharan) NMW Natural History Museum, Vienna, Austria (H. Schillhammer) ZIN Zoological Institute, Russian Academy of Sci ences, St. Petersburg, Russia (B.A. Korotyaev) ZMLU Zoological Museum (part of the Biolog ical Museum, Lund University), Sweden (C. Fägerström) ZMMU Zoological Museum of Moscow University, Moscow, Russia (N.B. Nikitsky) FMNH Field Museum of Natural History, Chicago, USA (C. Mayer, M. Thayer, A. Newton) ( y y ) HNHM Hungarian Natural History Museum, Budapest, Hungary (G. Makranczy) g y ( y) MNHN National Museum of Natural History, Paris, France (A. Taghavian) Where necessary, we have considered literature or ma terial from areas outside Middle Asia. However, species known only from outside this region were not included in this paper. One rather specialized and distinct group of species related to Quedius (Microsaurus) mutilatus, which comprises endemic Middle Asian species with nar row montane distributions, has been revised in a separate publication (Salnitska and Solodovnikov 2018). Howev er, species of the Q. mutilatus group are here included in the identification key to all species of Quedius currently known from Middle Asia. We hope that this taxonomic re vision and the first specialized key of Middle Asian Quedi us will stimulate further investigations of the genus in this and adjacent poorly known areas of the Palaearctic region. NHMD Natural History Museum of Denmark (for mer ZMUC, Zoological Museum of the Uni veristy of Copenhagen) (A. Solodovnikov, S. Selvantharan) NMW Natural History Museum, Vienna, Austria (H. Schillhammer) NMW Natural History Museum, Vienna, Austria (H. Schillhammer) ZIN Zoological Institute, Russian Academy of Sci ences, St. Petersburg, Russia (B.A. Korotyaev) ( ) ZIN Zoological Institute, Russian Academy of Sci ences, St. Petersburg, Russia (B.A. Korotyaev) g ( y ) ZMLU Zoological Museum (part of the Biolog ical Museum, Lund University), Sweden (C. Fägerström) ZMMU Zoological Museum of Moscow University, Moscow, Russia (N.B. Nikitsky) ZMMU Zoological Museum of Moscow University, Moscow, Russia (N.B. Nikitsky) Dtsch. Entomol. Z. Type material Where possible type material was examined and sup plied with our standard respective labels indicating the revised status or identity of the respective type speci mens. All original labels of the type specimens are cited verbatim in the ‘Material examined’ sections and, where available, photographed. Borders and geography of Middle Asia The term “Middle Asia” is somewhat fuzzy in the geo graphical or historical literature. For example, sometimes Kazakhstan is considered as a part of Middle Asia, some times an expression “Middle Asia and Kazakhstan” is used. Often the distinction between “Middle Asia” and “Central Asia” is not clear. English-language publications have used “Central Asia” to refer to areas of the former USSR, to areas of China and Mongolia and to areas that cross the former Soviet/Chinese border. To avoid this am biguity we follow Cowan (2007) and use “Middle Asia” to refer to Kazakhstan, Turkmenistan, Uzbekistan, Tajikistan and Kyrgyzstan collectively. The geographic area covered by these five countries is a subject of this paper (Fig.1). In Depositories of material 65 (2) 2018, 117–159 119 1988, 1992, 1995b, 1996, 2001, 2015a, 2017), species groups proposed for the fauna of China (Smetana 2017) are worth consideration, especially given that the large Xinjiang province of the north-western China borders with Middle Asia via Tajikistan, Kyrgyzstan and Kazakh stan. However, that large province of China seems to be one of the least explored areas there, what can be seen for example, from the lacking records for any wide-spread Middle Asian species from that province. Therefore, plac ing Middle Asian species in the species groups of Smetana (2017) was not possible, at least without extensive direct comparisons with the material from China. We can only propose that among the Middle Asian species, Q. hauseri and a species tentatively identified here as Q. fulvicollis may be related to the Chinese muscicola-group. Also, it should be noted that Smetana (2017) placed Q. koltzei in its own monotypic species group. We should also point to our disagreement with Smetana (2017) who considers Q. equus a member of the przewalskii-group, while we place it in the mutilatus-group (Salnitska and Solodovnikov 2018, and here). These disagreements are not essential for the taxonomic purposes of this paper and they once again call for a necessity of a large-scale phylogenetic study of Quedius. All species treated in this revision are also listed alphabetically in Table 1. Distribution maps Measurements were taken at X4.5 magnification using an ocular micrometer. They are abbreviated as follows: HL – head length (from base of labrum to neck constriction along the head midline); HW – head width (maximum, including eyes); PL – pronotum length (along midline); PW – pronotum width (maximum); EL – length of elytra (from humerus to the most distal part of the elytral poste rior margin); EW – width of elytra (maximum, with elytra closed along suture). Overall body length was measured from apex of labrum to apex of abdomen. All distributions were mapped using QGIS 2.12.0 and geographical coordinates indicated on the original locality labels of the specimens. In the case of older, non-georefer enced labels, we used approximate geographic coordinates that we were able to find for the respective toponyms with the aid of various printed maps or online systems (Google Maps, Google Earth, Global Gazetteer version 2.3 and oth ers). Ambiguously indicated localities are cited verbatim in the ‘Material examined’ sections and taken in quotation marks. All our interpretations for such localities are given in square brackets. Those of which that are mapped are also given with their approximate coordinates in Table 2. Preparation, examination and illustration of specimens Specimens were examined with Lomo MSP-2 ver. 2 and Leica M125 dissecting scopes. Habitus and genitalia pho tographs were obtained using a Nikon SMZ 1500 binocular microscope with a Nikon D700 digital SLR camera. Illus trations of the male genitalia were done from soft prepara tions of these structures in glycerin (after dissecting, mac eration in 10% KOH, and rinsing in distilled water) using a drawing tube attached to a Nikon SMZ 1500 binocular mi croscope. All dissected aedeagi are kept in glycerin in gen italia microvials pinned under their respective specimens. dez.pensoft.net Classification We use conventional subdivision of the genus Quedius into subgenera as in e.g. Schülke and Smetana (2015). Within the subgenera we list species so that those we pre sume to be closely related appear close to each other. Ex cept the recently defined coloratus-group (Assing 2017) and mutilatus-group (Salnitska and Solodovnikov 2018), we cannot use any of the hitherto proposed species groups in Quedius. Species groups of Coiffait (1978) for the West Palaearctic fauna are very outdated, inconsistent and even lack any diagnoses. Among those of Smetana (1971, dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 120 the west, Middle Asia is bordered by the Caspian Sea and the state border between Russia and Kazakhstan, nearly coinciding with the Volga River. In the north, Middle Asia is outlined by the long administrative border between Ka zakhstan and Russia. In the east, Middle Asia borders with north-western China through the eastern administrative borders of Kazakhstan, Kyrgyzstan and Tajikistan. In the south, it is outlined by the northern borders of Afghanistan and Iran. While large areas of Kazakhstan and Turkmeni stan are covered by more or less flat, desert landscape, east ern and south-eastern Kazakhstan, as well as Kyrgyzstan and Tajikistan are mainly montane countries with com plex relief and a diverse landscape mosaic. In north-east ern Kazakhstan, the Altai mountain chain stretches into Middle Asia from Russia. In eastern Kazakhstan, as well as in Kyrgyzstan and Tajikistan, the area is dominated by the vast mountain systems of Tien Shan and Pamir. Large lakes like the Aral Sea, Balkhash, Issyk-Kul, and rivers like Amu Darya or Syr Darya are significant elements in the geography of Middle Asia as well. earlier literature. For example, three species described from Middle Asia by Luze (1904), Q. (M.) fusicornis, Q. (M.) rufilabris and Q. (M.) solskyi, were entirely overlooked in the influential monograph of Coiffait (1978) and have not been studied since their original description. The most un fortunate flaw of Coiffait’s taxonomy was an artificial and over-splitting approach to species. As a result, all species of Quedius from Middle Asia he described as new, except Q. (M.) tadjikiscus, turned out to be synonyms here. Finally, some bionomic and distributional data on Mid dle Asian Quedius were published by local authors sta tioned in that region (Kascheev, 1984–2002; Kadyrov et al., 2014a, b; Gabdullina, 2016). History of the study of Quedius of Middle Asia Middle Asia is the region in the western Palaearctic where published data about Quedius remained the most fragmen tary and confusing, limited to a number of scattered and mostly outdated species descriptions. Eppelsheim (1888, 1892) was the first who studied Quedius material collect ed in Middle Asia by the early explorers such as Haus er, Staudinger, Akinin and described four new species: Q. (M.) mutilatus Eppelsheim, 1888, Q. (Raphirus) co haesus Eppelsheim, 1888, Q. (M.) capitalis Eppelsheim, 1892 and Q. (R.) novus Eppelsheim, 1892. Later, based on the material from Semenov and Hauser, Luze (1904) and Bernhauer (1918), respectively, described five more new species from Middle Asia: Q. (M.) solskyi Luze, 1904, Q. (M.) rufilabris Luze, 1904, Q. (M.) fusicornis Luze, 1904, Q. (R.) imitator Luze, 1904, Q. (M.) asiaticus Ber nhauer, 1918, Q. (M.) bucharensis Bernhauer, 1918 and Q. (R.) hauseri Bernhauer, 1918. These species descrip tions varied in quality and, in accordance with the time, were based exclusively on external morphology. Some of these species have been re-examined in the monograph by Gridelli (1924), while the first drawings of the aedeagi for some of them appeared in Wüsthoff (1938). Classification With the scattered, con fusing and then poorly accessible taxonomic literature on Quedius, no surprise that their local faunistic papers were greatly infested by incorrect species identifications. Ex amination of the material collected by Kascheev (1984– 2002), now deposited at ZIN, largely helped to reveal such misidentifications summed up in the Table 1 here. Overall, due to a hitherto lacking targeted contempo rary taxonomic investigation of the Middle Asian Quedius, identity of the majority of species described from, or re corded for, that region remained highly ambiguous. Most of the species described from Middle Asia needed broad er comparisons and a revision of the type material. At the same time, a number of widespread species from Middle Asia were misidentified or overlooked. A large amount of Quedius material from Middle Asia remained undeter mined and scattered in some institutional and private col lections. The revision of Q. (M.) mutilatus species group by Salnitska and Solodovnikov (2018) was the only recent taxonomic work that touched upon Middle Asian Quedius. dez.pensoft.net Genus Quedius Stephens, 1829 Type species. Quedius levicollis (Brullé, 1832). Type species. Quedius levicollis (Brullé, 1832). Among other Staphylinini in Middle Asia Quedius can be sometimes confused with Philonthus (sub tribe Philonthina), a genus with somewhat similar habitus and very abundant in the region. Species of Philonthus, however, do not have long infraorbital ridges, they lack empodial setae and mostly have a pronotal hypomeron well visible in lateral view. Also, Philonthus mostly pos sess multiple setiferous punctures in dorsal rows of pro notum (usually at most three in Quedius). Smaller species of Quedius may be confused with the genus Heterothops (subtribe Amblyopinina), but the latter have very thin acicular apical segments of maxillary palps, and a very different aedeagus without sensory peg setae and reduced median lobe giving the appearance of an absent paramere). Diagnosis. Medium to large size (body length 3.5– 24.0 mm) rove beetles with glossy forebody, infraorbital ridges extended from neck to base of mandibles and pro notal hypomera strongly inflexed under pronotal disk (not visible in lateral view). First segment of antennae at most slightly longer than second and third segments together. Last segment of maxillary palps fusiform, not densely se tose. Tarsal formula 5–5–5; anterior tarsi widened in both sexes, with pale adhesive setae ventrally, with pair of Table 1. Alphabetical list of Quedius species recorded for Middle Asia, with new synonyms. Boldfaced species are those confirmed by material in our study; species in regular font not given in square brackets are those known from literature only, presumably absent in Middle Asia; species in regular font and given in square brackets are those previously recorded for the region in literature based on misidentifications and here excluded from the fauna. Table 1. Alphabetical list of Quedius species recorded for Middle Asia, with new synonyms. Boldfaced species are those confirmed by material in our study; species in regular font not given in square brackets are those known from literature only, presumably absent in Middle Asia; species in regular font and given in square brackets are those previously recorded for the region in literature based on misidentifications and here excluded from the fauna. Species Subgenus Records from Middle Asia Notes Page here [Q. acuminatus acuminatus Hochhuth, 1849] Raphirus Kascheev 2001, 102; 2002, 181 Misidentification of Q. hauseri 150 Q. altaicus Korge, 1962 Quedius (s. str.) Toleutaev 2014, 44 128 [Q. auricomus Kiesenwetter, 1850] Raphirus Kascheev 1989, 36 Based on misidentification; here not confirmed by material – Q. balticus Korge, 1960 Quedius (s. Type species. Quedius levicollis (Brullé, 1832). Type species. Quedius levicollis (Brullé, 1832). According to the latest phylogenetic hypotheses (Solo dovnikov, 2006; Chatzimanolis et al., 2010; Brunke et al., 2016) the genus Quedius as it stands now in the taxonomic literature (e.g., summaries in Herman, 2001 or Schülke and Smetana, 2015) is a polyphyletic assem blage of species belonging to several different subtribes of Staphylinini. Within the Palaearctic or Middle Asia, all species of Quedius are members of the subtribe Quediina in the restricted sense of Brunke et al. (2016). Because of the polyphyly, Quedius in the current composition lacks synapomorphies and clear diagnosis. However, genus de scriptions and diagnostic combination of characters that can define any Palaearctic species as a member of the genus Quedius are available in Coiffait (1978), Smetana (1988), Assing and Schülke (2012) and other sources. The diagnosis of the genus Quedius and comparative notes we provide here are tuned for the fauna of Middle Asia. The next notable contribution to the study of Middle Asian Quedius was made in the papers by Coiffait (1954, 1955, 1963, 1967, 1969, 1970, 1975, 1978) devoted to the Western Palearctic fauna. Henry Coiffait added aedeagus illustrations for many Middle Asian species and integrated them in his identifications keys for the Western Palearctic Quedius. He also described Q. (R.) dzambulensis Coiffait, 1967, Q. (R.) ouzbekiscus Coiffait, 1969, Q. (R.) tschingan ensis Coiffait, 1969, Q. (R.) turkmenicus Coiffait, 1969, and Q. (M.) tadjikiscus Coiffait, 1975, all from Middle Asia. Unfortunately, Coiffait’s input was based on very limited material from Middle Asia and additionally suffered from inconsistent study of type material and omissions of the Adults and larvae of Quedius seem to be predators hunting small invertebrates in various, sufficiently hu Dtsch. Entomol. Z. 65 (2) 2018, 117–159 121 empodial setae. Males always with distinct apical emar gination on abdominal sternite VIII. Aedeagus varies in shape, paramere mostly with sensory peg setae. mid ground-based debris, mostly in forest leaf litter. In a largely arid region like Middle Asia, Quedius are mainly confined to humid open or forested habitats along creeks or rivers in the lowland or forests, meadows, snowfield margins and talus in the mountains. Some members of the subgenus Microsaurus are specialized inhabitants of mammal burrows. Overall, bionomics of the genus in Middle Asia remain largely unstudied. Comparison. Type species. Quedius levicollis (Brullé, 1832). str.) Klimenko 1996, 121 – [Q. boopoides Munster, 1923] Raphirus Kascheev 2002, 181 Apparently misidentification of Q. hauseri 150 [Q. boops boops Gravenhorst, 1802] Raphirus Eppelsheim 1892, 332; Kascheev 2001, 102; Toleutaev 2014, 44 Apparently misidentification of Q. hauseri 150 Q. brevis Erichson, 1840 Microsaurus Gabdullina 2016, 61 – Q. bucharensis Bernhauer, 1918 Microsaurus Bernhauer 1918, 93; Gridelli 1924, 56; Coiffait 1978, 186; Kadyrov et al. 2014a, 31; 2014b, 49 138 Q. capitalis Eppelsheim, 1892 Microsaurus Eppelsheim 1892, 329; Gridelli 1924, 40; Coiffait 1978, 148; Kadyrov et al. 2014a, 31; 2014b, 49 132 [Q. cincticollis cincticollis Kraatz, 1857] Raphirus Toleutaev 2014, 44 (cited as Q. cincticollis Kr.) Misidentification, likely of Q. hauseri 150 Q. cohaesus Eppelsheim, 1888 Raphirus Q. cohaesus: Eppelsheim 1888, 60; Gridelli 1925, 126; Coiffait 1963, 393; 1978, 248; Toleutaev 2014, 44; Q. turkmenicus: Coiffait 1969, 49; Q. afghanicus: Coiffait 1977: 139; Coiffait 1978, 232 142 =Q. turkmenicus Coiffait, 1969, syn. n. =Q. afghanicus Coiffait, 1977, syn. n. Q. sp. aff. Q. coloratus Raphirus 149 Q. curtipennis Bernhauer, 1908 Quedius (s. str.) Bernhauer 1908, 335 125 Q. equus Smetana, 2004 Microsaurus Salnitska and Solodovnikov 2018, 10 139 Q. fulgidus fulgidus Fabricius, 1792 Microsaurus Kascheev 2002, 181 (cited as Q. fulgidus F.) – Q. fuliginosus Gravenhorst, 1802 Quedius (s. str.) First record from Middle Asia 125 Q. fulvicollis Stephens, 1833 (tentative identification) Raphirus Klimenko 1996, 121 (based on uncertain reference) 155 [Q. fumatus Stephens, 1833] Raphirus Kascheev 2001, 102; Toleutaev 2014, 44; Gabdullina 2016, 61 Presumed misidentification – Q. fusicornis Luze, 1904 Microsaurus Luze 1904, 101; Gridelli 1924, 40 134 Q. hauseri Bernhauer, 1918 Raphirus Q. hauseri: Bernhauer 1918, 94; Tronquet 1981, 71; Klimenko 1996, 121; Q. peneckei: Bernhauer 1918, 95; Q. ouzbeciscus: Coiffait 1969, 52; 1970, 143; 1978, 278; Kascheev 2001, 102 Records of Q. acuminatus acuminatus, Q, boops and Q. boopoides likely belong to this species 150 =Q. peneckei Bernhauer, 1918, syn. n. =Q. ouzbekiscus Coiffait, 1969, syn. n. dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 122 Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 122 Species Subgenus Records from Middle Asia Notes Page here Q. humeralis Stephens, 1832 Raphirus Eppelsheim 1892, 332; Gridelli 1922, 130, 134 Presumed misidentification – Q. imitator Luze, 1904 Raphirus Q. imitator: Luze 1904, 102; Coiffait 1967, 406; 1978, 237; Bohác 1988, 556; Kadyrov et al. Type species. Quedius levicollis (Brullé, 1832). 2014a, 31; 2014b, 49; Q. tschinganensis: Coiffait 1969, 50; 1970, 143; 1978, 237; Klimenko 1996, 121; Kascheev 2001, 102 143 =Q. tschinganensis Coiffait, 1969, syn. n. Q. infuscatus Erichson, 1840 Microsaurus Kascheev 1984, 28; 1985, 46 – Q. limbatus Heer, 1839 Raphirus Eppelsheim 1892, 332; Smetana 1962, 146; Horion 1965, 284; 32; Kascheev 2001, 102; 2002, 181 139 [Q. longicornis Kraatz, 1857] Microsaurus Kascheev 2002, 181 Presumed misidentification – [Q. maurorufus Gravenhorst, 1806] Raphirus Toleutaev 2014, 44 Presumed misidentification of Q. pseudonigriceps 140 Q. meridiocarpathicus Smetana, 1958 Quedius (s. str.) Klimenko 1996, 121 (based on uncertain reference) – Q. mutilatus Eppelsheim, 1888 Microsaurus Eppelsheim 1888, 58; Gridelli 1924, 23; Coiffait 1978, 161; Klimenko 1996, 121; Smetana 1998, 118; Solodovnikov and Hansen 2016, 3–8; Salnitska and Solodovnikov 2018, 4 139 Q. kalabi Smetana, 1995 Microsaurus Smetana 1995a, 77; 1998, 119; Salnitska and Solodovnikov 2018, 9 139 Q. koltzei Eppelsheim, 1892 Microsaurus Coiffait 1978, 164 137 Q. molochinus Gravenhorst, 1806 Quedius (s. str.) Protopoyan 1967, 168 (cited as Q. ‘nittidipennis Steph. [sic!]’) – Q. kungeicus Solodovnikov & Salnitska Microsaurus Salnitska and Solodovnikov 2018, 13 139 [Q. nigriceps Kraatz, 1857] Raphirus Kascheev 2001, 102; 2002, 181; Kadyrov et al. 2014a, 31; 2014b, 49 Presumed misidentification – [Q. nigrocaeruleus Fauvel, 1876 ] Microsaurus Gabdullina 2016, 61 Presumed misidentification – Q. novus Eppelsheim, 1892 Raphirus Q. novus:Eppelsheim 1892, 331; Gridelli 1925, 125; Coiffait 1963, 389; 1970, 143; 1978, 228; Bohác 1988, 556; Smetana 1995a, 84; Klimenko 1996, 121; Kadyrov et al. 2014a, 31; 2014b, 49; Q. dzambulensis: Coiffait 1967, 403; Coiffait 1978, 229; Bohác 1988, 556; Kascheev 2001, 102 146 =Q. dzambulensis Coiffait, 1967, syn. n. Q. ochripennis Ménetries, 1832 Microsaurus Gridelli 1929, 21; Klimenko 1996, 121; Kascheev 2001, 102; Kadyrov et al. 2014a, 31; 2014b,49 131 [Q. persimilis Mulsant & Rey, 1876] Raphirus Kascheev 2001, 102; 2002, 181 (cited as Q. joyi Fagel) Presumed misidentification of Q. hauseri – Q. pseudonigriceps Reitter, 1909 Raphirus First record for Middle Asia 140 =Q. kirklarensis Korge, 1971, syn. n. [Q. picipes Mannerheim, 1830] Microsaurus Kascheev 2001, 102 Presumed misidentification – Q. puncticollis Thomson, 1867 Microsaurus Kascheev 2001, 102 132 Q. rufilabris Luze, 1904 Microsaurus Luze 1904, 100; Gridelli 1924, 72; Kadyrov et al. 2014a, 31; 2014b, 49 Type material not found 138 Q. scintillans Gravenhorst, 1806 Raphirus Eppelsheim 1892, 332; 155 [Q. scitus Gravenhorst, 1806] Microsaurus Kascheev 2001, 102 Presumed misidentification – Q. Key to species of Quedius of Middle Asia 1 Anterior margin of labrum entire so that labrum never bilobed or notched in the middle. Large species with body length 9.0–15.0 mm (fig. 187a in Assing and Schülke 2012)......................................................... 2 (Subgenus Quedius s. str.) – Anterior margin of labrum either with distinct notch in the middle, or with deep emargination so that labrum looks bi lobed. Mostly smaller species with body length 5.0–12.0 mm (fig. 187b–d in Assing and Schülke 2012)..................... 6 2 Scutellum without setiferous punctures, glabrous. Frons with additional setiferous punctures (that only occasionally maybe lost) between anterior frontal punctures.......................................................................................................... 3 – Scutellum with setiferous punctures, setose. Frons without additional setiferous punctures between anterior frontal punctures.................................................................................................................................................................. 4 3 Aedeagus (in parameral view): apical portion of paramere lanceolate, wider than its sinuate middle part; rows of sen sory peg setae on the parameral underside, in their basal half, extended more medially from parameral lateral margins (fig. 188c in Assing and Schülke 2012); lateral contours of apical part of median lobe not visible from under paramere (fig. 188a in Assing and Schülke 2012).......................................................................... Q. fuliginosus (Habitus Fig. 2A). – Aedeagus (in parameral view): apical portion of paramere gradually narrowing apicad, medially not sinuate and not narrower than its more apical part; rows of sensory peg setae on the parameral underside, in their basal half, extended more laterally, closer to parameral lateral margins (fig. 188f in Assing and Schülke 2012); lateral contours of median lobe apically visible from under paramere (fig. 188d in Assing and Schülke 2012).................................... Q. curtipennis 4 Body dark brown, with paler (sometimes reddish) elytra (Habitus as in Fig. 2C). Aedeagus (in lateral view): apex of paramere protruding beyond median lobe in the form of a distinct hook (fig. 1 in Hachikov 2003).................. Q. vicinus – Body and elytra black, or at most dark brown. Aedeagus (in lateral view): apex of paramere straight, not pointing out from median lobe as a distinct hook........................................................................................................................... 5 5 Elytra shortened, distinctly shorter than pronotum. Obviously brachypterous species, without whitish apical seam on ab dominal tergite VII. Smaller: body length 7.50–9.00 mm (Habitus Fig. 2B). Aedeagus as in figs 4–6 in Smetana 2002...... ......................................................................................................................................................................Q. sundukovi – Elytra normal, about as long as pronotum. Species with whitish apical seam on abdominal tergite VII. Larger: body length 8.6–12.5 mm. Habitus and aedeagus as in Fig. 5E–G.......................................................................... Q. altaicus 6 Eyes small or moderate in size, always distinctly shorter than temples (fig. Type species. Quedius levicollis (Brullé, 1832). solskyi Luze, 1904 Microsaurus Q. solskyi: Luze 1904, 99; Gridelli 1924, 72; Q. asiaticus: Bernhauer 1918, 92; Gridelli 1924, 57; Coiffait 1978, 183; Kascheev 2002, 181 135 =Q. asiaticus Bernhauer, 1918, syn. n. Q. sundukovi Smetana, 2003 Quedius (s. str.) First record for Middle Asia 130 Q. tadjikiscus Coiffait, 1975 Microsaurus Coiffait 1975, 32; 1978, 149; Kadyrov et al. 2014a, 31; 2014b, 49 138 Q. umbrinus Erichson, 1839 Raphirus Kascheev 1989, 36 149 Q. vicinus Ménetries, 1832 Quedius (s. str.) Boháč 1988, 554 131 Dtsch. Entomol. Z. 65 (2) 2018, 117–159 123 Table 2. Suggested georeferencing for ambiguous toponyms from old labels. Label data verbatim Locality Long Lat Country “ISKANDER-KUL ISKANDER-DARIA Glasunov 1892” Iskanderkul Lake, Iskander Darya river, Ayni Distr. [39°4.2’] [68°22.2’] Tajikistan “Seravschan Kumar Glasunov 1892” Kumar River valley, Ayni Distr. [68°31.8’] [39 16.2] Tajikistan “JAGNOB KARSAU Glasunov 1892” Yaghnob River valley, Sughd Distr. [68°32.4’] [39°11.4’] Tajikistan “JAGNOB CHISHARTOB Glasunov 1892” Yaghnob River valley, Sughd Distr. [68°32.4’] [39°11.4’] Tajikistan “Trkst. Jagnob Kol Schach-Sara Glasunov 1892” Yaghnob River valley, Sughd Distr. [68°32.4’] [39°11.4’] Tajikistan “SERAVSCHAN DARCH Glasunov 1892” Darg, Sughd Distr. [68°58.8’] [39°21’] Tajikistan “Seravschan Kschtut. Artutsch. Glasunov 1982” Kylali, Sughd Distr. [68°2.4’] [39°21.6’] Tajikistan “Seravschan Fl. Magian Glasunov 1892” Seravschan Mt. Ridge, Mogiyon, Panjakent Distr. [67°39.6’] [39°15’] Tajikistan “SERAVSCHAN OBBURDEN Glasunov 1892” Obburdon, Mastchoh Distr. [69°18’] [40°25.8’] Tajikistan “Mts. Karateghin Baldschuan 924 m. F. Hauser 1898” Karateghin Mts, Baljuvon, Baljuvon Distr. [69°40.2’] [38°18’] Tajikistan “Mt. Karateghin Sary-pul 1482 m. F. Hauser 1898” Karateghin Mts, Saripul, Khatlon Distr. [70°7.8’] [38°25.2’] Tajikistan ”PROV. KULIAB, Ak-sou-Tal, F. Hauser 1898/ Gift from Nat. Mus. Praha. 2009” Ak-Su, Khatlon Distr. [68°34.8’] [38°7.2’] Tajikistan “Gaudan, Transcaspian reg., 15.I.1898, E. Fimyanovich” Gaudan, Ashgabat Distr. [58°24’] [37°39’] Turkmenistan “Trkst. Mnt. Nurata UCHUN Glasunov 1892” Nurata Mt., Nurata Distr. [65°41.4’] [40°32.4’] Uzbekistan “Fergana valley, tract Aral, Achinsk, L. Arnoldi” Aral, Namangan Distr. [71°55.2’] [41°00’] Uzbekistan Table 2. Suggested georeferencing for ambiguous toponyms from old labels. Key to species of Quedius of Middle Asia 187d in Assing and Schülke 2012). Vertex (at least one side) with two basal punctures postero-medially from posterior frontal puncture (fig. 186b in Assing and Schülke 2012). Postero-lateral areas of pronotum somewhat explanate..................................7 (Subgenus Microsaurus) – Eyes large and convex, always longer than temples (fig. 187b, c in Assing and Schülke 2012). Vertex with one basal puncture postero-medially from posterior frontal puncture (fig. 186a in Assing and Schülke 2012). Postero-lateral areas of pronotum not explanate........................................................................................................18 (Subgenus Raphirus) 7 Elytra brownish, of about same or very similar coloration as the rest of the body. Eyes very small, 2.5–2.7 times as long as temples. Elytra shorter than pronotum. Distinctly brachypterous species without whitish apical seam on abdominal tergite VII (Fig. 2E)..................................................................................................................................................... 8 – Elytra reddish, always different in coloration from the rest of the body, which is black or at most dark brown. Eyes larger, ca. 0.5–1 times as long as temples. Elytra longer than, or as long as pronotum. Apical seam on abdominal tergite VII always distinct......................................................................................................................................................... 11 dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 124 y Q 8 Aedeagus (fig. 4G–N in Salnitska & Solodovnikov 2018), in parameral view: paramere apically deeply incised, appearing bilobed............................................................................................................................................................Q. equus – Aedeagus (in parameral view): paramere apically at most slightly incised.................................................................... 9 9 Aedeagus (fig. 4E, F Salnitska & Solodovnikov 2018), parameral view: apical portion of paramere ovoid (lanceolate), not rhomboid...................................................................................................................................................Q. kungeicus – Aedeagus, in parameral view: apical portion of paramere somewhat rhomboid (fig. 4B, D in Salnitska & Solodovnikov 2018)...................................................................................................................................................................... 10 10 Aedeagus (fig. 4C, D in Salnitska & Solodovnikov 2018), in lateral view: apical portion of median lobe relatively narrower and acute).......................................................................................................................................................Q. kalabi – Aedeagus (fig. 4A, B in Salnitska & Solodovnikov 2018), in lateral view: apical portion of median lobe relatively broader and blunt....................................................................................................................................................Q. mutilatus 11 Smaller species: body length around 6.0–9.3 mm. Aedeagus, underside of the paramere: peg setae arranged in rows with maximum 6–8 pegs in each row extending basad from pairs of lateral setae (Figs 7D, 8D)................................. 12 – Larger species: body length around 8.0–11.0 mm. Aedeagus, underside of the paramere: peg setae located at the apex of paramere only (Q. solskyi, Fig. 9C) or arranged in groups extending basad from the parameral apical margin, beyond the pairs of lateral setae (fig. 191j–k, t–v in Assing and Schülke 2012)...................................................................... 13 12 Aedeagus: underside of the paramere (Fig. 7D): with ca. Key to species of Quedius of Middle Asia 4–8 sensory peg setae in each of two rows curved along lateral sides of paramere........................................................................................................................................ Q. capitalis – Aedeagus: underside of paramere (Fig. 8D): with ca. 3 sensory peg setae in each of two linear rows............Q. fusicornis 13 Aedeagus, paramere (Fig. 9C) parallel-sided along most of its length, not lanceolate, with only 1–2 peg setae at apical margin on each side of the mid-apical incision......................................................................................................... 14 – Aedeagus, paramere not parallel-sided, with more or less lanceolate or rhomboid apical portion (fig. 191j, l in Assing and Schülke 2012); peg setae on parameral underside more numerous and arranged in irregular rows or groups. 15 14 Larger species with body length 8.9–9.7 mm; head distinctly wider than long (HL/HW ratio 0.7–0.8) with posterior frontal puncture situated in the middle of distance between posterior margin of eye and nuchal ridge. (Habitus and aedeagus as in Fig. 9A–D)...............................................................................................................................Q. solskyi – Smaller species with body length 8.5–9.4 mm; head from nearly as long as wide to longer that wide (HL/HW ratio 0.9–1.1) and posterior frontal puncture situated closer to posterior margin of eye than to nuchal ridge. Structure of the aedeagus unknown (for details see below)............................................................................................... Q. bucharensis 15 Pronotum with basalmost setiferous puncture of sublateral group (sometimes may be lost at one side) situated distinct ly behind the level of large lateral puncture (fig. 186a in Assing and Schülke 2012).................................................. 16 – Pronotum with punctures of sublateral group always situated before or at most at the same level as large lateral punc ture (fig. 186b in Assing and Schülke 2012)............................................................................................................. 17 16 Aedeagus, in parameral view: apical portion of the paramere lanceolate with bluntly pointed apical contour (fig. 191j, l in Assing and Schülke 2012)....................................................................................................................Q. ochripennis – Aedeagus, in parameral view: apical portion of the paramere not lanceolate, with broad and shallow apical emargination (fig. 191t, v in Assing and Schülke 2012)..................................................................................................Q. puncticollis 17 Pronotum with dorsal rows each with only two punctures. Aedeagus, underside of the paramere: peg setae arranged in four irregular groups: a pair of apical groups and a pair of subapical groups (fig. 11C in Coiffait, 1978)........Q. koltzei (externally Q. rufilabris, an ambiguous species described from ‘Zeravshan’ also fits here; for details see the section on this species) – Pronotum with dorsal rows each with three punctures. Aedeagus, underside of the paramere: peg setae arranged only in two subapical groups, the pair of apical groups absent (fig. Key to species of Quedius of Middle Asia 7K–M in Coiffait 1978)............................... Q. tadjikiscus 18 Scutellum with setiferous punctation; eyes large and convex, occupying almost entire lateral side of head; rather small species. Body not longer than 6.0 mm. Aedeagus as in (Figs 19B–D, 21B, C)............................................................ 19 – Scutellum glabrous, without setiferous punctation; eyes smaller and more flat; temples more distinct. Body length varies but includes larger species. Aedeagus different......................................................................................................... 20 19 Aedeagus: paramere almost parallel-sided, only slightly narrowing in the middle portion, rows of peg setae long and regular (Fig. 19C) Habitus and aedeagus as in Fig. 19A–D..............................................................................Q. hauseri – Aedeagus: paramere not parallel-sided, strongly narrowing in the middle portion, rows of peg setae shorter and irregu lar (Fig. 23C). Habitus and aedeagus as in Fig. 23A–C.................................................................................Q. fulvicollis 20 Frons with two additional punctures between anterior frontal punctures. Rather small species, body not longer than 6.0 mm.....................................................................................................................................................Q. scintillans – Frons without punctures between anterior frontal punctures. Species varying in size................................................. 21 21 Elytra shortened, slightly shorter than, or at maximum, as long as pronotum. Brachypterous species without whitish apical seam on abdominal tergite VII (Fig. 3F). Aedeagus as in Fig. 11...............................................Q. pseudonigriceps – Elytra longer than, or at minimum, as long as pronotum. Species with whitish apical seam on abdominal tergite VII (Fig. 3C). Aedeagus different.................................................................................................................................... 22 8 Aedeagus (fig. 4G–N in Salnitska & Solodovnikov 2018), in parameral view: paramere apically deeply incised, appearing bilobed............................................................................................................................................................Q. equus – Aedeagus (in parameral view): paramere apically at most slightly incised.................................................................... 9 9 Aedeagus (fig. 4E, F Salnitska & Solodovnikov 2018), parameral view: apical portion of paramere ovoid (lanceolate), not rhomboid...................................................................................................................................................Q. kungeicus – Aedeagus, in parameral view: apical portion of paramere somewhat rhomboid (fig. 4B, D in Salnitska & Solodovnikov 2018)...................................................................................................................................................................... 10 10 Aedeagus (fig. 4C, D in Salnitska & Solodovnikov 2018), in lateral view: apical portion of median lobe relatively narrower and acute).......................................................................................................................................................Q. kalabi – Aedeagus (fig. 4A, B in Salnitska & Solodovnikov 2018), in lateral view: apical portion of median lobe relatively broader and blunt....................................................................................................................................................Q. mutilatus 11 Smaller species: body length around 6.0–9.3 mm. Aedeagus, underside of the paramere: peg setae arranged in rows with maximum 6–8 pegs in each row extending basad from pairs of lateral setae (Figs 7D, 8D)................................. 12 – Larger species: body length around 8.0–11.0 mm. Aedeagus, underside of the paramere: peg setae located at the apex of paramere only (Q. solskyi, Fig. Key to species of Quedius of Middle Asia 9C) or arranged in groups extending basad from the parameral apical margin, beyond the pairs of lateral setae (fig. 191j–k, t–v in Assing and Schülke 2012)...................................................................... 13 12 Aedeagus: underside of the paramere (Fig. 7D): with ca. 4–8 sensory peg setae in each of two rows curved along lateral sides of paramere........................................................................................................................................ Q. capitalis – Aedeagus: underside of paramere (Fig. 8D): with ca. 3 sensory peg setae in each of two linear rows............Q. fusicornis 13 Aedeagus, paramere (Fig. 9C) parallel-sided along most of its length, not lanceolate, with only 1–2 peg setae at apical margin on each side of the mid-apical incision......................................................................................................... 14 – Aedeagus, paramere not parallel-sided, with more or less lanceolate or rhomboid apical portion (fig. 191j, l in Assing and Schülke 2012); peg setae on parameral underside more numerous and arranged in irregular rows or groups. 15 14 Larger species with body length 8.9–9.7 mm; head distinctly wider than long (HL/HW ratio 0.7–0.8) with posterior frontal puncture situated in the middle of distance between posterior margin of eye and nuchal ridge. (Habitus and aedeagus as in Fig. 9A–D)...............................................................................................................................Q. solskyi – Smaller species with body length 8.5–9.4 mm; head from nearly as long as wide to longer that wide (HL/HW ratio 0.9–1.1) and posterior frontal puncture situated closer to posterior margin of eye than to nuchal ridge. Structure of the aedeagus unknown (for details see below)............................................................................................... Q. bucharensis 15 Pronotum with basalmost setiferous puncture of sublateral group (sometimes may be lost at one side) situated distinct ly behind the level of large lateral puncture (fig. 186a in Assing and Schülke 2012).................................................. 16 – Pronotum with punctures of sublateral group always situated before or at most at the same level as large lateral punc ture (fig. 186b in Assing and Schülke 2012)............................................................................................................. 17 16 Aedeagus, in parameral view: apical portion of the paramere lanceolate with bluntly pointed apical contour (fig. 191j, l in Assing and Schülke 2012)....................................................................................................................Q. ochripennis – Aedeagus, in parameral view: apical portion of the paramere not lanceolate, with broad and shallow apical emargination (fig. 191t, v in Assing and Schülke 2012)..................................................................................................Q. puncticollis 17 Pronotum with dorsal rows each with only two punctures. Aedeagus, underside of the paramere: peg setae arranged in four irregular groups: a pair of apical groups and a pair of subapical groups (fig. 11C in Coiffait, 1978)........Q. koltzei (externally Q. dez.pensoft.net Key to species of Quedius of Middle Asia rufilabris, an ambiguous species described from ‘Zeravshan’ also fits here; for details see the section on this species) – Pronotum with dorsal rows each with three punctures. Aedeagus, underside of the paramere: peg setae arranged only in two subapical groups, the pair of apical groups absent (fig. 7K–M in Coiffait 1978)............................... Q. tadjikiscus 18 Scutellum with setiferous punctation; eyes large and convex, occupying almost entire lateral side of head; rather small species. Body not longer than 6.0 mm. Aedeagus as in (Figs 19B–D, 21B, C)............................................................ 19 – Scutellum glabrous, without setiferous punctation; eyes smaller and more flat; temples more distinct. Body length varies but includes larger species. Aedeagus different......................................................................................................... 20 19 Aedeagus: paramere almost parallel-sided, only slightly narrowing in the middle portion, rows of peg setae long and regular (Fig. 19C) Habitus and aedeagus as in Fig. 19A–D..............................................................................Q. hauseri – Aedeagus: paramere not parallel-sided, strongly narrowing in the middle portion, rows of peg setae shorter and irregu lar (Fig. 23C). Habitus and aedeagus as in Fig. 23A–C.................................................................................Q. fulvicollis 20 Frons with two additional punctures between anterior frontal punctures. Rather small species, body not longer than 6.0 mm.....................................................................................................................................................Q. scintillans – Frons without punctures between anterior frontal punctures. Species varying in size................................................. 21 21 Elytra shortened, slightly shorter than, or at maximum, as long as pronotum. Brachypterous species without whitish apical seam on abdominal tergite VII (Fig. 3F). Aedeagus as in Fig. 11...............................................Q. pseudonigriceps – Elytra longer than, or at minimum, as long as pronotum. Species with whitish apical seam on abdominal tergite VII (Fig. 3C). Aedeagus different.................................................................................................................................... 22 125 Dtsch. Entomol. Z. 65 (2) 2018, 117–159 22 Relatively large species, body length 8.1–11.7 mm. Aedeagus: paramere shorter, its apex far from reaching apex of median lobe (in lateral view) with subapical tooth located far basad from its apex (Fig. 18B). Habitus and aedeagus as in Fig. 18A–C................................................................................................................................Q. sp. aff. Q. coloratus – Mostly smaller species, body length 5.0–7.5 mm. Aedeagus different........................................................................ 23 23 Aedeagus: median lobe (in lateral view) distinctly curved; multiple sensory peg setae on the underside of the paramere arranged in one or two irregular longitudinal groups, never in clear straight rows along parameral margins (e.g., Fig. 17). Larger species 6.0–8.0 mm...................................................................................................................................... 24 – Aedeagus, in lateral view: median lobe straight, not curved dorso-ventrally (fig. 193r–t in Assing and Schülke 2012). Smaller species, body not longer than 6.5 mm......................................................................................................... Quedius (s. str.) curtipennis Bernhauer, 1908 Quedius fuliginosus var. curtipennis Bernhauer, 1908, 335 (original description) Quedius curtipennis: Herman 2001, 3134 (summary of lit erature); Assing and Schülke 2012, 457, 458 (diagno sis, distribution and bionomics, aedeagus illustration). Type material examined. Syntypes (all in FMNH): Faroe Islands: 1 ♂, “Suderö Faroer Ins./ Dr. Cornu 1907/ v. curti pennis Brh. Typus [handwritten]/fuliginosus Grav. Scheerp. [handwritten]/ Chicago NHMus M. Bernhauer Collection [printed]/ D. Drugmand det. 1994 Quedius (s. str.) curtipen nis Brnh. [preprinted handwritten]”; 1 ♂, “Nördl. Faroer Ins./ Dr. Cornu 1907/ v. curtipennis Brh. Typus [handwrit ten]/ fuliginosor Scheerp. [sic!] det. [illegible] [handwrit ten]/ Chicago NHMus M. Bernhauer Collection [printed]/ D. Drugmand det. 1994 Quedius (s. str.) curtipennis Brnh. [preprinted handwritten]”; Uzbekistan: 1 ♂, “v. curtipennis Buchara Bang Haas det. Bernh. [preprinted handwritten]/ Chicago NHMus M. Bernhauer Collection [printed]”. Key to species of Quedius of Middle Asia 25 24 Body brown to dark brown, sometimes elytra paler or reddish. Larger (body length 6.0–8.0 mm) and more robust spe cies (Fig. 4B). Aedeagus (fig. 194j–l in Assing and Schülke 2012): median lobe in lateral view with subapical tooth situ ated close to its apex (fig. 194k in Assing and Schülke 2012); underside of paramere with sensory peg setae arranged in wide irregular rows diverging from each other basad................................................................................Q. umbrinus – Body light-brown to brown, but never black, elytra brownish; larger (body length 6.0–7.0 mm) and more slender species (Figs 3E, 16A). Aedeagus (Figs 16B, C, 17): median lobe in lateral view with subapical tooth situated far basad from its apex; underside of paramere with ca. 40–50 peg setae arranged in one irregular median row...........................Q. novus 25 Aedeagus (fig. 193r–t in Assing and Schülke 2012): underside of paramere with ca. 4–5 sensory peg setae in each of two rows along its lateral margins. Habitus as in Fig. 3D...............................................................................Q. limbatus – Aedeagus (Figs 12C, D, 15): underside of paramere with many more (ca. 8–18) sensory peg setae in each of two rows along its lateral margins........................................................................................................................................... 26 26 Aedeagus (Figs 15, 14B, C, F, G): median lobe in lateral view with subapical tooth located distinctly more basad from its apex (Figs 14B, F; 15); underside of paramere with sensory peg arranged in two regular rows very close to each other (Figs 14C, G, 15)........................................................................................................................................... Q. imitator – Aedeagus (Figs 12C, D, 13B, C): median lobe in lateral view with subapical tooth located close to its apex (Figs 12C, 13B); underside of paramere with sensory peg setae arranged in two irregular rows more remote from each other (Figs 12D, 13C)...........................................................................................................................................Q. cohaesus Subgenus Quedius Stephens, 1829 mon widespread species since its original description. We have examined three male syntypes of Q. curtipennis kept at the FMNH, two from Faroe Islands and one from ‘Bu chara’ in Uzbekistan, all listed above. Our examination of the syntypes confirms that they are conspecific and match ing current interpretation of this species (e.g. in Assing and Schülke 2012). Quedius curtipennis is a common species widely distributed in the forests and humid microhabitats of the open landscapes of the Western Palaearctic (Herman 2001; Assing and Schülke 2012). Because of the strong morphological similarity, Q. curtipennis can be easily confused with Q. fuliginosus. As a result, current broad distributions for both species as recently summarized in Assing and Schülke (2012), especially outside Europe, need revision. A male syntype of Q. curtipennis from “Bu chara” (Uzbekistan) collected more than a century ago (see below) and overlooked in the subsequent literature is the only specimen of this species known from Middle Asia. Since the original description Q. curtipennis has not been recorded from any of the countries of Middle Asia. dez.pensoft.net Quedius (s. str.) fuliginosus Gravenhorst, 1802 Fig. 2A Comments on taxonomy, distribution and bionom ics. Bernhauer (1908) described Q. curtipennis as a variety of Q. fuliginosus without clear information on the type ma terial. In addition to the morphological diagnosis of a new variety Bernhauer (1908) mentioned that it is common on the Faroe Islands and also occurs in “Vorarlberg, Buchara and Böhmen (Wrana. Moldau)”. Interestingly, revision of the type material has never been published for this com Quedius fuliginosus: Herman 2001, 3155 (summary of lit erature); Assing and Schülke 2012, 457, 458 (diagno sis, distribution and bionomics, aedeagus illustration). Material examined. Kazakhstan: 1 ♂, Akshatau Mt., NW Ayaguz, Semipalat, forest leaf litter, 17.VII.1962, Material examined. Kazakhstan: 1 ♂, Akshatau Mt., NW Ayaguz, Semipalat, forest leaf litter, 17.VII.1962, dez.pensoft.net dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 126 Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia 126 Figure 2. Habitus of Quedius recorded in Middle Asia. A Q. fuliginosus (photo Lech Borowiec) B Q. sundukovi C Q. vicinus D koltzei E Q. mutilatus F Q. ochripennis. Scale bars: 1 mm. Figure 2 Habitus of Quedius recorded in Middle Asia A Q fuliginosus (photo Lech Borowiec) B Q sundukovi C Figure 2. Habitus of Quedius recorded in Middle Asia. A Q. fuliginosus (photo Lech Borowiec) B Q. sundukovi C Q. vicinus D Q. koltzei E Q. mutilatus F Q. ochripennis. Scale bars: 1 mm. dez.pensoft.net Dtsch. Entomol. Z. 65 (2) 2018, 117–159 127 Figure 3. Habitus of Quedius recorded in Middle Asia. A Q. puncticollis (photo http://danbiller.dk) B Q. hauseri C Q. imitator D Q. limbatus (photo Lech Borowiec) E Q. novus F Q. pseudonigriceps. Scale bars: 1 mm. Figure 3. Habitus of Quedius recorded in Middle Asia. A Q. puncticollis (photo http://danbiller.dk) B Q. hauseri C Q. imitator D Q. limbatus (photo Lech Borowiec) E Q. novus F Q. pseudonigriceps. Scale bars: 1 mm. in Middle Asia A Q puncticollis (photo http 3. Habitus of Quedius recorded in Middle Asia A Q puncticollis (photo http Figure 3. Habitus of Quedius recorded in Middle Asia. A Q. puncticollis (photo http://danbiller.dk) B Q. hauseri C Q. imitator D Q. limbatus (photo Lech Borowiec) E Q. novus F Q. pseudonigriceps. Scale bars: 1 mm. Figure 3. Habitus of Quedius recorded in Middle Asia. A Q. puncticollis (photo http://danbiller.dk) B Q. hauseri C Q. imitator D Q. dez.pensoft.net Quedius (s. str.) fuliginosus Gravenhorst, 1802 Fig. 2A Nesseby h:d, Nyborg, 35483, 04–09. VI.1963, Gom. Israelson leg.; Sweden: 1 ♀, Nb. [Norbot ten] Kihlangi, 10–17.VI.1947, T. Palm leg.; 1 ♀, Vittangi, 02–14.VIII.1963, Th. Palm leg.; 1 ♀, L. Lpm, Vittanger, 08.VI.1968, S. Lundberg leg.; 1 ♂, Lu. Lpm. Messaure, 09–16.VII.1973, K. Muller leg.; 2 ♂, 1 ♀, Jokkmokk, 21.V.1965, T.B. Engelmark leg. (ZMLU). Figure 4. Habitus of Quedius recorded in Middle Asia. A, Q. scintillans; B, Q. umbrinus (Lech Borowiec). Scale bars: 1 mm. L.V. Arnoldi leg. (ZIN); 2 ♂ Stepnyak, Zhukey Lake, 10.VII.2002, V.A. Kastcheev leg.; 1 ♂, Ivanovsky Mt. Ridge, 32 km S Leninogorsk, 1300 m a.s.l. 14.VIII.1986, I.I. Kabak leg. (ZIN); Uzbekistan: 1 ♂, 1 ♀, Tashkent, near railway station, plant residues, 24.V.1986, S.A. Kur batov leg. (cKur). Comments on taxonomy, distribution and bionom ics. Similarly to Q. curtipennis (see above), Q. fuligino sus is a widespread and common species in the Western Palaearctic, and subject of numerous publications. The most recent summary of its diagnostic characters, bion omics and distribution can be found in Assing and Schül ke (2012). For the same reasons as Q. curtipennis above, the exact distribution of Q. fuliginosus needs careful revi sion. Limited material from Kazakhstan and Uzbekistan examined here represents the first records of this species from Middle Asia. Comments on taxonomy and type material. The original description of Quedius altaicus was based on two female specimens (a holotype and a paratype) from “Central-Altai” without precise record of the type locali ty (Korge, 1962). Such ambiguity was stressed by Korge who noted that the status of Q. altaicus, which external ly appeared very similar to Q. unicolor and Q. subuni color, should be confirmed by the examination of male genitalia. Toleutaev (2014) recorded Q. altaicus from Saur Mountains (Eastern Kazakhstan), but that record needs verification. Quedius (s. str.) fuliginosus Gravenhorst, 1802 Fig. 2A limbatus (photo Lech Borowiec) E Q. novus F Q. pseudonigriceps. Scale bars: 1 mm. dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 128 Figure 4. Habitus of Quedius recorded in Middle Asia. A, Q. scintillans; B, Q. umbrinus (Lech Borowiec). Scale bars: 1 mm. graphed Kelsey Keaton 2014 Emu Catalog/ FMNHINS 2819427 Field Museum [printed]” (FMNH); Additional material. Kazakhstan: 1 ♂, West Al tai, Ivanovsky Mt. Ridge, 32 km Leninogorsk [Rid der], 14.VIII.1986, 1300 m a.s.l., I.I. Kabak leg. (ZIN); 1 ♂, SW Altai, E Narymsky Mt. Ridge, upper reach es of Shurshutsu River [Forpostnaya], lower forest zone, 21.VII.1997, 1300 m a.s.l., R.Yu. Dudko, V.K. Zinchenko leg.; 2 ♂, 1 ♀, SW Altai, E of Markakol Lake, Urunhayka, ground traps, 21.VI–07.VII.1997, 1500 m a.s.l., R.Yu. Dudko, V.K. Zinchenkо leg.; 1 ♂, same locality and collectors, but 8 km ESE Matabai, north slope of Matabai Mt. Ridge, forest, 10.VII.1997, 1600–2000 m a.s.l, (NHMD); 1 ♂, Manrak Mt. Ridge, 12 km Priozerny [Tugil], 16.VII.1986, I.I. Kabak leg. (ZIN); Russia: 1 ♂, Altai, Listvyaga Mt. Ridge, 10 km SSE Tesninskiy Belok Mt., Seredchiha River, forest, 27.VII.1997, 1200–1500 m a.s.l., R.Yu. Dudko, V.K. Zinchenko leg. (NHMD). Comparative material on Quedius subunicolor. Type material: Paratypes: Sweden: 1 ♂, “Häggenäs s-n Jtl. T. Palm 4–8, 8 1945 [printed]/ det. H. Korge Que dius subunicolor Korge [printed]/ Paratypus subunicolor Korge [pre-printed]/ Quedius subunicolor Korge [hand written]/ Type no. 1202:2 MZLU [printed]/ 2016 189 MZLU [printed]” (ZMLU); 3 ♀, same data, but two last labels as “Type no: 1202:3 MZLU/ 2016 190 MZLU [printed]” (Fig. 5D) (ZMLU); Additional material: Norway: 3 ♀, Fn. Nesseby h:d, Nyborg, 35483, 04–09. VI.1963, Gom. Israelson leg.; Sweden: 1 ♀, Nb. [Norbot ten] Kihlangi, 10–17.VI.1947, T. Palm leg.; 1 ♀, Vittangi, 02–14.VIII.1963, Th. Palm leg.; 1 ♀, L. Lpm, Vittanger, 08.VI.1968, S. Lundberg leg.; 1 ♂, Lu. Lpm. Messaure, 09–16.VII.1973, K. Muller leg.; 2 ♂, 1 ♀, Jokkmokk, 21.V.1965, T.B. Engelmark leg. (ZMLU). Comparative material on Quedius subunicolor. Type material: Paratypes: Sweden: 1 ♂, “Häggenäs s-n Jtl. T. Palm 4–8, 8 1945 [printed]/ det. H. Korge Que dius subunicolor Korge [printed]/ Paratypus subunicolor Korge [pre-printed]/ Quedius subunicolor Korge [hand written]/ Type no. 1202:2 MZLU [printed]/ 2016 189 MZLU [printed]” (ZMLU); 3 ♀, same data, but two last labels as “Type no: 1202:3 MZLU/ 2016 190 MZLU [printed]” (Fig. 5D) (ZMLU); Additional material: Norway: 3 ♀, Fn. Quedius (s. str.) altaicus Korge, 1962 Figs 5, 6 In spite of the ambiguous original description of Q. al taicus, new material from Altai including males exam ined here for the first time can be safely attributed to that species. This material perfectly matches Korge’s original description, and the information together with high qual ity photos of the holotype available from the Field Muse um online beetle type database (FMNH, 2018). Besides, there are no other species in the Altai region that could be misidentified as Q. altaicus. Quedus sundukovi, the only other similar species distributed from the Russian Far East to the South-Western Altai is distinctly different (for details see below). Quedius altaicus Korge, 1962, 152 (original description); Coiffait 1978 (characters), 194; Toleutaev 2014, 44 (distribution records). Material examined. Holotype: Russia: female, “Ze ntral-Altai, lg. Leder, det. Bang-Haag [handwritten]/ unicolor Kies. det. Bernhauer [handwritten]/ ♀ Holoty pus Quedius s. str. altaicus H. Korge [printed]/ Chicago NHMus M. Bernhauer Collection/ Holotype teste D.J. Clarke 2014 GDI Imaging Project [printed]/ Photo dez.pensoft.net 129 Dtsch. Entomol. Z. 65 (2) 2018, 117–159 Figure 5. Quedius subunicolor (paratype, male): A–D; Quedius altaicus (male): E–G. A, E, habitus; B, C, F, G, aedeagus. D, labels. B, F, median lobe, lateral view. C, G, paramere, underside; H, sclerite of internal sac. Scale bars: 1 mm. Figure 5. Quedius subunicolor (paratype, male): A–D; Quedius altaicus (male): E–G. A, E, habitus; B, C, F, G, aedeagus. D, labels. B, F, median lobe, lateral view. C, G, paramere, underside; H, sclerite of internal sac. Scale bars: 1 mm. The aedeagus of Q. altaicus (Fig. 5F, G) here exam ined for the first time is nearly identical with the aedea gus of the northern European Q. subunicolor (Fig. 5B, C). Both species slightly differ from each other in the shape of a large sclerite in the internal sac (labeled as H in Fig. 5B, F) and the degree of development of the subapical teeth of the median lobe (less pronounced in Q. altaicus, compare Fig. 5B, F). Comparison of the external morphology of the multiple specimens of Q. altaicus to each other and with the available specimens of Q. subunicolor, including its paratypes, demonstrates that the external characters provided by Korge (1962) as unique for Q. altaicus (microstructure of the head, proportions of the pronotum, chaetotaxy of the head and pronotum) do not hold. dez.pensoft.net Quedius (s. str.) altaicus Korge, 1962 Figs 5, 6 Given a subtle morphological difference between both species and poorly sampled ar eas of Russia, there remains a possibility that Q. subuni color may be a polytypic species continuously distribut ed from the Northern Europe to Altai. Or, Q. subunicolor and Q. altaicus may be a hitherto unrecorded case of the boreo-montane distribution. Both species should be subject to further sampling in the area which seems as a distribution gap between them. Also, a DNA-based phy logeographic investigation would be interesting. Below we provide a redescription of Q. altaicus. Body piceous black, only sometimes dark brownish; apical margin of abdominal segments vaguely paler; maxillary, labial palpi, and antennae dark-reddish; legs dark with paler brownish tarsi (Fig. 5E). Head with broadly rounded, but distinct hind angles with microsculpture consisting of transverse waves; eyes as a long as or slightly longer than tempora; posterior frontal puncture situated closer to posterior margin of head than to posteromedial margin of eye; two to four additional punctures present along medial margin of eye between anterior and posterior frontal punctures; tempo ral puncture situated close to posterior margin of eye at distance nearly equal to diameter of puncture. Antennae moderately long, segment 3 somewhat lon ger than 2, segments 4–8 longer than wide, each gradual ly becoming shorter towards apex, segments 7–11 about as long as wide. Pronotum wider than long PL/PW: 0.9–1.0 (1.0), widest at posterior third, narrowed anteriad; hind angles broadly rounded, but distinct; dorsal rows each with three punctures; sublateral rows each with two to three punc tures; waves of microsculpture transverse, similar to that on head. Scutellum finely punctured in its posterior half, with transverse or slightly isodiametric microsculpture. Elytra parallel-sided, as long as pronotum, at base nar rower than pronotum at widest point; shiny, punctation moderately dense and shallow, interspaces larger than di ameter of punctures, pubescence yellowish-grey.i Redescription. Measurements and ratios (range, arithmetic mean; n = 8): HL: 1.4–1.5 (1.5); HW: 1.4–1.5 (1.5); PL: 1.7–1.8 (1.8); PW: 1.9–2.0 (2.0); EL: 1.7–1.8 (1.8); EW: 1.8–2.0 (1.9); FB: 5.0–5.2 (5.1); TL: 8.6–11.4 (10.0); HL/HW: 0.9–1.0 (1.00); PL/PW: 0.9–1.0 (1.0); EL/EW: 0.9–1.1 (1.0). Abdomen with tergite VII (5th visible) with fine dis tinct whitish apical seam of palisade fringe; punctation dense and fine gradually becoming sparser towards apex dez.pensoft.net dez.pensoft.net 130 Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... Figure 6. Quedius (s. str.) altaicus Korge, 1962 Figs 5, 6 meridiocarpathicus) and in the shape of the medial sclerite of the internal sac that has rounded apex. Some authors stressed a strong similarity of Q. subunicolor (from which Q. altaicus is hardly distinct) with Q. unicol or, and the latter mainly Central European montane spe cies was incorrectly cited as Q. subunicolor in a number of the faunistic papers (e.g., Ciceroni and Zanetti 1995; Geiser et al. 2003; Boháč et al. 2004, 2005; Wojas 2006). Quedius subunicolor (and Q. altaicus), however, can be easily distinguished from Q. unicolor by transversal (not Bionomics. All clearly georeferenced specimens of Q. altaicus have been collected at the elevations between 1200 and 2000 m. Quedius (s. str.) altaicus Korge, 1962 Figs 5, 6 Distribution of sister species Quedius altaicus (Altai Mts. in Kazakhstan and Russia) and Q. subunicolor (Northern Europe). Dots indicate material examined in this paper, colored areas summarise literature data. Black dots indicate type localities. Figure 6. Distribution of sister species Quedius altaicus (Altai Mts. in Kazakhstan and Russia) and Q. subunicolor (Northern Europe). Dots indicate material examined in this paper, colored areas summarise literature data. Black dots indicate type localities. of abdomen, surface between punctures with very super ficial transverse irregularities, pubescence as on elytra. isodimetric) microsculpture of the frons and the structure of the aedeagus, especially by the internal sac with the obvious medial sclerite. From similar species that occur in Middle Asia Q. altaicus can be easily distinguished by the following characters: from Q. fuliginosus by the punctured (setose) scutellum and absence of addition al punctures between anterior frontal punctures; from Q. sundukovi by normally developed elytra (very short in distinctly brachypterous in Q. sundukovi), presence of fine whitish apical seam of palisade fringe on VII tergite (5th visible), and distinctly larger body. Male: aedeagus: median lobe with acute apex and small teeth on its parameral side near apex (Fig. 5B, F); paramere distinctly protruding over apex of median lobe, with two pairs of setae apically and two pairs of longer setae laterally below apex, its underside with numer ous sensory peg setae forming two subapical longitudi nal rows connected near apex (Fig. 5C, G). Internal sac (examined in situ) with two pairs of strongly sclerotized microstructures positioned laterally and one characteristi cally shaped medial sclerite (Fig. 5H) with rounded apex. Distribution. Quedius altaicus is known from “cen tral” (Korge, 1962) and southwestern Altai. Records from the southwestern Altai stretching across the border be tween Russia and Kazakhstan, provided here, are the first exact distributional data for this subspecies. We were not able to examine the material on which Toleutaev (2014) recorded this species from Saur Mountains, the latter re cords remains ambiguous. Comparison. Based on the structure of the aedeagus, especially the characteristic armature of the internal sac with the large middle sclerite ‘H’ (Fig. 5B, F; fig 189 in Assing and Schülke, 2012), Q. altaicus can be placed in the group with Q. subunicolor, Q. balticus, Q. molochi nus and Q. meridiocarpathicus. Quedius altaicus differs from Q. meridiocarpathicus in the unicolorus black col oration of the body (brown reddish with paler elytra in Q. dez.pensoft.net Quedius (s. str.) sundukovi Smetana, 2003 Fig. 2B Quedius sundukovi Smetana, 2003, 189 Quedius sundukovi Smetana, 2003, 189 Material examined. Kazakhstan: 1 ♂, SW Altai, East of Narymskij Mt. Ridge, upper course of Ozernaja River, dez.pensoft.net 131 Dtsch. Entomol. Z. 65 (2) 2018, 117–159 Lobanov leg.; 3 ♂, Kopetdag, Firjusa, 07.V.1968, G.S. Medvedev leg. (ZIN); 1 ♂, Kara-Kala, 29.IV–03.V.1989, S. Bečvář leg. (cSch). subalpine zone, 1900–2300 m a.s.l, 18.VII.1997, R.Yu. Dudko and V.K. Zinchenko leg. (NHMD); 3 ♂, 3 ♀, Sta novoe nagorje [highland], S part of Kodar Mt. Ridge, up per course of Chara River, 50 km WSW of village Novaja Chara, 1700–2000 m a.s.l., 26–27,VII.1995, A.Yu. and R.Yu. Dudko, and D.E. Lomakin leg (NHMD, ZIN); 1 ♀, same locality and collectors, but environs of lake Bolshoe Leprindo, 1000 m a.s.l., 23.VII.1995 (ZIN). subalpine zone, 1900–2300 m a.s.l, 18.VII.1997, R.Yu. Dudko and V.K. Zinchenko leg. (NHMD); 3 ♂, 3 ♀, Sta novoe nagorje [highland], S part of Kodar Mt. Ridge, up per course of Chara River, 50 km WSW of village Novaja Chara, 1700–2000 m a.s.l., 26–27,VII.1995, A.Yu. and R.Yu. Dudko, and D.E. Lomakin leg (NHMD, ZIN); 1 ♀, same locality and collectors, but environs of lake Bolshoe Leprindo, 1000 m a.s.l., 23.VII.1995 (ZIN). Comments on taxonomy, distribution and bionom ics. Gusarov (1993) clarified the identity and synonymy of Q. vicinus. A description and the illustrations of the aedeagus for this species can be found, for example, in Coiffait (1954, 1978, as Q. libanicus), Boháč (1988, as Q. libanicus), and Hachikov (2003). By the brown body with reddish elytra, frons without additional setiferous punc tures between anterior frontal punctures and by punctate scutellum, Q. vicinus can be distinguished from all other Middle Asian species of the nominal subgenus Quedius s. str. based on the external characters alone. The aedea gus of this species, with the parameral apex pointing out wards from the median lobe, is also very characteristic. Comments on taxonomy, distribution and bion omics. Quedius sundukovi was known from the Russian Far East (Smetana, 2003) and from Irkutsk Province and Zabaikalsky Territory (Smetana and Shavrin 2018). From the newly examined material it has become clear that Q. sundukovi is distributed even wider: from the Russian Far East through southern Siberia to Altai Mountains in Northeastern Kazakhstan. In the material examined we here provide only new records for Middle Asia, because the detailed documentation of its entire distribution will be published elsewhere. Quedius (Microsaurus) ochripennis Ménétriés, 1832 Fig. 2F Herman 2001, 3227 (summary of literature); Kascheev 2001, 102 (records); Assing and Schülke 2012, 466, 467 (diagnosis, distribution and bionomics, aedeagus illustration). Subgenus Microsaurus Dejean, 1833 All hitherto known specimens of Q. sundukovi were collected by pitfall traps (Smetana 2003). Based on the newly examined material here, Q. sundukovi inhabits ta lus-associated debris. Also it is found in regular leaf litter and moss on the ground. Quedius (s. str.) sundukovi Smetana, 2003 Fig. 2B Based on literature data it is a common species in Western Asia. In Middle Asia it was known only from Turkmenistan (Boháč 1988, as Q. libanicus). Based on the newly examined material Q. vicinus is recorded for the first time from southern Kazakhstan (Karatau Moun tains). The species prefers ground based debris and leaf litter, but also was found (Coiffait 1955, as Q. libanicus) in caves. Specimens from Middle Asia were collected along rivers, in ground traps and at light. Detailed description and illustration of the species is available in Smetana (2003). Quedius sundukovi is one of the smallest species in the nominative subgenus Que dius s. str. and the smallest in this subgenus in the fauna of Middle Asia. Additionally, it stands out from all other Quedius s. str. species in Middle Asia as the only distinct ly brachypterous species, with very short elytra and lack ing whitish apical seam on abdominal tergite VII. Quedius (Microsaurus) capitalis Eppelsheim, 1892 Fig. 7 Fig. 7 Quedius capitalis Eppelsheim 1892, 329 (original de scription); Gridelli 1924, 40 (characters); Coiffait 1978, 148 (characters and illustration of aedeagus); Kadyrov et al. 2014a, 31 and 2014b, 49 (distributional records). Comments on taxonomy, distribution and bionom ics. The diagnostic characters including illustrations of the aedeagus and the most recent summary of the bion omic and distribution data of this widespread and rath er common Western Palaearctic species can be found in Assing and Schülke (2012). From similar Middle Asian species Q. capitalis and Q. fusicornis, Q. ochripennis is distinguished by the larger body and shape of the aedea gus. From the larger Q. solskyi it can be safely distin guished by the characters of aedeagus. Material examined. Type material: Uzbekistan: Syn types: 1 ♂, “♂/ c.Epplsh. Steind. d. [printed]/ Qu. capi talis Epp. Type Taschkent, Leder [handwritten]/ Typus”; 1 ♂, “♂/ capitalis Epp. Taschkent Leder. [handwritten]/ Typus” (Fig. 7E, F) (NMW). Additional material. Uzbekistan: 2 ♀, Tien Shan, Aktasch, near Taschkent, 2000 m a.s.l., 13.VII.1984, D.W. Wrase leg. (cSch); Kazakhstan: 3 ♂, Karatau Mts, Khantagi River, 570 m a.s.l., 43°33’32.4N, 68°40’52.7E, 25.VI.2011, V.A. Kastcheev leg. (ZIN); Tajikistan: 3 ♂, Mountains near Kuljab, 1500–2000 m a.s.l., 20.VI.1963, A.V. Bogachev leg. (ZMMU). Quedius ochripennis is widely distributed in Europe and in the Mediterranean region. It is also known from Simla Hills in Himalaya, India (Smetana, 1988) and from Middle Asia where, based on earlier records (Ta ble 1) and material examined here, it occurs in south western Turkmenistan, entire territory of Tajikistan, and southern Uzbekistan. Comments on taxonomy and type material. In the original description, Eppelsheim (1892) mentioned mor phological characters of both males and females and stated that the species was known to him from a few specimens from ‘’Tashkent’’. As we learn from the introduction in his paper, specimens were collected by Hans Leder. Both male specimens from NMW labeled as “types” of Q. cap italis originally come from Eppelsheim’s collection and their morphology and label information fit the original description; therefore they are syntypes. Quedius ochripennis inhabits various ground based debris, often associated with decaying wood, also in nests of mammals, ants and wasps (Assing and Schülke 2012). Based on the material examined here, in Middle Asia Q. ochripennis prefers humid plant debris usually near water bodies, also it was found in caves and in tree foliage in an aphid nest. Quedius (s. str.) vicinus Ménétriés, 1832 Fig. 2C Material examined. Kasakhstan: 1 ♂, Karatau Mts, Byzhi River, Rynagus stream, 24.VII.2010, V.A. Kast cheev leg.; 1 ♂, Karatau Mts, near stream, 11.VII.2010, 42°53’41.42N, 70°42’56.6E, 600 m a.s.l., V.A. Kast cheev leg.; 1 ♂, 1 ♀, Aksu-Dzhabagly, Taldy-Bulak River, 10–20.IV.1979, B.V. Iskakov leg.; 2 ♂, same locality and collector, but, 04.V.1986; 1 ♂, 1 ♀, Ak su-Dzhabagly, Ulken-Kaindy, near water in moss, 18.VII.1986, B.V. Iskakov leg.; 1 ♂, S Kazakhstan, Boralday, 12–15.VI.1983, B.V. Iskakov leg.; 1 ♂, Zala tayskiy Alatau Mts, Krasnogorka [Sulutor], near stream, under tree, 75°13’50.4N, 43°23’45.7E, 28.VII.2010, V.A. Kastcheev leg.; Uzbekistan: 1 ♂, 1 ♀, Aruk-Tau Mt. Ridge, 25 km W Kyzyl-Kala, 04.IV.1966, O.L. Kry zhanovsky leg. (ZIN); 1 ♂, 1 ♀, Tashkent, near rail way station, plant debris, 24.V.1986, S.A. Kurbatov leg. (cKur); 1 ♂, Samarkand, Agalyk, 18.X.1935, Y.D. Kirschenblat leg.; 4 ♂, 4 ♀, Aman Kutan, 12.VI–06. VII.1932, V.V. Gussakovsky leg. (ZIN); 1 ♂, Yakka bag, Convulvulus sp. and thorny bushes, 02.XII.1941, K.V. Arnoldi leg. (cRyv); 1 ♂, 1 ♀, Yakkabag, hills S of the town, ravine in forest, cave, 30.XI.1941, K.V. Quedius vicinus Ménétriés, 1832, 144 (original descrip tion); Faldermann 1835, 129 (distribution records); Gusarov 1993, 73 (lectotype designation, = Q. liban icus Coiffait); Assing and Wunderle 2001, 37 (distri bution records); Hachikov 2003, 46 (illustration of ae deagus); Ghahari 2009, 2012, 5; Assing and Feldmann 2012; Özgen et al. 2016, 621.f Quedius libanicus Coiffait, 1954, 160 (original descrip tion); 1955, 427 (notes); 1978, 195 (characters); Jar rige 1971, 497; Korge 1971, 11; Boháč 1988, 554 [= 1989: 38] (characters, distribution records). Material examined. Kazakhstan: 1 ♂, 1 ♀, Karatau Mts, Khantagi River, 570 m a.s.l., 43°33’32.4N, 68°40’52.7E, 25.VI.2011, V.A. Kastcheev leg. (ZIN); Turkmenistan: 2 ♀, N Kopetdag, Firjusa-Cleft near Aschabad, 07.V.1989, D.W. Wrase leg. (cSch); 4 ♂, Firjusa, 25.V.1903, K.O. Anger, A. Jakovleva leg. (ZIN); 4 ♂, 2 ♂, Kopetdag, Chili River valley, 23–24.VIII.1935, L.V. Arnoldi leg.; 1 ♂, W Kopetdag, Kara-Kala [Garrygala], canyon Porchai, to light, village of the Nature Reserve, 05.IX.1986, A.L. dez.pensoft.net 132 Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... Material examined. Tajikistan: 3 ♂, 1 ♀, Pamir-Alai, Hisaar Mts, Adshuk-Cleft near Warsob, Bachufer, 01–03. VII.1990, M. Schülke leg. (cSch). Material examined. Tajikistan: 3 ♂, 1 ♀, Pamir-Alai, Hisaar Mts, Adshuk-Cleft near Warsob, Bachufer, 01–03. VII.1990, M. Schülke leg. (cSch). Arnoldi leg. (cRyv); 1 ♂, Ishkent, Kashkadarya River, 24.X.1947, K.V. Arnoldi leg. Quedius (s. str.) vicinus Ménétriés, 1832 Fig. 2C (ZMMU); Kyrgyzstan: 1 ♂, Kyrgyz-Alatoo Mts, 09.VII.2010, 72°28’38.6N, 42°48’49.2E, V.A. Kastcheev leg.; 1 ♂, 2 ♀, S Fergak skiy Mt. Ridge, Ak-Terek, 31.VIII–20.IX.1937, A.N. Kirichenko leg. (ZIN); 1 ♂, Kara-Alma, Ferganskiy Mt. Ridge, 26.VI.1945, K.V. Arnoldi leg. (ZMMU); Turkmenistan: 2 ♂, 3 ♀, Kopetdag Mts, 22.VI.1953, Ployvanova leg.; 5 ♂, 4 ♀, E Kopetdag Mts, Sunt Mt., in Ulmus sp. leaves rolled by aphids, 22–24.VI.1953, O.L. Kryzhanovsky leg.; same locality, but 1 ♂, Ul mus sp., Odyncova leg. (ZIN); 1 ♀, N Kopetdag Mts, Firjusa-Cleft, near Ashchabad, 07.V.1989, D.W. Wrase leg. (cSch); 3 ♂, 3 ♀, W Kopetdag Mts, N Karakala, 28.IX.1989, A.V. Puchkov (cSch); Tajikistan: 3 ♂, 2 ♀, 20 km S Danghara, 08.V.1962, Guryeva leg.; 1 ♂, Vakhsh Mt. Ridge, 10 km N Kangurt, 08.V.1970, G.S. Medvedev leg. (ZIN); 1 ♂, Pamir-Alai, Hisaar Mts, Adshuk-Cleft, near Warsob, 1200 m a.s.l., 01–03. VII.1990, M. Schülke & D.W. Wrase leg. (cSch); 1 ♂, 2 ♀, Gazimalyk Mt. Ridge, 15 km W Ganjin, 2000 m a.s.l, 17.V.1970, G.S. Medvedev leg. (ZIN); 2 ♂, Moun tains near Kuljab, 1500–2000 m a.s.l., 20.VI.1963, A.V. Bogachev leg. (ZMMU). Comments on taxonomy, distribution and bionom ics. Quedius puncticollis is widely distributed in North ern, Central and Eastern Europe for which the latest sum mary of diagnostic characters, distribution and biology can be found in Assing and Schülke (2012). It is most similar to Q. ochripennis from which it can be easily dis tinguished by the apically not lanceolate paramere with a broad and shallow apical emargination. Quedis puncticollis is commonly found in the burrows of small mammals, especially moles, also in bee and wasp nests (Assing and Schülke 2012). Based on literature data (Table 1) and material examined here, there are only few records of Q. puncticollis in Middle Asia: from southern Kazakhstan and southern Tajikistan. Quedius (Microsaurus) capitalis Eppelsheim, 1892 Fig. 7 In the mountains it was recorded at elevations up to 2000 m. We have examined aedeagi of both syntypes and con firm they are conspecific. Eppelsheim (1892) compared Q. capitalis with Q. ragusai Eppelsheim, 1889. Gridelli (1924), based on the examination of a syntype, provided additional morphological details for the species includ ing verbal description of its aedeagus (but no illustration) and placed it near Q. ochripennis. Based on a syntype male, Coiffait (1978) again redescribed this species and Herman 2001, 3249 (summary of literature); Kascheev 2001, 102 (distribution records); Assing and Schülke 2012, 466, 467 (diagnosis, distribution and bionomics, aedeagus illustration). Quedius (Microsaurus) puncticollis Thomson, 1867 Fig. 3A Herman 2001, 3249 (summary of literature); Kascheev 2001, 102 (distribution records); Assing and Schülke 2012, 466, 467 (diagnosis, distribution and bionomics, aedeagus illustration). dez.pensoft.net 133 Dtsch. Entomol. Z. 65 (2) 2018, 117–159 Figure 7. Quedius capitalis, syntypes, males. A, B, habitus; C, D, aedeagus of the syntype in the photo A. E (of the syntype in the photo A), F (of the syntype in the photo B), labels. C, median lobe, lateral view; D, paramere, underside. Scale bars: A, B = 1 mm; C = 0.5 mm. Figure 7. Quedius capitalis, syntypes, males. A, B, habitus; C, D, aedeagus of the syntype in the photo A. E (of the syntype in the photo A), F (of the syntype in the photo B), labels. C, median lobe, lateral view; D, paramere, underside. Scale bars: A, B = 1 mm; C = 0.5 mm. Antennae moderately long, antennal segments: 3rd longer than 2nd, 4th–10th gradually widening towards apex of antenna. Antennae moderately long, antennal segments: 3rd longer than 2nd, 4th–10th gradually widening towards apex of antenna. provided its first and hitherto the only available illustra tion of the aedeagus. Our examination of the syntypes of Q. capitalis confirms the correct identification of this species by both Gridelli (1924) and Coiffait (1978). In Schülke and Smetana (2015) Q. capitalis was erroneous ly placed in the subgenus Raphirus. Here we redescribe this poorly known species and provide further data on its distribution. Pronotum slightly wider than long, widest at about mid dle to posterior third; hind angles rounded but distinct; dorsal and sublateral rows each with three punctures; mi crosculpture with transverse waves as on posterior part of head. Scutellum impunctate with microsculpture slightly coarser than on pronotum. Elytra parallel-sided, slightly longer than wide, longer than pronotum, their punctation dense, interspaces shiny with distinct minute irregularities.i Redescription. Measurements and ratios (range, arith metic mean; n = 10): HL: 0.8–1.3 (1.0); HW: 0.8–1.5 (1.1); PL: 0.9–1.6 (1.3); PW: 1.1–1.8 (1.4); EL: 1.2–2.0 (1.6); EW: 1.2–1.9 (1.5); FB: 2.9–4.7 (3.9); TL: 6.5–9.3 (7.8); HL/HW: 0.8–1.1 (1.0); PL/PW: 0.8–1.0 (0.9); EL/EW: 1.0–1.2 (1.1). Body black to dark brown, hind margins of abdominal tergites slightly paler; elytra reddish; palpi and other ap pendages slightly lighter; body glossy (Fig. 7A, B). Abdomen: punctation fine and dense; interspaces with minute irregularities; posterior margin of tergite VII with palisade fringe. Male: protarsi with tarsomeres 1–4 dilated stronger than in females. dez.pensoft.net Quedius (Microsaurus) puncticollis Thomson, 1867 Fig. 3A ( ) ( ) ( ) Body length: 6.0–8.6 (7.3); head, scutellum and abdo men blackish, pronotum and hind margins of abdominal tergites slightly paler; elytra light red or orange; palpi, antennae and legs brown; body glossy (Fig. 8A, B). Head approximately as wide as long HL/HW: 0.9–1.1 (1.0); eyes small, not convex; temples slightly longer or as long as longitudinal diameter of eye; posterior frontal puncture closer to posterior margin of head than to anteri or frontal puncture; temporal puncture closer to posterior margin of head than to posterior margin of eye; two verti cal punctures behind posterior frontal puncture arranged as slightly oblique line between posterior margin of eye and dorsal part of neck; microsculpture of entire surface of head with transverse waves. Distribution. Based on the literature data (Table 1) and newly examined material, Q. capitalis is known from several localities near Tashkent (Uzbekistan), Karatau Mountains (southwestern Kazakhstan) and Hazratisho Mountains (southern Tajikistan). Distribution. Based on the literature data (Table 1) and newly examined material, Q. capitalis is known from several localities near Tashkent (Uzbekistan), Karatau Mountains (southwestern Kazakhstan) and Hazratisho Mountains (southern Tajikistan). Bionomics. Unknown. Quedius (Microsaurus) puncticollis Thomson, 1867 Fig. 3A Sternite VIII with weak triangular me dio-apical emargination; tergite X triangular with setae; sternite IX elongate, gradually narrowed apically, with moderately wide and long basal portion and obtuse ly rounded apical margin with numerous setae. Aedea gus (Fig. 7C, D): median lobe parallel-sided with broad and obtuse apex and tooth located near apex (Fig. 7 C). Paramere rhomboid sharply narrowing apicad; its apex almost reaching apex of median lobe, with two pairs of apical setae and two pairs of lateral setae below apex; Head approximately as wide as long or slightly longer; eyes small, not convex; temples as long as longitudinal diameter of eye; posterior frontal puncture closer to pos terior margin of head than to anterior frontal puncture; temporal puncture closer to posterior margin of head than to posterior margin of eye; two vertical punctures behind posterior frontal puncture arranged as slightly oblique line between posterior margin of eye and dorsal part of neck; microsculpture of head with transverse distinct wavelines. dez.pensoft.net 134 Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... Redescription. Measurements and ratios (range, arith metic mean; n = 6): HL: 1.0–1.2 (1.1); HW: 1.0–1.4 (1.1); PL: 1.1–1.5 (1.3); PW: 1.3–1.6 (1.4); EL: 1.5–1.7 (1.6); EW: 1.3–1.6 (1.5); FB: 3.7–4.4 (4.0); TL: 6.0–8.6 (7.3); HL/HW: 0.9–1.1 (1.0); PL/PW: 0.8–0.9 (0.9); EL/EW: 1.00–1.2 (1.1). Body length: 6.0–8.6 (7.3); head, scutellum and abdo men blackish, pronotum and hind margins of abdominal tergites slightly paler; elytra light red or orange; palpi, antennae and legs brown; body glossy (Fig. 8A, B). Redescription. Measurements and ratios (range, arith metic mean; n = 6): HL: 1.0–1.2 (1.1); HW: 1.0–1.4 (1.1); PL: 1.1–1.5 (1.3); PW: 1.3–1.6 (1.4); EL: 1.5–1.7 (1.6); EW: 1.3–1.6 (1.5); FB: 3.7–4.4 (4.0); TL: 6.0–8.6 (7.3); HL/HW: 0.9–1.1 (1.0); PL/PW: 0.8–0.9 (0.9); EL/EW: 1.00–1.2 (1.1). paramere (underside) with ca. 4–8 sensory peg setae in each of two sinuate lateral rows that extend basad over pairs of lateral setae (Fig. 7D). Comparison. Quedius capitalis seems to be closely related to Q. fusicornis and Q. ochripennis from which it can be easily distinguished externally by smaller body size and proportions, and by the structure of paramere with two sinuate lateral rows of peg setae (ca.4–8 in each row) extending basad over pairs of lateral setae. Quedius (Microsaurus) fusicornis Luze, 1904 Fig. 8 Antennae moderately long, antennal segments: 3rd longer than 2nd, 4th–10th gradually widening towards apex of antenna. Quedius fusicornis Luze, 1904, 28 (original description); Gridelli 1924, 69 (characters, notes) Pronotum slightly wider than long PL/PW: 0.8–0.9 (0.9), widest at about posterior third, gradually narrow ing anteriad; hind angles rounded but distinct; dorsal and sublateral rows each with three punctures; microsculpture with transverse waves similar to that on posterior part of head. Scutellum impunctate with microsculpture as on pronotum. Elytra parallel-sided, slightly longer than wide, as long as or slightly longer than pronotum and narrow er than maximum width of pronotum; punctation dense; setation gray; interspaces shiny, with distinct minute ir regularities Material examined. Type material: Tajikistan or Uz bekistan: Lectotype (here designated): ♂, “♂/ Seravchan Putchin Pass. Glasunov 1892 [printed]/ Type fusicornis Luze [handwritten]/ ex. coll. Luze [printed]/ ex. coll. Scheerpeltz [printed]/ Typus Quedius fusicornis Luze [pre-printed]’’; Paralectotypes: 1 ♀, “Seravchan Putchin Pass. Glasunov 1892 [printed]/ Type fusicornis Luze [handwritten]/ Que dius fusicornis Luze [handwritten]/ [square orange piece of paper]’’; 1 ♀, “Seravchan Boschara Glasunow 1892 [print ed]/ Type fusicornis Luze [handwritten]/ Quedius fusicor nis Luze [pre-printed]’’ (Fig. 8F, G) (NMW). Abdomen: punctation fine and moderately dense; in terspaces with vaguely distinct minute irregularities; pos terior margin of tergite VII with palisade fringe. Additional material. Uzbekistan: 1 ♂, Samarqand Region, Aman Kutan, 04.VII.1932, V.V. Gussakovsky leg.; Kyrgyzstan: 1 ♂, Kyrgyz-Alatoo Mts, 09.VII.2010, 72°28’38.6N, 42°48’49.2E, V.A. Kastcheev leg. (ZIN). Male: protarsi with tarsomeres 1–4 dilated stronger than in females. Aedeagus (Fig. 8C–E: median lobe par allel-sided along most of its length with broad and obtuse apex and tooth located near apex (Fig. 8C). Paramere par allel-sided, narrowing only in rhomboid apical portion; its apex almost reaching apex of median lobe, with two pairs of apical setae and two pairs of lateral setae below apex, with 6 peg setae arranged in two regular longitu dinal rows apically extending basad over pairs of lateral setae (Fig. 8D). Comments on taxonomy and type material. In the original description, Luze (1904) provided no information on the type material, but he indicated 7.7–8.5 mm body size range for the species. This suggests that he must have had more than one specimen to base a description on. He also in dicated “Seravschan: Putschin-Pass, Boschara’’ as a locality that his material was from. dez.pensoft.net Quedius (Microsaurus) fusicornis Luze, 1904 Fig. 8 Finally, we know from the intro duction in Luze’s paper that the material he examined was collected by Glasunov. Therefore, a single male (NMW) and two females (ZIN) that we examined and that match the orig inal description morphologically and in the label data, are syntypes. Luze (1904) compared Quedius fusicornis with his Q. solskyi and the widespread Q. cruentus Ol. Gridelli (1924) apparently based his short notes about this species exclusively on Luze’s description, without seeing any mate rial. Similarly to other species of Quedius described by Luze (1904), Q. fusicornis is missing in the monograph by Coif fait (1978) who apparently overlooked Luze’s publication. Here we provide a redescription and first illustrations of this poorly known species, including its aedeagus. Comments on taxonomy and type material. In the original description, Luze (1904) provided no information on the type material, but he indicated 7.7–8.5 mm body size range for the species. This suggests that he must have had more than one specimen to base a description on. He also in dicated “Seravschan: Putschin-Pass, Boschara’’ as a locality that his material was from. Finally, we know from the intro duction in Luze’s paper that the material he examined was collected by Glasunov. Therefore, a single male (NMW) and two females (ZIN) that we examined and that match the orig inal description morphologically and in the label data, are syntypes. Luze (1904) compared Quedius fusicornis with his Q. solskyi and the widespread Q. cruentus Ol. Gridelli (1924) apparently based his short notes about this species exclusively on Luze’s description, without seeing any mate rial. Similarly to other species of Quedius described by Luze (1904), Q. fusicornis is missing in the monograph by Coif fait (1978) who apparently overlooked Luze’s publication. Here we provide a redescription and first illustrations of this poorly known species, including its aedeagus. Comparison. Quedius fusicornis is similar to Q. cap italis. For comparison, see the latter species above. From other similar species such as Q. solskyi, Q. cruentus and Q. ochripennis, it can be easily distinguished by the struc ture of the apical part of the paramere with two medially situated short rows of peg setae (3 in each row) extending basad the pairs of lateral setae. Comparison. Quedius fusicornis is similar to Q. cap italis. For comparison, see the latter species above. From other similar species such as Q. solskyi, Q. cruentus and Q. Quedius (Microsaurus) fusicornis Luze, 1904 Fig. 8 ochripennis, it can be easily distinguished by the struc ture of the apical part of the paramere with two medially situated short rows of peg setae (3 in each row) extending basad the pairs of lateral setae. Distribution. We were not able to locate the type locality “Putchin Pass” situated somewhere along Zer avchan River that is extended from eastern Uzbekistan to western Tajikistan. Additional material was studied from eastern Uzbekistan (near Aman-Kutan) and north-west Dtsch. Entomol. Z. 65 (2) 2018, 117–159 135 Figure 8. Quedius fusicornis, types. A, lectotype, male, habitus; B, paralectotype, female, habitus. C–E, аedeagus of the lectotype: C, lateral view; D, paramere, underside; E, median lobe, ventral view. F (lectotype), G, (paralectotype), labels. Scale bars: 1 mm. Figure 8. Quedius fusicornis, types. A, lectotype, male, habitus; B, paralectotype, female, habitus. C–E, аedeagus of the lectotype: C, lateral view; D, paramere, underside; E, median lobe, ventral view. F (lectotype), G, (paralectotype), labels. Scale bars: 1 mm. ern Kyrgyzstan (Kyrgyz-Alatoo). Finally, one specimen was from ‘Tangi-Gharuh’, a toponym in Afghanistan that we could not locate. [illegible word] [handwritten]/ asiaticus Bernh. Cotypus. [handwritten]/ Chicago NHMus M. Bernhauer Collection [printed]’’ (Fig. 10G, H) (FMHN), Bionomics. Unknown. Additional material. Tajikistan: 1 ♂, Ramid [Ramit], Kafirnigan River, 27.VII.1939, A. Romanov leg. (ZMMU). Additional material. Tajikistan: 1 ♂, Ramid [Ramit], Kafirnigan River, 27.VII.1939, A. Romanov leg. (ZMMU). Comments on taxonomy, lectotype designation and new synonymy. In the original description of Q. solskyi, Luze (1904) did not specify the number of specimens he studied, but provided characters for both sexes and the lo cality “Jagnob: Kol, Schach-Sara’’ [Tajikistan, Yaghnob river, Sughd Distr.]. Therefore, a male from NMW with the locality label ’’Trkst. Jagnob Schach-Sara’’ is con sidered a syntype. We could not locate other syntypes. Gridelli (1924), similarly to the case with Q. fusicornis, based his notes about Q. solskyi only on Luze’s descrip tion, without checking type material. And as with other species of Quedius described by Luze (1904), Q. solskyi is missing in the monograph of Coiffait (1978). Under the circumstances of uncertain identity of other syntypes, we designate the only available male syntype as a lectotype to unambigiously fix the identity of Q. solskyi. dez.pensoft.net Quedius (Microsaurus) koltzei Eppelsheim, 1887 Fig. 2D Body dark brown to brown; apical margin of abdom inal tergites vaguely paler; elytra reddish; maxillary and labial palpi, as well as antennae dark-brownish; body glossy (Figs 9A, 10A–B). Quedius koltzei Eppelsheim, 1887, 420 (original de scription); Bernhauer and Schubert1916, 425 (cala log); Gridelli 1924, 24 (characters, new records); Scheerpeltz 1933, 1445 (catalog); Coiffait 1978, 164 (new records, characters, first illustration of the ae deagus); Smetana 1998, 115 (study of the holotype, redescription, comments); Smetana 2015b, (new re cords, characters). Quedius koltzei Eppelsheim, 1887, 420 (original de scription); Bernhauer and Schubert1916, 425 (cala log); Gridelli 1924, 24 (characters, new records); Scheerpeltz 1933, 1445 (catalog); Coiffait 1978, 164 (new records, characters, first illustration of the ae deagus); Smetana 1998, 115 (study of the holotype, redescription, comments); Smetana 2015b, (new re cords, characters). Head wider than long HL/HW: 0.7–0.8 (0.8), eyes very small, not convex; temples more than two times as long as longitudinal diameter of eye; posterior frontal punc ture in the middle between anterior puncture and posteri or margin of head; temporal puncture closer to posterior margin of head than to posterior margin of eye; two ver tical punctures arranged in almost straight line between posterior frontal puncture and neck; microsculpture with transverse waves. Antennae long: antennal segments: 3rd longer than 2nd; 4th-10th slightly widening towards apex of antenna. Material examined. Kazakhstan: 2 ♂, 1 ♀, Dzhungar skiy Alatau, Keskenterek River, 10–20.VII.1988, V.A. Kastcheev leg.; 3 ♂, same locality and collector, but 20– 30.VIII.1988 (ZIN); 1 ♂, Aksu-Dzhabagly, Taldy-Bulak River, 10–20.IV.1979, B.V. Iskakov leg. (ZIN); 3 ♂, Ter skey-Alatoo, VI.1957, Skopin leg. (MNHN). Pronotum slightly wider than long PL/PW: 0.8–0.9 (0.9), widest at its middle, slightly narrowing anteriad; hind angles rounded, barely distinct; dorsal and sublat eral rows each with three punctures; microsculpture with transverse waves similar to that on posterior part of head. Scutellum impunctate, with microsculpture as on prono tum. Elytra parallel-sided, as long as or longer than wide, narrower and longer than pronotum; punctation dense, setation brownish, interspaces shiny and with distinct minute irregularities.i Comments on taxonomy, type material and distri bution. Quedius koltzei was described by Eppelsheim (1887) from “Chabarovka” [Khabarovsk, Far East, Rus sia] based on a single female specimen. Gridelli (1924) basically repeated the original description. Coiffait (1978) interpreted a few males as that species from Ter skey-Alatoo, a mountain range in Kazakhstan very far from the type locality of Q. koltzei. Quedius (Microsaurus) solskyi Luze, 1904 Figs 9, 10 fusicornis but differs from the latter by the paramere with incised apex and two pairs of sensory peg setae. The aedeagi of Q. solskyi and Q. ochripennis differ in many ways. preserved male syntype (Fig. 10A, B) with the locality label “Ost. Buchara” exactly matching the data from the original description and the identification label “asiaticus Bernh. Typus” hand written by Bernhauer as a lectotype. Our examination of the mentioned types of both Q. solskyi and Q. asiaticus undoubtedly reveal they are con specific. Thus we place Q. asiaticus Bernhauer, 1918 in synonymy with Q. solskyi Luze, 1904 and provide a rede scription with the first illustration of the aedeagus of this poorly known species. Our examination of the mentioned types of both Q. solskyi and Q. asiaticus undoubtedly reveal they are con specific. Thus we place Q. asiaticus Bernhauer, 1918 in synonymy with Q. solskyi Luze, 1904 and provide a rede scription with the first illustration of the aedeagus of this poorly known species. Distribution. Vaguely recorded type localities for Q. solskyi and Q. asiaticus are located somewhere in north ern Tajikistan and in eastern Uzbekistan or western Ta jikistan. The only additional and better georeferenced specimen examined here comes from western Tajikistan: Ramid, Kafirnigan River. Redescription. Measurements and ratios (arithmetic mean = 4): HL: 1.4–1.6 (1.5); HW: 1.7–1.9 (1.9); PL: 1.6–1.8 (1.7); PW: 1.9–2.1 (2.1); EL: 2.0–2.2 (2.1); EW: 1.9–2.1 (2.0); FB: 5.1–5.6 (5.3); TL: 8.1–9.7 (9.1); HL/ HW: 0.7–0.8 (0.8); PL/PW: 0.8–0.9 (0.9); EL/EW: 1.0– 1.1 (1.1). Bionomics. Unknown. Quedius (Microsaurus) solskyi Luze, 1904 Figs 9, 10 Quedius asiaticus Bernhauer, 1918, syn. n. Quedius solskyi Luze, 1904, 99 (original description); Gridelli 1924, 72 (characteres, notes); Quedius asiaticus Bernhauer, 1918, 92 (original desrip tion); Gridelli 1924, 57 (characters); Coiffait 1978, 183 (characters); Kascheev 2002, 181 (distribution records). Material examined. Type material: Quedius solskyi: Ta jikistan: Lectotype (here designated): ♂, “♂/ Trkst. Jag nob Schach-Sara, Glasunov 1892 [printed]/ Type solskyi Luze [handwritten]/ ex. coll. Luze/ ex. coll. Scheerpeltz [printed]/ Typus Quedius solskyi Luze [pre-printed] “ (Fig. 9E) (NMW); Bernhauer (1918) described Q. asiaticus from ‘’Ost. Buchara’’ and compared it with Q. abietum distributed in southern Europe. Bernhauer (1918) did not even mention Luze’s Q. solskyi, even though his description matches the latter species. Both examined syntypes of Q. asiati cus are clearly conspecific in morphology. In order to fix the identity of the species, we designate here one better Quedius asiaticus: Tajikistan or Uzbekistan: Lec totype (here designated): ♂ “Ost. Buchara Rickmers. [handwritten ]/ Mus. Bremen [handwritten]/ asiaticus Bernh. Typus [handwritten]/ Chicago NHMus M. Bern hauer Collection [printed]’’; paralectotype: 1 ♂ “abietum dez.pensoft.net 136 Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... Figure 9. Quedius solskyi, lectotype. A, habitus. B–D, aedeagus: B, lateral view; C, paramere, underside. D, median lobe, ventral view. E, labels. Scale bars: 1 mm. Figure 9. Quedius solskyi, lectotype. A, habitus. B–D, aedeagus: B, lateral view; C, paramere, underside. D, median lobe, ventral view. E, labels. Scale bars: 1 mm. Figure 9. Quedius solskyi, lectotype. A, habitus. B–D, aedeagus: B, lateral view; C, paramere, underside. D, median lobe, ventral view E labels Scale bars: 1 mm Figure 10. Quedius asiaticus (new synonym of Q. solskyi), syntypes. A, B, habitus. C–F, aedeagus. C, E, median lobe, lateral view; D, F, paramere, underside. G, H, labels. Scale bars: 1 mm. Figure 10. Quedius asiaticus (new synonym of Q. solskyi), syntypes. A, B, habitus. C–F, aedeagus. C, E, median lobe, lateral view; D, F, paramere, underside. G, H, labels. Scale bars: 1 mm. dez.pensoft.net Dtsch. Entomol. Z. 65 (2) 2018, 117–159 137 preserved male syntype (Fig. 10A, B) with the locality label “Ost. Buchara” exactly matching the data from the original description and the identification label “asiaticus Bernh. Typus” hand written by Bernhauer as a lectotype. times as short as tempora. In the structure of the aedea gus Q. solskyi is more similar to Q. dez.pensoft.net Quedius (Microsaurus) rufilabris Luze, 1904 Quedius rufilabris Luze, 1904, 100 (original description); Gridelli 1924, 72 (characters, notes). Comments on taxonomy. Luze (1904) described Quedi us rufilabris from “Seravschan: Putschin Pass’’ [Mountain Range or river Zeravshan in Tajikistan or Uzbekistan]. The description was based on a single female specimen. Gridelli (1924) based his knowledge of this species on Solsky’s original description only and placed Q. ru filabris near Q. solskyi. Similarly to Luze’s other species, Coiffait (1978) overlooked this species in his monograph. Unfortunately, we were unable to find the holotype of Q. rufilabris, but based on its original description all diag nostic characters, especially chaetotaxy of the head and pronotum, match Q. koltzei. Since the presumed type lo cality of Q. rufilabris is rather remote from the distribu tion of Q. koltzei, if the latter even occurs in Middle Asia (see above), we treat the former species as different from Q. koltzei, at least until more material from relevant geo graphic areas will be studied. Comments on taxonomy. Quedius bucharensis was described from an unspecified number of specimens of both sexes coming from localities in Uzbekistan, Turk menistan and Tajikistan indicated as “Karateghingebirge (Baldschuan, 924 m, Hauser), Buchara (ohne nähere Fundortangabe, Bang-Haas) und Persien (Kopet-Dagh, Siaret, 1160 m, V. 1899, Hauser)’’ (Bernhauer 1918). Comments on taxonomy. Quedius bucharensis was described from an unspecified number of specimens of both sexes coming from localities in Uzbekistan, Turk menistan and Tajikistan indicated as “Karateghingebirge (Baldschuan, 924 m, Hauser), Buchara (ohne nähere Fundortangabe, Bang-Haas) und Persien (Kopet-Dagh, Siaret, 1160 m, V. 1899, Hauser)’’ (Bernhauer 1918). We have examined one male and one female from the FMNH which are clearly syntypes of Q. bucharensis. Of them, a male specimen was earlier dissected and its ae deagus must have been glued on the card point beside the specimen, but was obviously lost. Since there were no publications with the structure of Q. bucharensis aedea gus, the identity of this species remains ambiguous. An additional two females from NHMW with the same local ity labels as in the original description but without Ber nhauer’s handwritten type labels, seem conspecific with both mentioned syntypes even though they are somewhat smaller than the latter. Their earlier identifications as Q. Quedius (Microsaurus) koltzei Eppelsheim, 1887 Fig. 2D Based on that ma terial, he redescribed Q. koltzei again and provided the illustration of the aedeagus for the first time. Smetana (1998) also redescribed Q. koltzei, but based on the ho lotype. Later, Smetana (2015b) determined one male and one female from Heilongjiang province of China as Q. koltzei and illustrated their genital structures. Smetana’s comparison of the Chinese specimens with the type ma terial and geographic proximity of Heilongjiang province to the type locality of Q. koltzei corroborate his identi fication. Our examination of the male specimens from Terskey-Alatoo from Henry Coiffait’s collection that he identified as Q. koltzei revealed that they match as far as we can observe, with the illustrations of Q. koltzei from China in Smetana (2015b). But since Smetana (2015b) did not illustrate the lateral view of the aedeagus, only the re-examination of Chinese and, preferably, additional material may help to clarify the status of Middle Asian specimens from Terskey-Alatoo. In the absence of neg Abdomen: punctation fine and moderately dense; in terspaces with vaguely distinct minute irregularities; pos terior margin of tergite VII with palisade fringe. Male: head wider than long, larger than in females and with longer temples (Luze 1904). Aedeagus (Figs 9B–D, 10C, D, E, F): Median lobe (in parameral view) paral lel-sided along most of its length with obtusely pointed apex, with tooth located near apex (Figs 9B, 10C, E). Paramere parallel-sided, its apex almost reaching apex of median lobe; with two pairs of apical setae and two pairs of lateral setae below apex; underside with pair of peg setae close to apical margin on each side of medial emar gination (Figs 9C, 10D, F). Comparison. Quedius solskyi is similar to Q. fusi cornis and Q. ochripennis, but it can be externally dis tinguished from both by the larger body size, distinctly elongated elytra and smaller eyes with their diameter two Comparison. Quedius solskyi is similar to Q. fusi cornis and Q. ochripennis, but it can be externally dis tinguished from both by the larger body size, distinctly elongated elytra and smaller eyes with their diameter two dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 138 1978) suggest that this may be a species very similar to Q. koltzei. But Quedius tadjikiscus differs from Q. Quedius (Microsaurus) rufilabris Luze, 1904 solskyi, evident from the labels, are wrong because of the following characters: chaetotaxy of head with posterior frontal puncture closer to posterior margin of eye than to nuchal ridge, larger eyes, head longer than wide and as long and as wide as elytra. Quedius (Microsaurus) koltzei Eppelsheim, 1887 Fig. 2D koltzei in the presence of three punctures in the dorsal row of pronotum and the absence of apical groups of peg setae on the paramere. ative evidence, we consider Q. koltzei as a potentially widespread Asian species. It is also possible that Q. ru filabris, whose identity currently remains ambiguous, is conspecific with Q. koltzei (for details see the former species below). Quedius koltzei differs from other similar Middle Asian Microsaurus as follows: from Q. fusicornis, Q. capitalis and Q. solskyi in peg setae on paramere arranged in ir regular lines or groups; from Q. ochripennis, Q. puncti collis and Q. tadjikiscus in median lobe (in lateral view) narrowing into a blunt, but clear apex and peg setae on paramere arranged in four irregular groups. From Q. bu charensis, a species whose identity remains ambiguous (for details see that species below) Q. koltzei differs in the chaetotaxy of head (posterior frontal puncture situat ed closer to nuchal ridge than to posterior margin of eye) and pronotum (two punctures in dorsal row and sublateral group always situated before or at most at the same level as large lateral puncture). Quedius (Microsaurus) bucharensis Bernhauer, 1918 Quedius bucharensis Bernhauer, 1918, 93 (original de scription); Gridelli 1924, 56 (characters, distribution); Scheerpeltz 1933, 1435 (catalog); Coiffait 1978, 186 (external characters). Type material examined. Syntypes: Tajikistan: 1 ♂, “Mts. Karateghin Balfdschuan 924 m. F. Hauser 1898 [printed]/ bucharicus Bern. det. Bernh. det. Bernh. [handwritten]/ bucharensis Bernh. Typus [handwritten]/ Chicago NHmus M. Bernhauer Collection [printed]’’; 1 ♀, “Buchara Handiger [handwritten]/ ochripennis Asia centr. Handiger [handwritten]/ bucharensis Bernh. Co typus [handwritten]/ bucharensis Bernh. [handwritten]/ Chicago NHmus M. Bernhauer Collection [printed]’’ (FMNH); Turkmenistan: 1 ♀, “♀/ Pers. Kopet-Dagh. Siaret 1160 m 5.99. Coll. Hauser [printed]/ Quedius per sicus Brh. [handwritten]/ solskyi Luze [handwritten]/ ex. coll. Moczarski [printed]/ ex. coll. Scheerpeltz [print ed]’’; 1 ♀, “♀/ Pers. Kopet-Dagh. Siaret 1160 m 6.99. Coll. Hauser [printed]/ solskyi ? [sic!] Luze [handwrit ten]/ ex. coll. Moczarski [printed]/ ex. coll. Scheerpeltz [printed]’’ (NMW). Type material examined. Syntypes: Tajikistan: 1 ♂, “Mts. Karateghin Balfdschuan 924 m. F. Hauser 1898 [printed]/ bucharicus Bern. det. Bernh. det. Bernh. [handwritten]/ bucharensis Bernh. Typus [handwritten]/ Chicago NHmus M. Bernhauer Collection [printed]’’; 1 ♀, “Buchara Handiger [handwritten]/ ochripennis Asia centr. Handiger [handwritten]/ bucharensis Bernh. Co typus [handwritten]/ bucharensis Bernh. [handwritten]/ Chicago NHmus M. Bernhauer Collection [printed]’’ (FMNH); Turkmenistan: 1 ♀, “♀/ Pers. Kopet-Dagh. Siaret 1160 m 5.99. Coll. Hauser [printed]/ Quedius per sicus Brh. [handwritten]/ solskyi Luze [handwritten]/ ex. coll. Moczarski [printed]/ ex. coll. Scheerpeltz [print ed]’’; 1 ♀, “♀/ Pers. Kopet-Dagh. Siaret 1160 m 6.99. Coll. Hauser [printed]/ solskyi ? [sic!] Luze [handwrit ten]/ ex. coll. Moczarski [printed]/ ex. coll. Scheerpeltz [printed]’’ (NMW). Based on the material examined here, we have addi tional records for Q. koltzei from Kazakhstan. Bionomics remains unknown. dez.pensoft.net Quedius (Microsaurus) tadjikiscus Coiffait, 1975 Quedius tadjikiscus Coiffait, 1975, 32 (original descrip tion); 1978, 149 (notes). Quedius tadjikiscus Coiffait, 1975, 32 (original descrip tion); 1978, 149 (notes). Comments on taxonomy. We could not locate and ex amine the type material of Q. tadjikiscus described from “Tadjikabad, Daran-Nazaran” in Tajikistan, and did not come across any material that could be identified as that species. The description and the illustrations of the ae deagus of Q. tadjikiscus available from Coiffait (1975, The material used by Bernhauer (1918) in the original description of Q. bucharensis comes from localities rath er remote from each other. Given that and the body size dez.pensoft.net dez.pensoft.net Dtsch. Entomol. Z. 65 (2) 2018, 117–159 139 variation among the examined specimens from different localities, it is possible that they are not conspecific. On the other hand, significant intraspecific variability in body size and proportions is usual in some Microsaurus spe cies. More extensive material including males is needed to clarify the case. paramere and degree of its incision Q. kalabi displays a transition between Q. mutilatus having lesser incised paramere with more peg setae in lateral groups, and Q. equus having deeper incised paramere with lesser peg se tae in lateral groups. variation among the examined specimens from different localities, it is possible that they are not conspecific. On the other hand, significant intraspecific variability in body size and proportions is usual in some Microsaurus spe cies. More extensive material including males is needed to clarify the case. Distribution. Quedius kalabi replaces Q. mutilatus in the eastern part of Terskey-Alatoo Mountains in Kyrgyz stan. Quedius (Microsaurus) equus Smetana, 2014 figs 1, 4G–N in Salnitska and Solodovnikov 2018 Quedius (Microsaurus) equus Smetana, 2014 figs 1, 4G–N in Salnitska and Solodovnikov 2018 Comments. We have proposed the mutilatus-group for several Middle Asian species in Salnitska and Solodovnikov (2018), where we revised all available ma terial. Thus only brief information for each of these spe cies is provided with reference to the revision for details. Diagnosis. Quedius equus distinctly differs from all other species of the mutilatus-group by the deep apical incision of the paramere and by low number (1–3) of sensory peg setae on its underside arranged in lateral longitudinal rows. Diagnosis. The mutilatus-group is characterized by the following: brown to dark brown dorso-ventrally flat tened body, notably small eyes, short elytra, absence of palisade fringe on abdominal tergite VII; aedeagus ro bust, with apical portion of median lobe slightly curved towards paramere with characteristic tooth near apex (in lateral view), with paramere widest shortly before apex (in parameral view) having four distinct groups of sensory peg setae on the underside: two apical and two lateral. Distribution. Quedius equus is known from north- east Terskey-Alatoo Mountains in Kazakhstan and from Xinjiang province of China. Presumably it has a broader continuous distribution in this area. Quedius (Raphirus) limbatus Heer, 1839 Fig. 3D Quedius (Raphirus) limbatus Heer, 1839 Fig. 3D Herman 2001, 3187 (summary of literature); Kascheev 2001, 102; 2002, 181 (distribution records); Assing and Schülke 2012, 473, 474 (diagnosis, distribution and bionomics, aedeagus illustration). Distribution. Quedius mutilatus is restricted to the central part of Terskey-Alatoo Mountains south from Is syk-Kul lake in Kyrgyzstan. Material examined. Kazakhstan: 2 ♂, 1 ♀, 7 Almaty area, Dzhungarskiy Alatau, 7 km E Lepsinsk, Chornaya River canyon, 1200–1400 m a.s.l., Betula sp., Malus, Populus etc. forest, 45°31’N, 80°43’E, 13–15.VI.2001, S.I. Golovatch leg. (cRyv); 3 ♂, 6 km SE Rudnichnyi, Koksu River canyon, 1300–1400 m a.s.l., 44°41’N, 78°58’E, Betula sp., Populus, Picea etc. forest, 09–10. VI.2001, S.I. Golovatch leg. (cRyv); 2 ♂, 3 km SSE Lepsinsk, Bulinka River canyon, 1100–1800 m a.s.l., 45°30’N, 80°38’E, 16–17.VI.2001, S.I. Golovatch leg. Material examined. Kazakhstan: 2 ♂, 1 ♀, 7 Almaty area, Dzhungarskiy Alatau, 7 km E Lepsinsk, Chornaya River canyon, 1200–1400 m a.s.l., Betula sp., Malus, Populus etc. forest, 45°31’N, 80°43’E, 13–15.VI.2001, S.I. Golovatch leg. (cRyv); 3 ♂, 6 km SE Rudnichnyi, Koksu River canyon, 1300–1400 m a.s.l., 44°41’N, 78°58’E, Betula sp., Populus, Picea etc. forest, 09–10. VI.2001, S.I. Golovatch leg. (cRyv); 2 ♂, 3 km SSE Lepsinsk, Bulinka River canyon, 1100–1800 m a.s.l., 45°30’N, 80°38’E, 16–17.VI.2001, S.I. Golovatch leg. figs 1, 4E–F, 5 in Salnitska and Solodovnikov 2018 figs 1, 4E–F, 5 in Salnitska and Solodovnikov 2018 figs 1, 4E–F, 5 in Salnitska and Solodovnikov 2018 Diagnosis. Among all species of the group, Quedius kungeicus can be distinguished by the ovoid apical part of the paramere without a distinct apical incision (in param eral view) and by the distinctly curved apical portion of the median lobe (in lateral view) with longer tip and with out distinct sub-apical tooth. figs 1–2, 4A–B in Salnitska and Solodovnikov 2018 figs 1–2, 4A–B in Salnitska and Solodovnikov 2018 Diagnosis. Quedius mutilatus is most similar to Q. kunge icus from which it can be distinguished by the rhomboid shape of the paramere with slight apical incision; by the less curved apical portion of the median lobe (lateral view) with more stronger ventral sub-apical tooth. From Q. kalabi and Q. equus it differs by the not so deeply in cised apex of paramere and distinctly larger number of sensory peg setae in lateral groups on the paramere. Diagnosis. Quedius mutilatus is most similar to Q. kunge icus from which it can be distinguished by the rhomboid shape of the paramere with slight apical incision; by the less curved apical portion of the median lobe (lateral view) with more stronger ventral sub-apical tooth. From Q. kalabi and Q. equus it differs by the not so deeply in cised apex of paramere and distinctly larger number of sensory peg setae in lateral groups on the paramere. Quedius (Microsaurus) mutilatus Eppelsheim, 1888 Distribution. Quedius kungeicus is known only from the holotype collected in the Kungey-Alatoo Mountains of Kazakhstan. figs 1–2, 4A–B in Salnitska and Solodovnikov 2018 Quedius (Microsaurus) kungeicus Solodovnikov & Salnitska, 2018 Distribution and biology. The mutilatus-group is re stricted to the Tien-Shan Mountains where all species of the group are confined to high elevations, up to 3600 m. Based on the morphology and limited bionomic data, all species of the group are hypogean and are mostly found under stones or deep in leaf litter. Distribution and biology. The mutilatus-group is re stricted to the Tien-Shan Mountains where all species of the group are confined to high elevations, up to 3600 m. Based on the morphology and limited bionomic data, all species of the group are hypogean and are mostly found under stones or deep in leaf litter. figs 1, 4E–F, 5 in Salnitska and Solodovnikov 2018 figs 1, 3, 4C–D in Salnitska and Solodovnikov 2018 Ryvkin leg.; 1 ♂, Terskey-Alatoo Mts, Barskoon Valley, Chuli Riv er, 15.VII.1983, S.K. Alekseev leg. (cRyv); 1 ♂, Terskey Alatoo Mts, Kochevnikov field research station, meadow, 19.VI.1984, N. Turtseva leg. (cRyv). (cRyv); 1 ♂, Zailiysky Alatau Mts, ca. 20 km Turgen, Tur gen River canyon, near Batan , 1750 m a.s.l., Picea, Bet ula sp., Salix etc. forest, 25.V.2001, 43°14’N, 77°46’E, S.I. Golovatch leg. (cRyv); 1 ♂, Urjar Distr., Tarbagatay River valley, ca. 1000 m a.s.l., highly disturbed Populus forest with Salix, Rosa, Lonicera, Crataegus, 47°17’N, 81°34’E, 24–25.VI.2001, S.I. Golovach leg. (cRyv); 3 ♂, Makanchi Distr., Tarbagatay Mts, 4 km NE Petrovskoe (=Kyzylbulak), Kyzylbulak River valley, 1100–1200 m a.s.l., riverine, Populus, Malus, Salix forest, 22.VI.2001, 47°03’N, 82°18′E, S.I. Golovatch leg. (cRyv). Comments on taxonomy, distributon and bionom ics. The latest summary of diagnostic characters, bionom ics and distribution of Q. limbatus, a common Western Palearctic species can be found in Assing and Schülke (2012). Based on earlier records (Table 1) and newly ex amined material in Middle Asia it is known from southern Kazakhstan and Turkmenistan. Among all Middle Asian species Q. limbatus is more similar to Q. cohaesus from which it can be easily distin guished by the structure of aedeagus with a sharper apex of the median lobe (in lateral view) and sensory peg se tae of the paramere (underside) arranged in short regular rows, slightly diverging from each other basally. Usually this species occurs in lowlands up to the sub alpine zone, but is mostly confined to forests and humid ground-based debris, often near streams (Assing and Schülke 2012). In Middle Asia Q. limbatus was collected at elevations up to 1750 m near rivers in forested land scapes. figs 1, 3, 4C–D in Salnitska and Solodovnikov 2018 figs 1, 3, 4C–D in Salnitska and Solodovnikov 2018 figs 1, 3, 4C–D in Salnitska and Solodovnikov 2018 Diagnosis. Quedius kalabi differs from all other species of the mutilatus-group by its narrower and somewhat curved apical portion of the median lobe of the aedeagus with relatively short blade of its subapical tooth (aedea gus in lateral view). In shape of the apical portion of the dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 140 of Mynteke River, leaf litter, 2100 m a.s.l., 11.VIII.1991, A.V. Tishechkin leg. (cRyv); 2 ♂, Lle-Alatau NP Tal gar env., SW slope, leaf litter sifting, 2745 m a.s.l., 43.24846N, 77.40380E, 10–11.V.2014, M. Kocián leg. (cKoc); 1 ♂, Almaty Area, Talgar district, Ak-Bulak, 2700 m a.s.l., 43.1454N, 77.2404E, 24.V.2014, O. Na kladal leg. (cKoc); 3 ♂, Zailiyskiy Alatau Mts, 2300 m a.s.l., Levyi Talgar River, 22.VIII.2009, V.A. Kastcheev leg. (cRyv); 2 ♂, Almaty Area, Zailiyskiy Alatau Mts, ca. 20 km Turgen, Turgen River canyon, near Batun, 1750 m a.s.l., 43°14’N, 77°46’E, Picea, Betula, Salix, etc. forest, 25.V.2001, S.I. Golovatch leg. (cRyv); 1 ♂, Almaty Area, Uygurskiy District, Ketmen Mts, 5 km SE Kyrghyzsay (=Podgornoye), 1500–1900 m a.s.l., 43°17’N, 79°31’E, Picea, Betula, Populus, etc. forest, 01–02.VI.2001, S.I. Golovatch leg. (cRyv); 1 ♂, Tastau, 2–3 km up-stream of river mouth, leaf litter, 09.VIII.1991, A.V. Tishechkin leg.(cRyv); 1 ♂, 1 ♀, Ketmen Mts, near Ketmen, 2500 m a.s.l., 28.VII.1991, S.V. Saluk leg. (cRyv); 1 ♂, E Zailyiskiy Alatau Mts., Belshabdar River, 2600 m a.s.l., 26.VI.2002, A.V. Puchkov leg. (cSch); 2 ♂, Zailiyskiy Alatau, Semirechye, Kargalinka valley, 2000–2350 m a.s.l., 01–07.VI.1907, A.[sic!] Jacobson leg.; same locali ty and collector, but 1 ♀, 1800–2350 m a.s.l., 05.VI.1907 (ZIN); 1 ♂, Kungey-Alatoo Mts, Kulbastau canyon, 20–27.VII.1988, V.A. Kastcheev leg. (ZIN); 2 ♂, Kung ey-Alatoo, Chilik River, Sarybastau, 12–15.VI.1988, V.A. Kastcheev leg. (ZIN); Kyrgyzstan: 2 ♂, 2 ♀, Kung ey-Alatoo Mts, upper reaches of Tschon-Kemin River, 2200–2500 m a.s.l., VII.1999, J. Frisch leg. (cKoc); 1 ♂, Kungey-Alatoo, Kurmenti River, 09–11.VII.1987, V.A. Kastcheev leg. (ZIN); 2♂, Issyk-Kul’ Area, Kung ey-Alatoo Mts, valley of left confluent of Chon-Uryukty River, leaf litter in slope forest with Picea schrenkiana, Sorbus tianschanica, etc, 10.IX.1983, A.B. Ryvkin leg. (cRyv); 1♂, Issyk-Kul’ Area, Terskey-Alatoo Mts, Chon- Kyzyl-Suu River valley near Geographical Field Re search Station, 2500 m a.s.l., moss in forest with Picea schrenkiana (He+Hm+C), 02.IX.1983, A.B. Quedius (Raphirus) pseudonigriceps Reitter, 1909 Fig. 3F, 11 Quedius (Raphirus) pseudonigriceps Reitter, 1909 Fig. 3F, 11 Quedius kirklarensis Korge, 1971, syn. n. Quedius kirklarensis Korge, 1971, syn. n. Quedius pseudonigriceps: Herman 2001, 3247 (summary of literature); Assing and Schülke 2012, 473, 474 (di agnosis, distribution and bionomics, aedeagus illustra tion); Solodovnikov 2004, 223 (characters, synonymy, notes). Quedius kirklarensis Korge, 1971, 52 (original descrip tion); Coiffait 1978, 257 (notes, distribution records). Comments on taxonomy, distribution and bionom ics. The latest summary about Quedius pseudonigriceps can be found in Solodovnikov (2004) and Assing and Schülke (2012). Quedius pseudonigriceps is widespread in Southern Europe and Western Asia. We here record it for the first time from Middle Asia: from southern Ka zakhstan and northern Kyrgyzstan. It can be easily dis tinguished from all similar Middle Asian species by the shortened elytra and absence of fine whitish apical seam of palisade fringe on tergite VII. Comments on taxonomy, distribution and bionom ics. The latest summary about Quedius pseudonigriceps can be found in Solodovnikov (2004) and Assing and Schülke (2012). Quedius pseudonigriceps is widespread in Southern Europe and Western Asia. We here record it for the first time from Middle Asia: from southern Ka zakhstan and northern Kyrgyzstan. It can be easily dis tinguished from all similar Middle Asian species by the shortened elytra and absence of fine whitish apical seam of palisade fringe on tergite VII. In Middle Asia Q. pseudonigriceps is brachypterous (Fig. 3F) and characterized by the significant variability in the structure of aedeagus which nevertheless has no geographical pattern and leaves no doubt about species identity (Fig. 11). Solodovnikov (2004) noted that Que Material examined. Kazakhstan: 1 ♀, Altai, Bukhtar ma River, Uryl-Chingistai, 13.VI.1987, V.A. Kastcheev leg. (ZIN); 3 ♂, 1 ♀, Saur Mt. Ridge, 15 km S Kindir lik, 2000 m a.s.l., 10.VII.1962, L.V. Arnoldi leg. (ZIN); 1 ♂, Almaty Area, Dzhungarskiy Alatau Mts, 7 km E Lepsinsk, Chyornaya River canyon, 1200–1400 m a.s.l., 45°31’N, 80°43’E, Betula sp., Malus, Populus etc. forest, 13–15.VI.2001, S.I. Golovatch leg. (cRyv); 1 ♂, Dzhun garskiy Alatau Mts, upper reaches of Sarydzhaz River, 3500 m a.s.l., 13.VII.1991, V.A. Kastcheev leg. (cRyv); 1 ♂, 1 ♀, W part of Dzhungarskiy Alatau Mts, N slope, up per reaches of Aktau River S of Glinovka, 2500–2800 м, 06.VIII.1991, A.V. Tishechkin leg. (cRyv); 1 ♂, Dzhun garskiy Alatau Mts, Е slope of Sandyktas Mt., right side In Middle Asia Q. pseudonigriceps is brachypterous (Fig. Quedius (Raphirus) pseudonigriceps Reitter, 1909 Fig. 3F, 11 Quedius cohaesus: Lecto type, ♂, “Turcmenia Leder. Reitter [printed]/ c. Eppelsh. Steind. d. [printed]/ ♂ [handwritten]/ cohaesus mihi [handwritten]/ Lectotype Quedius cohaesus Eppelsheim, 1888 A. Solodovnikov des. 2003 [printed]’’ (NMW); Paralectotype, ♀, “Turcmenia Leder. Reitter [printed]/ c. Eppelsh. Steind. d. [printed]/ cohaesus mihi/ ♀ [hand written]/ Paralectotypus Quedius cohaesus Eppelsheim, 1888 A. Solodovnikov des. 2013 [printed]’’ (Fig. 12E, F) (NMW). Type material examined. Quedius cohaesus: Lecto type, ♂, “Turcmenia Leder. Reitter [printed]/ c. Eppelsh. Steind. d. [printed]/ ♂ [handwritten]/ cohaesus mihi [handwritten]/ Lectotype Quedius cohaesus Eppelsheim, 1888 A. Solodovnikov des. 2003 [printed]’’ (NMW); Paralectotype, ♀, “Turcmenia Leder. Reitter [printed]/ c. Eppelsh. Steind. d. [printed]/ cohaesus mihi/ ♀ [hand written]/ Paralectotypus Quedius cohaesus Eppelsheim, 1888 A. Solodovnikov des. 2013 [printed]’’ (Fig. 12E, F) (NMW). In Middle Asia Q. pseudonigriceps usually inhabits moist leaf litter in deciduous and mixed forests and wet ground debris near streams in the mountains at the alti tudes up to 2800 m. Quedius afghanicus: Holotype, ♂, “Khat Chaї 2600 m. 22.VIII.74 [handwritten]/ Paktui Afghan. [handwritten]/ G.M.uG.L. [handwritten]/ Type [printed]/ Museum Paris Coll. H. Coiffait [printed]/ Q. (Sauridus) afghanicus H. Coiffait 1977 [pre-printed’’ (Fig. 13D) (MNHN). Quedius (Raphirus) pseudonigriceps Reitter, 1909 Fig. 3F, 11 3F) and characterized by the significant variability in the structure of aedeagus which nevertheless has no geographical pattern and leaves no doubt about species identity (Fig. 11). Solodovnikov (2004) noted that Que In Middle Asia Q. pseudonigriceps is brachypterous (Fig. 3F) and characterized by the significant variability in the structure of aedeagus which nevertheless has no geographical pattern and leaves no doubt about species identity (Fig. 11). Solodovnikov (2004) noted that Que dez.pensoft.net Dtsch. Entomol. Z. 65 (2) 2018, 117–159 141 Figure 11. Quedius pseudonigriceps, distribution, median lobe of the aedeagus laterally, and variability of the paramere (as exam ples specimens from various localities numbered respectively on the map). Scale bars: 0.5 mm. Figure 11. Quedius pseudonigriceps, distribution, median lobe of the aedeagus laterally, and variability of the paramere (as exam ples specimens from various localities numbered respectively on the map). Scale bars: 0.5 mm. Coiff., notes, distribution records); Toleutaev 2014, 44 (distribution records).f dius kirklarensis from Turkey is almost identical with Q. pseudonigriceps from South Europe and Western Asia except for the absence of palisade fringe on abdominal tergite VII in the former. Therefore he suggested that Q. kirklarensis may be a brachypterous form of Q. pseud onigriceps. A new synonymy could not be established back then because of the limited material and also due to the similar species Quedius cohaesus and Quedius turk menicus from Middle Asia. With more material available here for all relevant taxa we can undoubtedly place Que dius kirklarensis Korge, 1971 in synonymy to Q. pseudo nigriceps Reitter, 1909. For details on Quedius cohaesus and Quedius turkmenicus, see below. dius kirklarensis from Turkey is almost identical with Q. pseudonigriceps from South Europe and Western Asia except for the absence of palisade fringe on abdominal tergite VII in the former. Therefore he suggested that Q. kirklarensis may be a brachypterous form of Q. pseud onigriceps. A new synonymy could not be established back then because of the limited material and also due to the similar species Quedius cohaesus and Quedius turk menicus from Middle Asia. With more material available here for all relevant taxa we can undoubtedly place Que dius kirklarensis Korge, 1971 in synonymy to Q. pseudo nigriceps Reitter, 1909. For details on Quedius cohaesus and Quedius turkmenicus, see below. Quedius afghanicus Coiffait, 1977, 139 (original descrip tion).f Quedius turkmenicus Coiffait, 1969, 49 (original descrip tion); Coiffait 1978, 245 (characters, notes). Type material examined. Q. turkmenicus Coiffait, 1969, syn. n. Quedius (Raphirus) cohaesus Eppelsheim, 1888 Fig. 12 Q. afghanicus Coiffait, 1977, syn. n. (Fig. 13)f Additional material. Turkmenistan: 1 ♂, Asia. centr., N-Kopet-Dagh, Firjusa-Cleft, near Ashchabad, 07.V.1989, D.W. Wrase leg. (cSch); 1 ♂, Kopetdag Mts, Karakala env., 28.IX.1989, A.V. Puchkov leg. (cSch); Ta jikistan: 1 ♂, Gazimalyk Mt. Ridge, 15 km NW Ganjin, 2000 m a.s.l, 14.V.1970, G.S. Medvedev leg. (ZIN). Q. turkmenicus Coiffait, 1969, syn. n. Q. turkmenicus Coiffait, 1969, syn. n. Quedius cohaesus Eppelsheim, 1888, 60 (original des cription); Bernhauer and Schubert 1916, 421 (catalog); Gridelli 1925, 26 (characters, distribution records); Coiffait 1963, 393 (characters); Korge 1964, 122 (dis tribution records); Smetana 1967, 558 (distribution records); Coiffait 1978, 248 (characters, distribution records); Solodovnikov 2004, 227 (=Q. meurguesae Comments on taxonomy and new synonymy. Coif fait (1969, 1977) described Q. turkmenicus and Q. afghan icus from Turkmenistan and Afghanistan, respectively. dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 142 Figure 12. Quedius cohaesus, types: A (lectotype, male), B (paralectotype, female), habitus. C, D, aedeagus of the lectotype: C, median lobe, lateral view; D, paramere, underside. E, F, labels. Scale bars: A, B = 1 mm; C, D = 0.3 mm. Figure 12. Quedius cohaesus, types: A (lectotype, male), B (paralectotype, female), habitus. C, D, aedeagus of the lectotype: C, median lobe, lateral view; D, paramere, underside. E, F, labels. Scale bars: A, B = 1 mm; C, D = 0.3 mm. We were able to study the type material for Q. afghanicus only (Fig. 13), which turns out to be conspecific with Q. cohaesus and therefore is placed here into synonymy with the latter. Unfortunately, we were unable to examine the type material of Q. turkmenicus which, according to Coif fait (1969) is deposited in the collection of the Paul Sabat ier University at Toulouse, France. Nevertheless, because it is obvious from the original descriptions and illustra tions that Q. turkmenicus is conspecific with Q. cohaesus, the former is also placed into synonymy with the latter. These new synonymies are consistent with the earlier revealed synonymy of Q. cohaesus with Q. meurguesae Coiffait, 1977 from Iran (Solodovnikov 2004). Below we redescribe this insufficiently known widespread species and provide data on its distribution and bionomics. Quedius (Raphirus) imitator Luze, 1904 Figs 3C, 15 Quedius tschinganensis Coiffait, 1969, syn. n. (Fig. 14) Quedius tschinganensis Coiffait, 1969, syn. n. (Fig. 14) Quedius imitator Luze, 1904, 102 (original description); Bernhauer 1905, 596 (notes); Bernhauer and Schubert 1916, 429 (list with synonyms); Gridelli 1924, 135 (characters, notes); Coiffait 1967, 406 (characters); Coiffait 1978, 237 (characters, distribution records); Boháč 1988, 556 (distribution records); Klimenko 1996, 121;f , ; Quedius tschinganensis Coiffait, 1969, 50 (original de scription); Coiffait 1970, 143 (list); Coiffait 1978, 237 (characters); Kascheev 2001, 102 (distribution records); Quedius tschinganensis var. gracilicornis Coiffait, 1977, 139 (original description); Quedius tschinganensis var. debilicornis Coiffait, 1978, 237 ( l f ili i ) Quedius tschinganensis Coiffait, 1969, 50 (original de scription); Coiffait 1970, 143 (list); Coiffait 1978, 237 (characters); Kascheev 2001, 102 (distribution records);f scription); Coiffait 1970, 143 (list); Coiffait 1978, 237 (characters); Kascheev 2001, 102 (distribution records); Quedius tschinganensis var. gracilicornis Coiffait, 1977, 139 (original description); Quedius tschinganensis var debilicornis Coiffait 1978 Figure 13. Quedius afghanicus (new synonym of Q. cohaesus), holotype, male. A, habitus. B, aedeagus, lateral view; C, param ere, underside. D, labels. Scale bars: 0.5 mm. Quedius tschinganensis var. gracilicornis Coiffait, 1977, 139 (original description);f Quedius tschinganensis var. gracilicornis Coiffait, 1977, 139 (original description);f Quedius tschinganensis var. debilicornis Coiffait, 1978, 237 (replacement name for gracilicornis). punctures; sublateral rows each with two punctures; mi crosculpture with shallow hardly visible transverse waves. Type material examined. Quedius imitator: Tajikistan or Uzbekistan: Lectotype (here designated): 1 ♂, “Ser avschan Darch Glasunov 1892 [printed]/ Q. imitator Luze J. Boháč det. 1983 [pre-printed]”; paralectotypes: 1 ♂, “[square orange piece of paper]/ Seravschan Putchin Pass. Glasunov, 1892 [printed]/ Quedius imitator Luze [hand written]/ Q. imitator Luze J. Boháč det. 1983 [pre-print ed]”; paralectotypes: 1 ♂, “Seravschan Putchin Pass. Glasunov, 1892 [printed]/ Quedius imitator Luze [hand written]/ Q. imitator Luze J. Boháč det. 1983 [pre-print ed]/ Quedius sp.1. cf. suturalis Ksw. A. Solodovnikov 1997 [handwritten]”; 3 ♂, “Seravschan Putchin Pass. Glasunov 1892 [printed]/ Q. imitator Luze J. Boháč det. 1983 [pre-printed]/ Quedius sp.1. cf. suturalis Ksw. A. Solodovnikov 1997 [handwritten]”; 2 ♀, “Seravschan Putchin Pass. Glasunov 1892[printed]”; 2 ♂, “Seravschan Obburden Glasunov, 1892 [printed]/ Q. imitator Luze J. Boháč det. 1983 [pre-printed]/ Quedius sp.1. cf. sutura lis Ksw. A. Solodovnikov 1997 [handwritten]”; 1 ♀, “Iskander-Kul Iskander-Darja Glasunov 1892 [printed]/ Q. imitator Luze J. Boháč det. 1983 [pre-printed]” (ZIN). Scutellum punctate with microsculpture distinctly denser as on pronotum. Quedius (Raphirus) cohaesus Eppelsheim, 1888 Fig. 12 Body light to dark brownish; head black, pronotum dark brown to brown; elytra brownish with hind angles paler; abdomen dark brown with posterior margins dis tinctly lighter; hind legs yellowish, antennae, maxillary and labial palps darker, body glossy (Figs 12A; 13A). Head slightly wider than long HL/HW: 0.9–1.1 (1.0), eyes large and convex; temples distinctly shorter than eyes (ratio 0.2–0.3 (0.3); with shallow, but dense trans verse microsculpture; punctation: one puncture at ante rior margin near antennal pit, anterior frontal puncture at posterior margin of antennal pit, posterior frontal and temporal punctures closer to posterior margin of eye than to posterior margin of head; vertical punctures (ca. 1–2) closer to neck than to posterior margin of eye. Antennae long: antennal segments: 3rd longer than 2nd; 4th–10th distinctly widening towards apex of antennae. Redescription.Measurements and ratios (range, arith metic mean; n = 3): HL: 0.7–0.9 (0.8); HW: 0.8–0.9 (0.9); PL: 0.9–1.2 (1.0); PW: 0.9–1.1 (1.0); EL: 1.2–1.5 (1.4); EW: 1.2–1.3 (1.3); FB: 2.9–3.6 (3.2); TL: 5.6–6.7 (6.2); HL/HW: 0.9–1.1 (1.0); PL/PW: 0.9–1.1 (1.0); EL/EW: 1.0–1.2 (1.1). Pronotum slightly wider than long or transverse PL/ PW: 0.9–1.1 (1.0), widest at its posterior half, vaguely narrowing anteriad, wider and longer than head; hind an gles rounded barely distinct; dorsal rows each with three dez.pensoft.net 143 Dtsch. Entomol. Z. 65 (2) 2018, 117–159 Figure 13. Quedius afghanicus (new synonym of Q. cohaesus), holotype, male. A, habitus. B, aedeagus, lateral view; C, param ere, underside. D, labels. Scale bars: 0.5 mm. modern sense, but certainly some locality in Middle Asia (Eppelsheim, 1888). Based on the literature (Table 1) and material examined here, Q. cohaesus is known from Iran (material not recorded here), Turkmenistan, Tajikistan and Afghanistan (most of the material not recorded here). Bionomics. It is only known that Q. cohaesus can be found at rather high elevations, up to 2600 m (Coiffait, 1977). dez.pensoft.net Quedius (Raphirus) imitator Luze, 1904 Figs 3C, 15 River canyon, near Batun, 1750 m a.s.l., 43°14’N, 77°46’E, Picea, Betula sp., Salix, etc. forest, 25.V.2001, S.I. Golo vatch leg. (cRyv); 1 ♂, Zailiysky Alatau, Chilik River, Sa rybastau, 15.VI.1988, V.A. Kastcheev leg. (ZIN); 4 ♂, 1 ♀, Almaty Area, Uygurskiy Distr., Ketmen Mts, 5 km SE Kyrghyzsay (=Podgornoye), 1500–1900 m a.s.l., 43°17’N, 79°31’E, Picea, Betula sp., Populus, etc. forest, 01–02. VI.2001, S.I. Golovatch leg. (cRyv);1 ♂, Ketmen Mts, Ma lyi Kyrgisai, 28.VII.1987, V.A. Kastcheev leg., (ZIN); 3 ♂, Karatau Mts, 660 m a.s.l., 42°53’41.42N, 70°42’56.6E, leaf litter along stream, 11.VII.2010, V.A. Kastcheev leg. (ZIN); 8 ♂, 1 ♀, Karatau Mts, Byzhi River, Rynagus stream, 757 m a.s.l., 43°57’08.7N, 68°12’04.2E, 24–25.VII.2010, V.A. Kastcheev leg. (ZIN); 2 ♂, 1, Karatau Mts, Aktobe River, grove, 25.VII.2010, V.A. Kastcheev leg. (ZIN); 6 ♂, 1 ♀, Karatau Mts, Khantagi River, 570 m a.s.l., 43°33’32.4N, 68°40’52.7E, 25.VI.2011, V.A. Kastcheev leg. (ZIN); same locality and collector, but 1 ♂, leaf litter under Salix sp., 536 m a.s.l., 43°32’46.5N, 68°39’50.6E, 21.VII.2010 (ZIN); ♂, 1 ♀, 27 km S Chulak-Kurgan, 04.VI.1983, B.V. Iska kov leg. (ZIN); 1 ♂, 1, Chimkent, Aksukent, Aksu River, 29.VI.1983, V.A. Kastcheev leg. (ZIN); 2 ♂, Aksu-Zhabag ly Nature Reserve, Tokmak River, near border, under sto nes, 1600 m a.s.l., 30.V.1974, E.V. Ishkov leg. (ZIN); 1 ♂, 2 ♀, Aksu-Dzhabagly, Taldy-Bulak River, 15–25.VI.1983, B.V. Iskakov leg. (ZIN); 1 ♂, Aksu-Dzhabagly, Isbala Ri ver, 18.VII.1986, (ZIN); 1 ♂, Aksu-Djabagly, Djabagly Ri ver, tract Ulken-Kaindy, IV.1986, B.V. Iskakov leg. (ZIN); same locality and collector, but 1 ♂, Kshi-Kaindy River, 01.V.1986 (ZIN); Uzbekistan: 1 ♂, Kitab, 30.VII.1933, V.V. ridus) tschinhganensis v. gracilicornis H. Coiffait det. [sic!] 1977 [pre-printed]” (MNHN). Additional material. Tajikistan: 3 ♂, Zeravshan Mt. Ridge, Chap-Dara River valley, 2500 m a.s.l., 26.VI.1983, S.K. Alekseev leg. (cRyv); 1 ♂, Pamir-Alai, Zeravshan Mt Ridge, Zavron valley, 2100–3000 m a.s.l., 12–13.VII.1990, M. Schülke & D.W. Wrase leg (cSch); 1 ♂, Zeravshan Mt. Ridge, near Mazor, 14.VIII.1989, K.G. Michailov leg. (NHMD); Kazakhstan: 1 ♂, Makanchi District, Tar bagatay Mts, 6 km NE Kirovka (=Karatuma), Sholakterek River valley, ca. 1200 m a.s.l., 47°10’N, 82°06’E, highly disturbed Populus forest with Salix, Rosa, Lonicera, Cra taegus, etc., 23–24.VI.2001, S.I. Golovatch leg. (cRyv); 1 ♂, Dzhungarskiy Alatau, S Koktuma, Alakol Lake, 05.VI.1962, L.V. Arnoldi leg. (ZIN); 2 ♂, Almaty Area, Dz hungarskiy Alatau Mts, 6 km NE Rudnichnyi, Koksu Riv er canyon, 1300–1400 m a.s.l., 44°41’N, 78°58’E, Betula sp., Populus, Picea etc. Quedius (Raphirus) imitator Luze, 1904 Figs 3C, 15 Elytra parallel-sided, hardly narrowing anteriad, as long as wide or slightly longer than wide EL/EW: 1.0–1.2 (1.1); wider and slightly longer than pronotum; puncta tion dense with interspaces wider than diameter of punc tures, interspaces shiny, with distinct minute irregulari ties; setation brownish.i Abdomen: punctation fine and dense; interspaces with minute irregularities; posterior margin of tergite VII with palisade fringe. Male: protarsi with tarsomers 1–4 dilated stronger than in females. Aedeagus (Figs 12C, D; 13B, C): Me dian lobe parallel-sided with moderately acute apex, tooth situated close to its apex (Figs 12C, 13B). Paramere parallel-sided, slightly narrowing basad; its apex almost reaching apex of median lobe; sensory peg setae arranged in two irregular and wide longitudinal rows along each lateral margin of apical portion extending over pairs of lateral setae below apex (Figs 12D, 13C). Comparison. Among other Raphirus that occurs in Middle Asia, Q. cohaesus is most similar to Q. pseud onigriceps from which it can be easily distinguished by the presence of an apical seam of palisade fringe VII and normally developed elytra, as well as by the characters of the aedeagus. Quedius tschinganensis: Uzbekistan: Holotype: ♂, “Ouzbekistan 8-68 Mts Tschingan 1500 m. H.C. [print ed]/ Q. (Sauridus) tschinganus [sic!] Coiff. H. Coiffait det. 1968 [pre-printed]/ Holotype [printed]”; 5 ♂, 1 ♀, “Ouzbekistan 8-68 Mts Tschingan 1500 m. H.C. [print ed]/ Paralectotype [printed]” (Fig. 14D, H) (MNHN). Distribution. Quedius cohaesus was described from “Turcmenia” which is not necessarily Turkmenistan in the Quedius tschinganensis gracilicornis: Tajikistan: ♂, “Karatak Buchara [printed]/ Type [printed]/ Q. (Sau Quedius tschinganensis gracilicornis: Tajikistan: ♂, “Karatak Buchara [printed]/ Type [printed]/ Q. (Sau dez.pensoft.net 144 Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... Figure 14. Quedius tschinganensis (new synonym of Q. imitator), types. A–D, holotype; E–H, paratype. A, E, habitus. B, C, F, G, aedeagus: B, lateral view; C, dorsal view; F, median lobe, lateral view; G, paramare, underside. D, H, labels. Scale bars: A, E = 1 mm; B, C, F, G = 0.5 mm. Figure 14. Quedius tschinganensis (new synonym of Q. imitator), types. A–D, holotype; E–H, paratype. A, E, habitus. B, C, F, G, aedeagus: B, lateral view; C, dorsal view; F, median lobe, lateral view; G, paramare, underside. D, H, labels. Scale bars: A, E = 1 mm; B, C, F, G = 0.5 mm. Quedius (Raphirus) imitator Luze, 1904 Figs 3C, 15 forest, 09–10.VI.2001, S.I. Golo vatch leg. (cRyv); 2 ♂, Zalataysky Alatau, Krasnogorka [Sulutor], stream beach under tree, 75.13504E, 43.23457N, 28.VII.2010, V.A. Kastcheev leg. (ZIN); 2 ♂, 1 ♀, Lle- Alatau NP Talgar env., Ak-Bulak Resort horse and cow dung, 1750 m a.s.l., 43.26897N, 77.37145E, 08.V.2014, M. Kocián leg. (cKoc); 2 ♀, Lle-Alatau NP Talgar env., Ak-Bu lak Resort, horse and cow dung, 1690 m a.s.l., 43.27039N, 77.37137E, 12–15.V.2014, M. Kocián leg. (cKoc); 1 ♂, Almaty Area, Zailiyskiy Alatau Mts, Medeo near Almaty, 1500–1600 m a.s.l., 43°10’N, 77°04’E, Picea, Betula, etc. forest, 27.V.2001, S.I. Golovatch leg. (cRyv); 3 ♂, Almaty Area, Zailiyskiy Alatau Mts, ca. 20 km SE Turgen, Turgen dez.pensoft.net Dtsch. Entomol. Z. 65 (2) 2018, 117–159 145 Figure 15. Quedius imitator, distribution, median lobe of the aedeagus laterally, and variability of the paramere (as an example specimens from one locality, indicated by black dot). Scale bars: 1 mm. Dtsch. Entomol. Z. 65 (2) 2018, 117–159 145 Figure 15. Quedius imitator, distribution, median lobe of the aedeagus laterally, and variability of the paramere (as an example specimens from one locality, indicated by black dot). Scale bars: 1 mm. Gussakovsky leg. (ZIN); Kyrgyzstan: 1 ♂, Terskey Ala too Mts, Barskoon Valley, Chuli River, 15.VII.1983, S.K. Alekseev leg. (cRyv); 1 ♂, 1 ♀, Osh Area, Sary-Chelek Bio sphere Reserve, near Arkit, Bakay-say Tract, 14.VII.1983, K.G. Mikhailov leg. (cRyv); 1 ♂, Chatkal Mt. Ridge, near Arkit, nut-fruit forest, 16.V.1961, V.A. Zaslavsky leg. (ZIN); 2 ♂, Tien Shan, Kichik-Alai Mt. Ridge, upper reach es of Kyrghyz-Ata River, Kara-Goy, 2400–2850 m a.s.l., Juniperus stand, 21–23.V.1993, S.I. Golovatch leg. (cRyv); 1 ♂, Osh Area, environs of Sary-Chelek Biosphere Reserve, confluent of Aflatun River, Batrakhan (=Baltyrkan) Tract, moss and leaf litter in birch forest & under Picea schrenki ana at stream bank, 31.VII.1983, A.B. Ryvkin leg. (cRyv); 3 ♂, Tien Shan, Chatkal Mt. Ridge, Sary-Chelek Bio sphere Reserve, 1550–2200 m a.s.l., forests, 29–31.V.1993, S.I.Golovatch leg. (cRyv); 2 ♂, 1 ♀, Osh Area, W Tien Shan, Ferganskiy Mt. Ridge, near Yarodar, 1100–1200 m a.s.l., Juglans forest, in leaf litter, 27–28.IX.1983, K.Yu. Es kov leg. (cRyv); 1 ♂, Osh Area, W Tien Shan, Ferganskiy Mt. Ridge, Yarodar, 1300 m a.s.l., rill bank, in leaf litter and under stones, 24–25.IX.1983, K.Yu. Eskov leg. (cRyv); 3 ♂, Tien Shan, Baubash-Ata Mt. Ridge, near Arslanbob, 1800– 1900 m a.s.l., scrub, litter & under stones. 19.V.1993. S.I. Golovatch leg. Quedius (Raphirus) imitator Luze, 1904 Figs 3C, 15 (cRyv); 10 ♂, 1 ♀, Tien Shan, Baubash-Ata dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 146 Mt. Ridge, near Yarodar, 1400–1700 m a.s.l., Juglans for est, litter & under bark. 16–17.V.1993. S.I. Golovatch leg. (cRyv); 4 ♂, 1 ♀, Ferganskiy Mt. Ridge, Kara-Alma, 1800 m a.s.l., 22–24.VI.1945, K.V. Arnoldi leg. (ZMMU); ♂ 1 ♂, 3 ♀, Gava, Fergana Valley, Jalal-Abad Region, near station of Forest Institute of Russian Academy of Sciences, 04.IX.1950, L.V. Arnoldi leg. (ZIN); 1 ♂, Fergana Valley, Kulun Lake, 3000 m a.s.l., 07.V.1993, I.I. Kabak leg. (ZIN); 2 ♂, 1 ♀, S Fergana Valley, Ak-Terek, 25.IX.1937, A.N. Kirichenko leg. (ZIN). from the apex, to the state with obtuse apex and with more sensory peg setae arranged denser and closer to the apex (Figs 14C, G; 15). Shape of the median lobe is more stable and varies only slightly in length and degree of sharpness of its apex (Figs 14B, F; 15). Mapping of this variability across the species distribution does not show any geograph ical patterns. Externally all specimens including females, also show no traits that would correspond to variants differ ent in the shape of the paramere. Thus we place Q. tschin ganensis Coiffait, 1969 in synonymy with Q. imitator Luze, 1904. Our study of the type specimen of Q. tschinganensis debilicornis also shows it to be conspecifc with Q. imitator. g ( ) Comments on the lectotype designation. In the origi nal description of Q. imitator, Luze (1904) did not specify the number of syntypes but provided geographical data that indicated multiple syntypes collected in the localities “Ser avschan, Putchin-Pass, Darch, Obburden, Urmitan, Kumar; Jagnob, Varsaut; Iskander-Kul, Iskander-Darja” (approxi mate coordinates as we interpret these localities are given in the Table. 2). Also, the syntype series must have included both sexes because male characters were specified sepa rately in the description. In the ZIN collection we found 11 specimens from several localities along Zeravchan and Iskander Darya Rivers matching those in the original de scription (for details see ‘Material examined’ above). Based on that and additional information from the specimen la bels, there is no doubt that they are syntypes. Earlier they were identified by Boháč (1988) as Q. imitator without rec ognizing them as syntypes. Quedius (Raphirus) imitator Luze, 1904 Figs 3C, 15 In order to fix the identity of the species, we designate here one male syntype with more preciselocality “Seravschan Darch Glasunov 1892” (Darg, Sughd Distr.) as the lectotype. Bernhauer (1905) consid ered Q. imitator as a “rough form” of Q. oblitteratus (now synonym of Q. humeralis Stephens, 1832). Gridelli (1924) not seeing types or any other material of Q. imitator was not sure about the status of this species. Based on the non-type material, Coiffait (1967, 1978) illustrated its aedeagus for the first time that here is shown to be the correct species interpretation. Boháč (1988) provided new records for the species from Uzbekistan and Turkmenistan that are reliable because he examined syntypes.f Comments on taxonomy, distribution and bionom ics. Quedius imitator can be diagnosed by the following character combination: body dark brown with darker head and abdomen; elytra with slightly yellowish anterior angles; antennae usually pale; scutellum without setifer ous punctation; aedeagus with ventral tooth of median lobe located remotely from its apex, with median lobe and paramere very narrow, apex of paramere obtusely sharpened and sensory peg setae arranged in two regu lar rows convergent to each other. Among other Raphirus that occur in Middle Asia, Q. imitator is most similar to Q. cohaesus from which it can be easily distinguished by the mentioned diagnostic characters of the aedeagus. Based on the examined material and literature (Ta ble 1), Q. imitator is widely distributed in all countries of Middle Asia (Fig. 15). According to the label data of the examined material, Q. imitator inhabits ground based debris and leaf litter of mainly deciduous forests along rivers and streams at various elevations, up to 3000 m. Also it can be found in dung or under stones. Quedius (Raphirus) novus Eppelsheim, 1892 Figs 3E, 17 Quedius dzambulensis Coiffait, 1967, syn. n. (Fig. 16) Quedius novus Eppelsheim, 1892, 331 (original descrip tion); Gridelli 1925, 125; Wüsthoff 1938 (illustration of aedeagus); Coiffait 1963, 389 (characters); Coiffait 1970, 143 (distribution records); Coiffait 1978, 228 (notes); Boháč 1988, 556 (distribution records; notes); Smetana 1995a, 84 (distribution records); Klimenko 1996, 121 (distribution records); Kadyrov et al. 2014a, 31; 2014b, 49 (distribution records).f Comments on the new synonym. Coiffait (1969) de scribed Quedius tschinganensis (Fig. 14) from Uzbeki stan and separated it from Q. imitator by darker body coloration, antennal segment 3 longer than 2, presence of ‘lateral’ puncture on pronotum, denser punctation of the elytra and more elongated median lobe. Additionally Coiffait (1977) described Q. tschinganensis gracilicor nis, a variety of Q. tschinganensis from Tajikistan based on some differences in coloration of the body and the pro portions of antennae. Later (1977), he replaced the preoc cupied name gracilicornis by the new name debilicornis. Both are unavailable names due to ICZN Article 15.2 as already noted in Herman (2001). Quedius dzambulensis Coiffait, 1967, 403 (original de scription); Coiffait 1978, 229 (characters, distribution records); Boháč 1988, 556 (notes); Kascheev 2001, 102 (distribution records). dez.pensoft.net Material examined. (ZIN); 2 ♂, 2 ♀, Kar zhantau, 30 km E Leninskoe, Karabau River valley, 01–05. VII.1983, B.V. Iskakov leg. (ZIN); 1 ♂, Urochishe Shilikti, 05.VI.2010, K.V. Makarov, A.V. Matalin leg. (cSch); Kyr gyzstan: 1 ♂, Kyrgyz Alatau, under Salix sp., 09.VII.2010, 72°28’38.6N, 42°48’49.2E, V.A. Kastcheev leg. (ZIN); 3 ♂, Osh Area, Sary-Chelek Biosphere Reserve, “head” of Sary-Chelek Lake, 1940–1945 m a.s.l., lake shore and bottom of partly dried rill with Carex spp., Equisetum sp., Juncus sp., Phragmites australis, etc., 12.VIII.1983, A.B. Ryvkin leg. (cRyv); 1 ♂, Tien Shan, Baubash-Ata Mt. Ridge, near Arslanbob, 1800–1900 m a.s.l., scrub, litter & under stones, 19.V.1993, S.I. Golovatch leg. (cRyv);1 ♂, Aruktau 25 km Kyzyl-Kiya, IV.1966, O.L. Kryzhanovsky leg. (ZIN); Tajikistan: 1 ♀, “Seravshan Kumar Glasun ov 1892/ Q. dzambulensis J.Boháč det. 1983” (ZIN); 1 ♂, “Seravshan Kschtut. Artutsch. Glasunov 1892/ Q. dzam bulensis J.Boháč det. 1983/ Quedius dzambulensis Coiff. A. Solodovnikov det. 1997” (ZIN); 1 ♀, “Seravshan Fl. Magian Glasunov 1892/ Q. dzambulen sis J. Boháč det. 1983/ Quedius dzambulensis Coiff. A. Solodovnikov det. 1997” (ZIN); 1 ♂, “Trkst. Jagnob Kol Glasunov 1892/ Q. dzambulensis Epp. J. Boháč det. 1983” (ZIN); 3 ♂, Zeravshan Mt. Ridge, Chap-Dara River valley, 2500 m a.s.l., 26.VI.1983, S.K. Alekseev leg. (cRyv); 1 ♂, Konda ra, under stones near aryk, 03.VI.1973, V.V. Yanushev leg. (cRyv); 1 ♂, Warsob, 03.V.1988, S.V. Saluk leg. (cRyv); 1 ♂, Pamir-Alai, Hisaar Mts, Adshuk-Cleft near Warsob, 1200 m a.s.l., 01–03.VII.1990, M. Schülke & D.W. Wrase leg. (CSch); 1 ♂, 2 ♀, “♀ or ♂/ Mts Karateghin Baldschuan 924 m. F. Hauser 1898/ novus/ ex. coll. Scheerpleltz” (NMW); 2 ♂, 1 ♀, “♀ or ♂/ Mts. Karateghin Baldschuan 924 m. F. Hauser 1898/ novus Epp./ ex. coll. Breit/ ex. coll. Scheerpleltz” (NMW); 1 ♂, ”♀/ Mts. Karateghin Balds chuan 924 m. F. Hauser 1898/ Quedius novus Epp. det. Bernhauer/ ex. coll. Scheerpleltz” (NMW); 2 ♂, 1 ♀,“♀/ Mts. Karateghin Baldschuan 924 m. F. Hauser 1898/ Col lect. Hauser /Q. novus Epp. Bernh.d.” (NMW); 3 ♀, “♀/ Mts. Karateghin Baldschuan 924 m. F. Hauser 1898/ Col lect. Hauser” (NMW); 3 ♀, “♀/ Mt. Karateghin Sary-pul 1482 m. F. Hauser 1898/ Collect. Hauser” (NMW); 1 ♂, Figure 16. Quedius dzambulensis (new synonym of Q. novus), holotype, male. A, habitus. B, median lobe, lateral view; C, paramere, unserside. D, labels. Scale bars: 1 mm. but “♂/ novus Epp. Deutsch. ent. Zeit. 1892. P. Material examined. Type material examined. Quedius novus: Uzbekistan: Lectotype (here designated), ♂, “novus Epp. Taschkent Leder. [handwritten]/ c. Epplsh. Steind. d. [printed]/ Typus [printed]” (NMW); Paralectotypes, 2 ♀, same data as in lec totype; 2 ♂, 2 ♀, same data as in lectotype, but without “no vus Epp. Taschkent Leder.”; 1 ♂, same data as in lectotype, Our examination of the material from Middle Asia, in cluding types, showed continuous variability in the exter nal morphology and aedeagus that connects the states of Q. imitator and Q. tschinganensis. The shape of the paramere varies from the state with narrow and sharp apex with lesser number of sensory peg setae arranged in regular rows away 147 Dtsch. Entomol. Z. 65 (2) 2018, 117–159 same locality and collector, leaf litter near stream, 1600– 1700 m a.s.l., 17–19.V.1984 (cRyv); Kazakhstan: 1 ♂, Almaty Area, Dzhungarskiy Alatau Mts, 6 km NE Rud nichnyi, Koksu River canyon, 1300–1400 m a.s.l., 44°41’N, 78°58’E, Betula sp., Populus, Picea etc. forest, 09–10. VI.2001, S.I. Golovatch leg. (cRyv); 1 ♂, Dzhungarskiy Alatau Mts, S slope, E ridge, middle reaches of Ispul Riv er, 1900 m a.s.l., litter in Abies forest, 14.VIII.1991, A.V. Tishechkin leg. (cRyv); 1 ♂, Almaty Area, Talgar District, Ak-Bulak, 2700 m a.s.l., 43.1613N, 77.2404E, 24.V.2014, O. Nakladal leg. (cKoc); 3 ♂, 1 ♀, Aksu-Dzhabagly, Isbala River, 15–25.VI.1983, B.V. Iskakov leg. (ZIN); 2 ♂, 1 ♀, Aksu-Dzhabagly, Taldy-Bulak River, 10–20.IV.1979, B.V. Iskakov leg. (ZIN); 1 ♂, Aksu-Dzhabagly, Ulken-Kaindy River, 15.VI.1991, V.A. Kastcheev leg. (ZIN); 1 ♂, SW slopes of Ugamskij Range, Silbili River, 18.VI.2004, A.V. Matalin leg. (cSch); 2 ♂, 1 ♀, Karzhantau, Kaskasu River, 10–12.VII.1983, B.V. Iskakov leg. (ZIN); 2 ♂, 2 ♀, Kar zhantau, 30 km E Leninskoe, Karabau River valley, 01–05. VII.1983, B.V. Iskakov leg. (ZIN); 1 ♂, Urochishe Shilikti, 05.VI.2010, K.V. Makarov, A.V. Matalin leg. (cSch); Kyr gyzstan: 1 ♂, Kyrgyz Alatau, under Salix sp., 09.VII.2010, 72°28’38.6N, 42°48’49.2E, V.A. Kastcheev leg. (ZIN); 3 ♂, Osh Area, Sary-Chelek Biosphere Reserve, “head” of Sary-Chelek Lake, 1940–1945 m a.s.l., lake shore and bottom of partly dried rill with Carex spp., Equisetum sp., Juncus sp., Phragmites australis, etc., 12.VIII.1983, A.B. Ryvkin leg. (cRyv); 1 ♂, Tien Shan, Baubash-Ata Mt. Ridge, near Arslanbob, 1800–1900 m a.s.l., scrub, litter & under stones, 19.V.1993, S.I. Golovatch leg. (cRyv);1 ♂, Aruktau 25 km Kyzyl-Kiya, IV.1966, O.L. Kryzhanovsky leg. (ZIN); Tajikistan: 1 ♀, “Seravshan Kumar Glasun ov 1892/ Q. dzambulensis J.Boháč det. Material examined. 1983” (ZIN); 1 ♂, “Seravshan Kschtut. Artutsch. Glasunov 1892/ Q. dzam bulensis J.Boháč det. 1983/ Quedius dzambulensis Coiff. A. Solodovnikov det. 1997” (ZIN); 1 ♀, “Seravshan Fl. Magian Glasunov 1892/ Q. dzambulen sis J. Boháč det. 1983/ Quedius dzambulensis Coiff. A. Solodovnikov det. 1997” (ZIN); 1 ♂, “Trkst. Jagnob Kol Glasunov 1892/ Q. dzambulensis Epp. J. Boháč det. 1983” (ZIN); 3 ♂, Zeravshan Mt. Ridge, Chap-Dara River valley, 2500 m a.s.l., 26.VI.1983, S.K. Alekseev leg. (cRyv); 1 ♂, Konda ra, under stones near aryk, 03.VI.1973, V.V. Yanushev leg. (cRyv); 1 ♂, Warsob, 03.V.1988, S.V. Saluk leg. (cRyv); 1 ♂, Pamir-Alai, Hisaar Mts, Adshuk-Cleft near Warsob, 1200 m a.s.l., 01–03.VII.1990, M. Schülke & D.W. Wrase leg. (CSch); 1 ♂, 2 ♀, “♀ or ♂/ Mts Karateghin Baldschuan 924 m. F. Hauser 1898/ novus/ ex. coll. Scheerpleltz” (NMW); 2 ♂, 1 ♀, “♀ or ♂/ Mts. Karateghin Baldschuan 924 m. F. Hauser 1898/ novus Epp./ ex. coll. Breit/ ex. coll. Scheerpleltz” (NMW); 1 ♂, ”♀/ Mts. Karateghin Balds chuan 924 m. F. Hauser 1898/ Quedius novus Epp. det. Bernhauer/ ex. coll. Scheerpleltz” (NMW); 2 ♂, 1 ♀,“♀/ Mts. Karateghin Baldschuan 924 m. F. Hauser 1898/ Col lect. Hauser /Q. novus Epp. Bernh.d.” (NMW); 3 ♀, “♀/ Mts. Karateghin Baldschuan 924 m. F. Hauser 1898/ Col Figure 16. Quedius dzambulensis (new synonym of Q. novus), holotype, male. A, habitus. B, median lobe, lateral view; C, paramere, unserside. D, labels. Scale bars: 1 mm. same locality and collector, leaf litter near stream, 1600– 1700 m a.s.l., 17–19.V.1984 (cRyv); Kazakhstan: 1 ♂, Almaty Area, Dzhungarskiy Alatau Mts, 6 km NE Rud nichnyi, Koksu River canyon, 1300–1400 m a.s.l., 44°41’N, 78°58’E, Betula sp., Populus, Picea etc. forest, 09–10. VI.2001, S.I. Golovatch leg. (cRyv); 1 ♂, Dzhungarskiy Alatau Mts, S slope, E ridge, middle reaches of Ispul Riv er, 1900 m a.s.l., litter in Abies forest, 14.VIII.1991, A.V. Tishechkin leg. (cRyv); 1 ♂, Almaty Area, Talgar District, Ak-Bulak, 2700 m a.s.l., 43.1613N, 77.2404E, 24.V.2014, O. Nakladal leg. (cKoc); 3 ♂, 1 ♀, Aksu-Dzhabagly, Isbala River, 15–25.VI.1983, B.V. Iskakov leg. (ZIN); 2 ♂, 1 ♀, Aksu-Dzhabagly, Taldy-Bulak River, 10–20.IV.1979, B.V. Iskakov leg. (ZIN); 1 ♂, Aksu-Dzhabagly, Ulken-Kaindy River, 15.VI.1991, V.A. Kastcheev leg. (ZIN); 1 ♂, SW slopes of Ugamskij Range, Silbili River, 18.VI.2004, A.V. Matalin leg. (cSch); 2 ♂, 1 ♀, Karzhantau, Kaskasu River, 10–12.VII.1983, B.V. Iskakov leg. Material examined. uedius novus, distribution, median lobe of the aedeagus laterally, and variability of the paramere (as examples speci rious localities numbered respectively on the map). Scale bars: 0.5 mm. 2 ♀, “Collect. Hauser” (NMW); 1 ♀, near Muminobod, 1300 m a.s.l., 15.V.1962, O.L. Kryzhanovsky leg. (ZIN); Tajikistan or Uzbekistan: 1 ♂, “Seravshan Boscha ra Glasunov 1892/ Q. dzambulensis J. Boháč det. 1983” (ZIN); 4 ♂, 2 ♀, “Seravshan Putchin pass. Glasunov 1892/ Q. dzambulensis J. Boháč det. 1983” (ZIN). 2 ♀, “Collect. Hauser” (NMW); 1 ♀, near Muminobod, 1300 m a.s.l., 15.V.1962, O.L. Kryzhanovsky leg. (ZIN); Tajikistan or Uzbekistan: 1 ♂, “Seravshan Boscha ra Glasunov 1892/ Q. dzambulensis J. Boháč det. 1983” (ZIN); 4 ♂, 2 ♀, “Seravshan Putchin pass. Glasunov 1892/ Q. dzambulensis J. Boháč det. 1983” (ZIN). “Taschkent Leder. Reitter” indicating that they are likely to be syntypes as well. Examination of all syntypes confirms that previous authors correctly interpreted this species. In order to fix its identity, here we designate one male syn type from NMW as the lectotype. Due to the intraspecific variability (Figs 16, 17) and resulting new synonymy Q. novus = Q. dzhambulensis (see below), we chose a syntype for lectotypification which has a more narrow longitudinal row of sensory peg setae on the paramere, best matching Coiffait’s (1967) illustration for Q. novus. “Taschkent Leder. Reitter” indicating that they are likely to be syntypes as well. Examination of all syntypes confirms that previous authors correctly interpreted this species. In order to fix its identity, here we designate one male syn type from NMW as the lectotype. Due to the intraspecific variability (Figs 16, 17) and resulting new synonymy Q. novus = Q. dzhambulensis (see below), we chose a syntype for lectotypification which has a more narrow longitudinal row of sensory peg setae on the paramere, best matching Coiffait’s (1967) illustration for Q. novus. Comments on taxonomy and lectotype designa tion. In the original description of Q. novus, Eppelsheim (1892) stated that he had examined numerous specimens from Tashkent and one from Margelan [Margilan in Uz bekistan]. He also stated in the introduction of that study that he received material from ‘Turkestan’ from multiple collections of Hauser, Staudinger and Reitter. In partic ular, he mentioned that the material from Tashkent from Reitter’s collection was collected by Leder. In NMW al together we found 11 conspecific specimens, all originally from Eppelsheim’s collection (with printed label “c.Ep plsh. dez.pensoft.net Material examined. 331 [hand written]”; 1 ♀, “Taschkent Leder.Reitter. [printed]/ Quedi us novus Epph. n.sp. [handwritten]/ 95”; 1 ♀, “Tasckkend [sic!] Reitter. [printed]/ Collect. Hauser [printed]”; 1 ♀, “Taschkend Leder. Reitter. [printed]” (NMW); 1 ♂, “Tash kent, Leder, Reitter [printed]/ Q. novus Epp. J. Boháč det. 1983 [handwritten]” (ZIN); 1 ♀, “Tashkent, Leder, Reitter [printed]/ Q. novus Epp. [handwritten]/ Q. novus Epp. J. Boháč det. 1983 [handwritten]/ Quedius dzambulensis Coiff. A. Solodovnikov det. 1997 [handwritten]” (ZIN) but “♂/ novus Epp. Deutsch. ent. Zeit. 1892. P. 331 [hand written]”; 1 ♀, “Taschkent Leder.Reitter. [printed]/ Quedi us novus Epph. n.sp. [handwritten]/ 95”; 1 ♀, “Tasckkend [sic!] Reitter. [printed]/ Collect. Hauser [printed]”; 1 ♀, “Taschkend Leder. Reitter. [printed]” (NMW); 1 ♂, “Tash kent, Leder, Reitter [printed]/ Q. novus Epp. J. Boháč det. 1983 [handwritten]” (ZIN); 1 ♀, “Tashkent, Leder, Reitter [printed]/ Q. novus Epp. [handwritten]/ Q. novus Epp. J. Boháč det. 1983 [handwritten]/ Quedius dzambulensis Coiff. A. Solodovnikov det. 1997 [handwritten]” (ZIN) Quedius dzambulensis: Holotype, “Turkestan Aulie Ata [printed]/ Aulie [handwritten]/ Quedius pyrenae us Coll. Reitter [pre-printed]/ Holotype [printed]/ Q. (Sauridus) dzambulensis Coiff. H. Coiffait det. 1967” (Fig. 16D) (HNHM). Additional material examined. Uzbekistan: 2 ♂, 1 ♀, Chatkal Mt. Ridge, Ters River bank up-stream of Yangiba zar, 27.IV.1986, I.A. Belousov leg. (cRyv);1 ♂, Chatkal Nature Reserve, bank of small rill, wet ground, Poaceae gen. sp., Equisetum sp., moss, 19.IX.1983, K.Yu. Eskov leg. (cRyv); 1 ♂, 60 km W Jizzakh, near Asmansay, by the stream, 15.V.1986, B.V. Iskakov leg. (ZIN); 1 ♂, 2 ♀, 60 km W Jizzakh, by the stream, Nuratau Mt., 14.V.1986, B.V. Iskakov leg. (ZIN); 1 ♂, “Trkst., Mnt. Nurata, UCHUN Glasunov 1892” [Nurata Distr., Nurata] (ZIN); 3 ♂, 1 ♀, Aman Kutan River 12.VI.1932, V.V. Gussakovsky leg. (ZIN); same locality and collector, but 1 ♀, 05.VII.1932 (ZIN); 4 ♂, 3 ♀, Agalyk, Samarkand, 22–23.Х.1935, Y.D. Kirschenblat leg. (ZIN); 4 ♂, 2 ♂, Kugitangtau Mts, near Kampyrtepa, Kampyrtepa say, under stones near stream, 1400 m a.s.l., 10.V.1984, A.V. Tanasevitch leg. (cRyv); dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 148 Figure 17. Quedius novus, distribution, median lobe of the aedeagus laterally, and variability of the paramere (as examples speci mens from various localities numbered respectively on the map). Scale bars: 0.5 mm. Quedius (Raphirus) sp. aff Q. coloratus Fauvel, 1875 Fig. 18 Quedius coloratus: Herman 2001, 3129 (summary of literature); Assing 2017, 207 (characters, distribution records, bionomics). Distribution. Based on the literature data (Table 1) that proved to be reliable for this species and the material examined here, Q. novus is widely distributed in Middle Asia and appears the most common in southern Kazakh stan, eastern Uzbekistan, western Kyrgyzstan and north eastern Tajikistan (Fig.17). Figure 18. Quedius sp. aff Q. coloratus (specimen from Kygyz stan). A, habitus. B, median lobe, lateral view; C, paramere, un derside. Scale bars: A = 1 mm; B, C = 0.5 mm. Bionomics. Quedius novus prefers various wet ground based plant debris or moss usually near water bodies. It seems to occur both in forested and open habi tats, up to 2700 m. Occasionally it was also found under stones and in dung. Material examined. Sensory peg setae on the paramere vary in arrangement, from denser (as in Coiffait’s illustration for Q. novus) to sparser (as in Coiffait’s illustration for Q. dzambulenisis) witin a longi tudinal group (Fig. 17). The mentioned variability has no geographic pattern. Therefore, we consider Q. dzambulen sis Coiffait, 1967 to be a junior synonym of Q. novus Epp. As a common and widespread species in Europe, Q. umbrinus was noted and illustrated in numerous papers. The latest summary can be found in Assing & Schülke (2012). Based on Kascheev (1989) and material exam ined here, Q. umbrinus occurs in the mountains of south ern Kazakhstan where it can be found in leaf litter and dung at elevations up to 1845 m. Diagnosis. Body dark brown; elytra with lighter col ored humeri and shallow micropunctation between punc tures; antennae slightly paler; scutellum without setifer ous punctation. (Figs 3E, 16A) Aedeagus (Figs 16B, C; 17): ventral tooth of median lobe located remotely from its apex; median lobe and paramere very broad (Figs 16B, 17); apex of paramere obtusely pointed and sensory peg setae arranged in long wide band in the middle of param ere (Figs 16C, 17). Quedius novus can be easily distin guished from the similar Middle Asian species Quedius umbrinus by the coloration and micropunctation of elytra and also by the mentioned above aedeagal characters. Material examined. Steind.”), whose morphology and label data match with the original description. We consider all of them to be syntypes. Of them, 8 specimens (on 5 pins) were earli er supplied with the curatorial printed red labels “types”; only two specimens have what we consider Eppelsheim’s hand written labels “novus Epp. Taschkent Leder.” and one specimen having “novus Epp. Deutsch. ent. Zeit. 1892. P. 331” label in a different handwriting probably attached by somebody later, after the species description was published. Also in the ZIN collection we found two more specimens conspecific with the syntypes at NMW and with the label Comments on the new synonym. The aedeagus of Q. novus was first illustrated by Wüsthoff (1938) based on non-type material. Coiffait (1963, 1970, 1978) redescribed the species, also illustrated the aedeagus and provided more records for Q. novus from Uzbekistan. Our examination of syntypes proved both Wüsthoff’s and Coiffait’s interpre tation of this species was correct. Also Coiffait (1967) de scribed Q. dzambulensis from Dzambul (Kazakhstan) (Fig. 16), a species which seemed to be very similar to Q. novus even from the illustrations of the aedeagi for both species. Later, Boháč (1988) examined material from the ZIN collection and provided new records from Uzbekistan, Tajikistan, Kyrgyzstan of Q. dzambulensis and only one record from Uzbekistan for Q. novus. He also stated that Q. novus is very closely related to Q. dzambulensis with which it can be easily confused. We checked all material from ZIN studied by Boháč (1988) and found that, with out knowing it, the only specimens he identified as Q. no 149 Dtsch. Entomol. Z. 65 (2) 2018, 117–159 vus were the syntypes of that species. All other specimens he identified as Q. dzambulensis. vus were the syntypes of that species. All other specimens he identified as Q. dzambulensis. is most similar to Q. novus from which it can be distin guished by the structure of aedeagus: median lobe with distinct ventral tooth near its apex and apical portion slightly curved dorso-ventrally (in lateral view); param ere (underside) with sensory peg setae arranged in wide lateral rows merging at parameral anterior margin. Our examination of a broader sample from Middle Asia, including types of both species, showed continuous vari ability in the structure of the aedeagus connecting the state of Q. novus with the state of Q. dzambulensis. Quedius (Raphirus) umbrinus Erichson, 1839 Fig. 4B Quedius umbrinus: Herman 2001, 3287 (summary of literature); Kascheev 1989, 36 (records); Assing and Schülke 2012, 475, 477 (diagnosis, distribution and bionomics, aedeagus illustration). Material examined. Kazakhstan: 1 ♂, Almaty Area, Dz hungarskiy Alatau Mts, 3 km SSE Lepsinsk, Bulinka River canyon, 1100–1800 m a.s.l., 45°30’N, 80°38’E, Betula sp., Malus, Populus etc. forest, 16–17.VI.2001, S.I. Golovatch leg. (cRyv); 1 ♂, Almaty Area, Talgar District., Ak-Bu lak, 43.1613N, 77.2214E, 10–15.V.2014, O. Nakladal leg. (cKoc); 1 ♂, Lle-Alatau NP Talgar env., Ak-Bulak Resort, horse and cow dung, 1690 m a.s.l., 43.27039N, 77.37137E, 12–15.V.2014, M. Kocián leg. (cKoc); 1 ♂, 1 ♀, Lle-Alatau NP Talgar env., SW slope, leaf litter sifting, 1845 m a.s.l., 43.25851N, 77.38501E, 09.V.2014, M. Kocián leg. (cKoc). Figure 18. Quedius sp. aff Q. coloratus (specimen from Kygyz stan). A, habitus. B, median lobe, lateral view; C, paramere, un derside. Scale bars: A = 1 mm; B, C = 0.5 mm. Comments on taxonomy, distribution and bion omics. Among all Middle Asian Raphirus, Q. umbrinus dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 150 Syntype teste D.J. Clarke 2014 GDI Imaging Project [printed]/ Photographed Kelsey Keaton 2014 Emu Cata log [printed]/ FMNHINS 2819453 Field Museum [print ed]” (Fig. 20B) (FMNH). Material examined. Kyrgyzstan: 1 ♂, N Tien-Shan, Kyrgyz Alatoo Mts, S Tokmak, near Kegety Pass, left tributary of Tuyuk River, 3000 m a.s.l., 42°24’43”N, 75°00’52”E, 13.V.1986, I.A. Belousov leg. (cRyv). Syntype teste D.J. Clarke 2014 GDI Imaging Project [printed]/ Photographed Kelsey Keaton 2014 Emu Cata log [printed]/ FMNHINS 2819453 Field Museum [print ed]” (Fig. 20B) (FMNH). Quedius ouzbekiscus: Holotype: Uzbekistan: ♂: “Ou zbekistan 8-68 Mts Tschingan 1500 m. H.C. [printed]/ Q. (Raphirus) ouzbekiscus Coiff. H. Coiffait det. 1968 [pre-printed]/ Holotype [printed]” (Fig. 21D); paratypes, 3 ♂, 35 ♀: same data, but “paratype [printed]” (MNHN) (one of the male paratypes is Q. fulvicollis, see that spe cies below). Comments on taxonomy, distribution and bionom ics. Externally and by the structure of the aedeagus, a sin gle male specimen from Kyrgyzstan (Fig.18) examined here seems to be a new species from the coloratus-group, recently revised by Assing (2017). Quedius coloratus and allied species forming that group are regional Mediterra nean endemics with allopatric distributions, altogether ex tending from Greece, through Turkey to Jordan. Quedius (Raphirus) umbrinus Erichson, 1839 Fig. 4B Iskakov leg.; 2 ♂, same locality and data, but in moss, 17.VII.1988, (ZIN); 1 ♂, 4 ♀, Ak su-Dzhabagly Nature Reserve, Taldy-Bulak River, 10– 20.IV.1979, B.V. Iskakov leg.; 1 ♂, 2 ♀, same locality and collector, but same locality and collector, but 10– 20.V.1979; 3 ♂, same locality and collector, but 04.V.1986 (ZIN); 2 ♂, Aksu-Dzhabagly Nature Reserve, Dzha tanskoi River., 18.V.1985, V.A. Kastcheev leg. (ZIN); 1 ♀, NW slopes of Ugamskiy Mt. Ridge, left tributary of Boldabrek River, 18.VI.2004, A.V. Matalin leg. (ZIN); 1 This specimen from the high elevations of Kyrgyz Ala too, far from the Mediterranean region, is a noteworthy find ing for the coloratus-group. More material is needed for a clearer understanding of its identity and formal description. Quedius (Raphirus) umbrinus Erichson, 1839 Fig. 4B Our spec imen differs from all known species of the coloratus-group in the structure of aedeagus (sharp apex of median lobe, subapical tooth located much further away form the apex, peg setae of the paramere less distinctly arranged in lon gitudinal rows and situated more medially (Fig. 18B, C). Quedius ouzbekiscus: Holotype: Uzbekistan: ♂: “Ou zbekistan 8-68 Mts Tschingan 1500 m. H.C. [printed]/ Q. (Raphirus) ouzbekiscus Coiff. H. Coiffait det. 1968 [pre-printed]/ Holotype [printed]” (Fig. 21D); paratypes, 3 ♂, 35 ♀: same data, but “paratype [printed]” (MNHN) (one of the male paratypes is Q. fulvicollis, see that spe cies below). ) Additional material examined. Kazakhstan: 1 ♂, Almaty Area, Dzhungarskiy Alatau Mts, 6 km NE Rud nichnyi, Koksu River canyon, 1300–1400 m a.s.l., 44°41’N, 78°58’E, Betula sp., Populus, Picea etc. forest, 09–10.VI.2001, S.I. Golovatch leg (cRyv).; 1 ♂, Kolbas tau, under bark in Abies forest, spruce logs, 04.VI.1988, V.A. Kastcheev leg. (ZIN); 5 ♂, Karatau Mts, Byzhi Riv er, Rynagus stream,757 m a.s.l., 43°57’08.7N, 68°12’04.2E, 24–25.VII.2010, V.A. Kastcheev leg. (ZIN); 1 ♂, 2 ♀, NW Karatau, 15 km NW Babai kurgan, 09.VI.1983, B.V. Iskakov leg. (ZIN); 1 ♂, Karatau Mts, 660 m a.s.l., 42°53’41.42N, 70°42’56.6E, 11.VII.2010, V.A. Kastcheev leg. (ZIN); 1 ♂, Karatau Mts, Khantagi River, 570 m a.s.l., 43°33’32.4N, 68°40’52.7E, 25. VI.2011, V.A. Kastcheev leg. (ZIN); 1 ♂, S Kazakhstan, near Merke, 10–15.VI.1988, B.V. Iskakov leg. (ZIN); 2 ♂, near Almaty, Zailiyskiy Alatau Mts, 2500–2800 m a.s.l, 29.VIII–03.IX.1992, K.Yu. Eskov leg.; 1 ♂, Almaty Area, Zailiyskiy Alatau Mts, Medeo near Almaty, Picea, Betula etc. forest, 1500–1600 m a.s.l., 43°10’N, 77°04’E, 27.V.2001, S.I. Golovatch leg.; 2 ♂, 2 ♀, S of Alma-Ata, upper reaches of Bolshaya Almatinka River, 2300–2500 m a.s.l., Picea schrenkiana forest, 06.VI.1993, S.I. Golo vatch leg. (cRyv); 2 ♂, Almaty Area, Talgar district, Ak-Bulak, 2700 m a.s.l., 43.1454N, 77.2404E, 10.V.2014, O. Nakladal leg. (cKoc); 1 ♂, Lle-Alatau, NP Talgar env., Ak-Bulak, resort horse and cow dung, 1750 m a.s.l., 12– 15.V.2014, 77.37145N, 43.26897 E.M. Kocián leg. (cKoc); 4 ♂, 1 ♀, Zailiysky Alatau, Sarybastau Valley, Chilik River, 12–15.VI.1988, V.A. Kastcheev leg. (ZIN); 5 ♂, 1 ♀, Ketmen Mts, Dolayty Valley, 15.VII.1988, V.A. Kastcheev leg. (ZIN); 2 ♂, same locality and collector, but 24.VIII.1987 (ZIN); 1 ♂, Kyrgyz Alatoo, 42°48’49.2E, 72°28’38.6N, 09.VII.2010, V.A. Kastcheev leg. (ZIN); 1 ♂, Aksu-Dzhabagly Nature Reserve, 27. VI.2004, A.V. Matalin leg. (ZIN); 1 ♂, Aksu-Dzhabagly Nature Reserve, Kish-Koyandy-Tau, meadow-steppe belt, 17.VII.1986, B.V. Quedius (Raphirus) hauseri Bernhauer, 1918 Figs 19, 22 Quedius (Raphirus) hauseri Bernhauer, 1918 Figs 19, 22 Quedius peneckei Bernhauer, 1918, syn. n. (Fig. 20)f Quedius peneckei Bernhauer, 1918, syn. n. (Fig. 20)f Quedius ouzbekiscus Coiffait, 1969, syn. n. (Fig. 21) Quedius hauseri Bernhauer, 1918, 94 (original descrip tion); Gridelli 1925, 154 (characters); Scheerpeltz 1933, 1443 14 (= Q. peneckei Bern.); Wüsthoff 1938, 221 (illustration of aedeagus); Coiffait 1978, 264 (characters, distribution records); Tronquet 1981, 71 (distribution records); Klimenko 1996, 121 (distribu tion records). Quedius peneckei Bernhauer, 1918, 95 (original de scription); Gridelli 1925, 154 (variety of Q. hauseri); Scheerpeltz 1933, 1443 (variety of Q. hauseri); Coif fait 1978, 264 (variety of Q. hauseri, characters).f Quedius ouzbekiscus Coiffait, 1969, 52 (original de scription); Coiffait 1970, 143 (list); Coiffait 1978, 278 (characters, notes); Kascheev 2001, 102 (distribution records). Type material examined. Quedius hauseri: Lectotype (here designated): Tajikistan: 1 ♂, “Mts. Karateghin Baldschuan 924 m. F. Hauser 1898. [printed]/ hauseri Bern. Typus [handwritten]/ Chicago NHMus M. Ber nhauer [printed]/ Syntype teste D.J. Clarke 2014 GDI Imaging Project [printed]/ Photographed Kelsey Keaton 2014 Emu Catalog [printed]/ FMNHINS 2819454 Field Museum [printed]” (Fig. 19E) (FMNH). Quedius peneckei: Syntype: Kyrgyzstan: 1 ♀, “Tien- schan. [sic!] Przewalsk. Karakolthal [printed]/ picipen nis Hr. Turkest. Penecke det. Bernhauer [pre-handwrit ten]/ acuminatus Hoch. var. elytris brevibus det. Bernh. [pre-printed]/ var. Peneckei Bern. Typus. [handwritten]/ Chicago NHMus M. Bernhauer Collection [printed]/ dez.pensoft.net 151 Dtsch. Entomol. Z. 65 (2) 2018, 117–159 Figure 19. Quedius hauseri, lectotype, male. A, habitus. B–D, aedeagus: B, median lobe, lateral view; C, paramere, underside, D, median lobe, ventral view. E, labels. Scale bars: A = 1 mm; B–D = 0.2 mm. Figure 19. Quedius hauseri, lectotype, male. A, habitus. B–D, aedeagus: B, median lobe, lateral view; C, paramere, underside, D, median lobe, ventral view. E, labels. Scale bars: A = 1 mm; B–D = 0.2 mm. (cRyv); Kyrgyzstan: 1 ♂, Chuy Region, S Bishkek, SE Kashkasu, Kyzyl-Beles, 2010 m a.s.l., 74.3226N, 42.3904E, 05.VII.2011, J. Frisch leg. (cKoc); 1 ♂, Tien Shan, Ala Archa, 2000 m a.s.l., 09.VII.1984, D.W. Wrase leg. (cSch); 4 ♂, 1 ♀, Kungey-Alatoo, Chilik River, tract Sarybastau, 12–15.VI.1988, V.A. Kastcheev leg. (ZIN); 1 ♂, Issyk-Kul’ Area, Tyupskiy District, Kungey-Alatoo Mts, valley 3–4 km N of Shaty, banks of river and rill: under stones, in moss, and among sedges, 30.VIII.1983, A.B. Ryvkin leg. (cRyv); 1 ♂, 1 ♀, Issyk-Kul’, Kyzyl- Tuu-Kyzyl-Suu, Barskoon Barskaun Pass, Picea schren kiana-forest, 2200 m a.s.l., 77.3551N, 42.0242E, 23. Quedius (Raphirus) hauseri Bernhauer, 1918 Figs 19, 22 VI.2011, J. Frisch leg. (cKoc); 3 ♂, Issyk-Kul’ Area, Ter skey-Alatoo Mts, Chon-Kyzyl-Suu River basin, Kashka tor River canyon near Geographical Field Research Sta tion, 2900 m a.s.l., in moss & under stones near swampy rill in slope forest with Picea schrenkiana & Juniperus turcomanica, 25.VIII.1983, A.B.Ryvkin leg. (cRyv); 2 ♂, 1 ♀, Terskey-Alatoo Mts, Chon-Kyzyl-Suu River, spruce litter, motley grass, 2150 m a.s.l., 08.VII.1988, V.V. Yanushev leg. (cRyv); 1 ♂, Terskey-Alatoo, Chon- Kyzyl-Suu River, 2500 m a.s.l., 28.VI.1959, Panfilov leg. (ZIN); 1 ♂, 1 ♀, Terskey-Alatoo, Lahol River, 3300 m a.s.l., 25.VII.1992, D. Milko leg. (cSch); 1 ♂, East ♂, 2 ♀, Karzhantau, Kaskasu River, 10–12.VII.1983, B.V. Iskakov leg. (ZIN); Uzbekistan: 1 ♂, Chimgan Val ley, bank of stream with Equisetum sp., 1984, А.V. Tana sevitch leg. (cRyv); 1 ♂, Tien Schan, Aktasch, Taschkent, 2000 m a.s.l., 13.VII.1984, D.W. Wrase leg. (cSch); 1 ♂, 2 ♀, Samarkand, Agalik, 23.X.1935, Y.D. Kirschenblat leg. (ZIN); 1 ♂, 1 ♀, Aman Kutan, 12.VI.1932, leg. V.V. Gussakovsky (ZIN); 1 ♂, same locality, but in dung, 29.V.1965, Guryeva leg. (ZIN); 1 ♂, ”789[.] 24.5.62. Ré gion de Boukhara. Ouest de Zeravchan [,] pris d’un ruis seau’[=789. 24.V.1962. Bukhara Area, Eastwards of Zer avshan, captured from a rill]/ ‘24.5.62. Китабский перевал [.] У ручья [.] пр [.] 121-124 [.]’ [=24.V.1962. Kitab Pass. Near rill. Samples 121–124.] “ (ZMMU); 5 ♂, 1 ♀, “797 [.] 11.5.62. Région de Boukhara. Ouest de Zeravchan. Pente marécageuse.” [=797. 11.V.1962. Bukhara Area. Eastwards of Zeravshan. Swampy slope.] (ZMMU); 1 ♂, Shahrisabz, near city wall, 14.XII.1941, K.V. Arnoldi leg.; 1 ♂, 1 ♀, same locality and collector, gardens along road to Kitab, 27.XI.1941 (cRyv); 1 ♂, Kugitangtau Mts, near Kampyrtepa, Kampyrtepa say, un der stones along stream, 1400 m a.s.l., 15.V.1984, A.V. Tanasevitch leg.; 3 ♂, same locality and collector, but in litter along stream, 1600–1700 m a.s.l., 17–19.V.1984 (cRyv); Kyrgyzstan: 1 ♂, Chuy Region, S Bishkek, SE Kashkasu, Kyzyl-Beles, 2010 m a.s.l., 74.3226N, 42.3904E, 05.VII.2011, J. Frisch leg. (cKoc); 1 ♂, Tien Shan, Ala Archa, 2000 m a.s.l., 09.VII.1984, D.W. Wrase leg. (cSch); 4 ♂, 1 ♀, Kungey-Alatoo, Chilik River, tract Sarybastau, 12–15.VI.1988, V.A. Kastcheev leg. (ZIN); 1 ♂, Issyk-Kul’ Area, Tyupskiy District, Kungey-Alatoo Mts, valley 3–4 km N of Shaty, banks of river and rill: under stones, in moss, and among sedges, 30.VIII.1983, A.B. Ryvkin leg. Quedius (Raphirus) hauseri Bernhauer, 1918 Figs 19, 22 (cSch); 1 ♂, same locali ty and collectors, but snowfiled edge at km 55, 1800 m a.s.l., 28.VI.1990 (cSch); 8 ♂, 1 ♀, same locality and Tien Shan, Turgen valley, Tshon Ashu Pass, 2900–3500 m a.s.l., VII.2001, V.G. Dolin & S. Andreeva leg. (cSch); 1 ♂, 1 ♀, Osh Area, Sary-Chelek Biosphere Reserve, shore of Iri-Kyol Lake, mosses and peat among Carex sp., Juncus sp., and Phragmites australis, 04–05. VIII.1983, A.B. Ryvkin leg.; 1 ♂, Osh Area, Sary-Chelek Biosphere Reserve, combe under pass from Khodzha- Ata River to the “head” of Sary-Chelek Lake, 2000 m a.s.l., at slope with Carex spp., Brachypodium sp., Poa sp., etc., 11.VIII.1983, A.B. Ryvkin leg.; 1 ♂, Osh Area, Sary-Chelek Biosphere Reserve, “head” of Sary-Chelek Lake, 1940–1945 m a.s.l., lake shore and bottom of part ly dried rill with Carex spp., Equisetum sp., Juncus sp., Phragmites australis, etc., 12.VIII.1983, A.B. Ryvkin leg.; 2 ♂, 1 ♀, Sary-Chelek Biosphere Reserve, 04–10. VII.1983, S.K. Alekseev leg.; 1 ♂, Tien Shan, Chatkal Mt. Ridge, Sary-Chelek Biosphere Reserve, 1550–2200 m a.s.l., forests, 29–31.V.1993, S.I. Golovatch leg. (cRyv); 2 ♂, Osh Area, Sary-Chelek Biosphere Reserve, Keltesay stream valley, leaf litter in slope Juglans forest, 28.VI.1983, A.B.Ryvkin leg. (cRyv); 1 ♂, Ala Kul Pass, Kara Kol, 3860 m a.s.l., VII.1998, C. Reuter leg. (cSch); 1 ♀, Bajduly Mt., Dolon Pass, 1600 m a.s.l., 15–18. VII.2001, V.G. Dolin & S. Andreeva leg. (cSch);1 ♂, Tien Shan, Dolon Pass, 2500–3200 m, 23–25.VII.1991, J. Turna leg. (cSch); 1 ♂, Naryn Area, 50 km W Naryn City, Karatoo Mts, Ala-Myshik Tract, leaf litter in birch forest at river bank, 22.VIII.1983, A.B. Ryvkin leg.; 2 ♂, Ferganskiy Mt. Ridge, near Arslanbob, dry subalpine meadow, under stones, 2200 m a.s.l., 1.X.1983, K.Yu. Eskov leg. (cRyv); 2 ♂, Osh Area, W Tien Shan, Fer ganskiy Mt. Ridge, Yarodar, 1300 m a.s.l., rill bank, in leaf litter and under stones, 24–25.IX.1983, K.Yu. Eskov leg. (cRyv); 1 ♂, Tien Shan, E slope Ferghanskiy Mt. Ridge, upper reaches of Urumbash River 2000 m a.s.l., 19.VII.2001, A.V. Puchkov leg. (cSch); 1 ♂, Tien Shan, Ferghanskiy Mt. Ridge, Burgut Pass, 3200 m a.s.l., 20. VII.2001, A.V. Puchkov leg. (cSch); 1 ♂, Ferganskiy Mt. Ridge, Kara-Unkur valley, 2150 m, 22–24.VII.2001, V.G. Dolin & S. Andreeva leg. (cSch); 1 ♂, Alay valley, Nyra, 19.VII.1960, Lopatin leg. (ZIN); Tajikistan: 1 ♂, 2 ♀, “Seravshan Kschtut. Artutsch. Quedius (Raphirus) hauseri Bernhauer, 1918 Figs 19, 22 (cRyv); 1 ♂, 1 ♀, Issyk-Kul’, Kyzyl- Tuu-Kyzyl-Suu, Barskoon Barskaun Pass, Picea schren kiana-forest, 2200 m a.s.l., 77.3551N, 42.0242E, 23. VI.2011, J. Frisch leg. (cKoc); 3 ♂, Issyk-Kul’ Area, Ter skey-Alatoo Mts, Chon-Kyzyl-Suu River basin, Kashka tor River canyon near Geographical Field Research Sta tion, 2900 m a.s.l., in moss & under stones near swampy rill in slope forest with Picea schrenkiana & Juniperus turcomanica, 25.VIII.1983, A.B.Ryvkin leg. (cRyv); 2 ♂, 1 ♀, Terskey-Alatoo Mts, Chon-Kyzyl-Suu River, spruce litter, motley grass, 2150 m a.s.l., 08.VII.1988, V.V. Yanushev leg. (cRyv); 1 ♂, Terskey-Alatoo, Chon- Kyzyl-Suu River, 2500 m a.s.l., 28.VI.1959, Panfilov leg. (ZIN); 1 ♂, 1 ♀, Terskey-Alatoo, Lahol River, 3300 m a.s.l., 25.VII.1992, D. Milko leg. (cSch); 1 ♂, East dez.pensoft.net 152 Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... Figure 20. Quedius peneckei (new synonym of Q. hauseri), syntype, female. A, habitus. B, labels. Scale bar: 1 mm. Figure 20. Quedius peneckei (new synonym of Q. hauseri), syntype, female. A, habitus. B, labels. Scale bar: 1 mm. City, Karatoo Mts, Ala-Myshik Tract, leaf litter in birch forest at river bank, 22.VIII.1983, A.B. Ryvkin leg.; 2 ♂, Ferganskiy Mt. Ridge, near Arslanbob, dry subalpine meadow, under stones, 2200 m a.s.l., 1.X.1983, K.Yu. Eskov leg. (cRyv); 2 ♂, Osh Area, W Tien Shan, Fer ganskiy Mt. Ridge, Yarodar, 1300 m a.s.l., rill bank, in leaf litter and under stones, 24–25.IX.1983, K.Yu. Eskov leg. (cRyv); 1 ♂, Tien Shan, E slope Ferghanskiy Mt. Ridge, upper reaches of Urumbash River 2000 m a.s.l., 19.VII.2001, A.V. Puchkov leg. (cSch); 1 ♂, Tien Shan, Ferghanskiy Mt. Ridge, Burgut Pass, 3200 m a.s.l., 20. VII.2001, A.V. Puchkov leg. (cSch); 1 ♂, Ferganskiy Mt. Ridge, Kara-Unkur valley, 2150 m, 22–24.VII.2001, V.G. Dolin & S. Andreeva leg. (cSch); 1 ♂, Alay valley, Nyra, 19.VII.1960, Lopatin leg. (ZIN); Tajikistan: 1 ♂, 2 ♀, “Seravshan Kschtut. Artutsch. Glasunov 1892” (ZIN); 1 ♀, “Jagnob Karsau Glasunov 1892” (ZIN); 3 ♂, “Jagnob Chishartob Glasunov 1892” (ZIN); 1 ♂, 1 ♀, “Iskander-Kul/ Glasunov 1892”; 1 ♂, same collector, but “Iskander-Daria” (ZIN); 1 ♀, Pamir-Alai, Seravshan Val ley, near Navobod, 10–01.VII.1990, M. Schülke & D.W. Wrase leg. (cSch); 3 ♂, 1 ♀, Pamir-Alai, Hisaar Mts, Ad shuk-Cleft near Warsob, 1200 m a.s.l., 01–03.VII.1990, M. Schülke & D.W. Wrase leg. Quedius (Raphirus) hauseri Bernhauer, 1918 Figs 19, 22 collectors, but Bachufer, 01–03.VII.1990 (cSch); 2 ♀, “Gissaar: Karatag. (stgr.) E. Willberg” (ZIN); 5 ♂, 1 ♀, Dushanbe, Charangon River, 03.VI.1934, V.V. Gussa kovky leg. (ZIN); 1 ♂, Dushanbe, foothills, 16.V.1963, A.V.Bogachev leg. (ZMMU); 1 ♂, “Prov. Kuliab, Ak- sou-Tal, F.Hauser 1898/ Gift from Nat. Mus. Praha. 2009” (ZMMU); 1 ♀, Schugnan, Sardym, Gunt River, 16.VII.1897, A. Kaznakov leg. (ZIN); Uzbekistan or Tajikistan: 1 ♂, ”Buchara./ Staudinger./ 825./ boops/ Quedius (Raphirus) acuminatus” (ZMMU); 1 ♂, “Putchin Pass Glasunov 1892” (ZIN). We were able to study a male specimen from the FMNH (for details see above) which is clearly a syntype and which we designate as the lectotype to fix the identity of that species. Our examination of the type specimen of Q. hauseri confirms the correct identification of this spe cies by both Wüsthoff (1938) and Coiffait (1978). In the same paper, Bernhauer (1918) described Quedius peneckei as a brachypterous variation of Q. hauseri from ’Tien-Shan, Przewalsk, Karakoltal’ [now Karakol, Issyk-Kul region, Kyrgyzstan], also not specifying either a number or sex of the material he studied. He only stated that Q. peneckei was similar to Q. fulvicollis from which it could be distinguished by the elongate pronotum and more densely punctured abdomen. Gridelli (1924) and Coiffait (1978) also considered Q. peneckei as a variation of Q. hauseri. Of them, Gridelli (1924) stated that he had studied the type material but without details on sex or number of specimens. In catalogs Q. peneckei is given as a variation (Scheerpeltz, 1933; Hermann, 2001) or synonym (Schülke & Smetana, 2015) of Q. hauseri. There was not a single illustration of Q. peneckei ever published. We were able to study one female specimen from the FMNH which is clearly a syntype of Q. peneckei. It is conspecific with Q. hauseri and does not look to be distinctly brachypterous. Based on that and the fact that there is only one species of this type in Middle Asia, Comments on taxonomy, lectotype designation and new synonymy. In the original description of Q. hau seri, Bernhauer (1918) did not specify the type materi al but he mentioned localities “Baldschuan [Baljuvon], 924 m, Sary-pul, 1482 m” [Tadjikistan: Karateghin Mts.] (Fig.19E) and “Ost-Buchara: Tschitschantan, Karatag und Repetek, vor.” [Tadjikistan: Vorukh jamoat, accord ing to Frisch 2015] where his material came from. Also it is clear from the original description that he studied both sexes. All this suggests multiple syntypes. Quedius (Raphirus) hauseri Bernhauer, 1918 Figs 19, 22 Glasunov 1892” (ZIN); 1 ♀, “Jagnob Karsau Glasunov 1892” (ZIN); 3 ♂, “Jagnob Chishartob Glasunov 1892” (ZIN); 1 ♂, 1 ♀, “Iskander-Kul/ Glasunov 1892”; 1 ♂, same collector, but “Iskander-Daria” (ZIN); 1 ♀, Pamir-Alai, Seravshan Val ley, near Navobod, 10–01.VII.1990, M. Schülke & D.W. Wrase leg. (cSch); 3 ♂, 1 ♀, Pamir-Alai, Hisaar Mts, Ad shuk-Cleft near Warsob, 1200 m a.s.l., 01–03.VII.1990, M. Schülke & D.W. Wrase leg. (cSch); 1 ♂, same locali ty and collectors, but snowfiled edge at km 55, 1800 m a.s.l., 28.VI.1990 (cSch); 8 ♂, 1 ♀, same locality and Tien Shan, Turgen valley, Tshon Ashu Pass, 2900–3500 m a.s.l., VII.2001, V.G. Dolin & S. Andreeva leg. (cSch); 1 ♂, 1 ♀, Osh Area, Sary-Chelek Biosphere Reserve, shore of Iri-Kyol Lake, mosses and peat among Carex sp., Juncus sp., and Phragmites australis, 04–05. VIII.1983, A.B. Ryvkin leg.; 1 ♂, Osh Area, Sary-Chelek Biosphere Reserve, combe under pass from Khodzha- Ata River to the “head” of Sary-Chelek Lake, 2000 m a.s.l., at slope with Carex spp., Brachypodium sp., Poa sp., etc., 11.VIII.1983, A.B. Ryvkin leg.; 1 ♂, Osh Area, Sary-Chelek Biosphere Reserve, “head” of Sary-Chelek Lake, 1940–1945 m a.s.l., lake shore and bottom of part ly dried rill with Carex spp., Equisetum sp., Juncus sp., Phragmites australis, etc., 12.VIII.1983, A.B. Ryvkin leg.; 2 ♂, 1 ♀, Sary-Chelek Biosphere Reserve, 04–10. VII.1983, S.K. Alekseev leg.; 1 ♂, Tien Shan, Chatkal Mt. Ridge, Sary-Chelek Biosphere Reserve, 1550–2200 m a.s.l., forests, 29–31.V.1993, S.I. Golovatch leg. (cRyv); 2 ♂, Osh Area, Sary-Chelek Biosphere Reserve, Keltesay stream valley, leaf litter in slope Juglans forest, 28.VI.1983, A.B.Ryvkin leg. (cRyv); 1 ♂, Ala Kul Pass, Kara Kol, 3860 m a.s.l., VII.1998, C. Reuter leg. (cSch); 1 ♀, Bajduly Mt., Dolon Pass, 1600 m a.s.l., 15–18. VII.2001, V.G. Dolin & S. Andreeva leg. (cSch);1 ♂, Tien Shan, Dolon Pass, 2500–3200 m, 23–25.VII.1991, J. Turna leg. (cSch); 1 ♂, Naryn Area, 50 km W Naryn 2 dez.pensoft.net Dtsch. Entomol. Z. 65 (2) 2018, 117–159 153 Figure 21. Quedius ouzbekiscus (new synonym of Q. hauseri), holotype, male. A, habitus; B, median lobe, lateral view; C, param ere, underside; D, labels. Scale bars: A = 1 mm; B, C = 0.2 mm. Figure 21. Quedius ouzbekiscus (new synonym of Q. hauseri), holotype, male. A, habitus; B, median lobe, lateral view; C, param ere, underside; D, labels. Scale bars: A = 1 mm; B, C = 0.2 mm. dez.pensoft.net Material examined. eastern Kazakstan (southern border through Dzhungaskiy Alatau) to southern Tajikistan (Pamir Mountains, Schug nan) (Fig. 22). It was also recorded from Afganistan (Schülke and Smetana, 2015). Additional material. Kazakhstan: 3 ♂, Karatau Mts, 660 m a.s.l., 42°53’41.42N, 70°42’56.6E, 11.VII.2010, V.A. Kastcheev leg. (ZIN); Uzbekistan: 1 ♂, Chatkal Nature Reserve, bank of small rill, wet ground, Poaceae gen. sp., Equisetum sp., moss, 19.IX.1983, K.Yu. Eskov leg. (cRyv); 1 ♂, Golodnaya Step [Sirdaryo Reg., Guliston], 17.V.1903, G.G. Jacobson (ZIN); 1 ♂, “Trkst. Mnt. Nurata UCHUN Glasunov 1892” (ZIN); 1 ♂, Samarkand Reg., Kattakur gan, 18.V.1932, V.V. Gussakovsky leg. (ZIN); 1 ♂, Aman Kutan, shady wet say, near forestry building, 31.V.1942, K.V. Arnoldi leg. (ZMMU); 2 ♂, Qashqadaryo Reg., Ki tab, 30.VII.1933, V.V. Gussakovsky leg. (ZIN); Turkmen istan: 1 ♂, Kov-Ata [Bacharden] 110 km NW Ashchabad, 10.V.1989, D.W. Wrase leg. (cSch); 2 ♂, 5 ♂, N Kopetdag, Firjusa-Cleft, near Ashchabad, 07.V.1989, D.W. Wrase leg. (cSch); 1 ♂, Kugitangtau Mts, near Svintsovyi Rud nik, 1300 m a.s.l., under stones, 11.V.1984, A.V. Tanase vitch leg. (cRyv); Tajikistan: 3 ♂, Warsobob, 03.VI.1988, S.V. Saluk leg. (cRyv); 1 ♂, Dushabe, Kharangon River, 03.VI.1934, V.V. Gussakovsky leg. (ZIN); 2 ♂, 15 km SE Shaahrtuz, Tuyntau Mt., 02–03.VI.1982, G.S. Medvedev Bionomics. Based on the material examined here Q. hauseri usually inhabits various humid ground based plant debris or moss near water bodies. It occurs both in forested and open habitats. It also can be found under stones, bark and in dung, mostly at the medium to high elevations up to 3300 m. Quedius (Raphirus) fulvicollis Stephens, 1833 Fig. 23 Quedius fulvicollis: Herman 2001, 3159 (summary of lit erature); Assing and Schülke 2012, 481, 482 (diagno sis, distribution and bionomics, aedeagus illustration); Klimenko 1960, 121 (distribution records) Material examined. One of the male paratypes of Q. ou zbekiscus (new synonym of Q. hauseri, see above), for details see material examined for Q. hauseri and Fig. 23 Comments on taxonomy, distribution and bionomics. One of the male paratypes of Q. ouzbekiscus (new synonym of Q. hauseri) was in fact a different species that we tenta tively identify as Q. fulvicollis. It can be easily distinguished from Q. hauseri by the shape of the paramere (compare Fig. 23C and Figs 19C, 21C, respectively). Quedius fulvicollis is considered a widely distributed Holarctic species, in Asia confined to Siberia and Russian Far East (Schülke and Smetana, 2015). The specimen from Chatkal Mountains in Uzbekistan examined here would be a distinct southernmost record for this species in the Palaearctic region and the first record for Middle Asia. In this respect it is noteworthy that it comes from ca. 1500 m of elevation. Also it is remarkable that this specimen from Middle Asia stands out from the variability range of Q. fulvicollis by the very narrow middle portion of the paramere and shorter and more irregular rows of peg setae. It well may be that our specimen represents a species new to science. Given the poorly studied variation of Q. fulvicollis, which itself maybe a complex of species and very limited material from Middle Asia, a decision on this matter is pending further study. Figure 23. Quedius 'fulvicollis' (specimen from Uzbekistan). A, habitus. B, median lobe, lateral view; C, paramere, under side. Scale bars: A = 1 mm; B, C = 0.2 mm. In general Q. fulvicollis prefers forest landscapes and usually can be found in wet ground-based debris, at banks of ponds, forest lakes and in swampy areas. Apart from the elevation, no bionomic data is available for the Mid dle Asian specimen. An earlier record of Q. fulvicollis from Tajikistan in Klimenko (1996) was based on uncer tain material and needs verification. Quedius (Raphirus) hauseri Bernhauer, 1918 Figs 19, 22 Bernhauer (1918) compared Q. hauseri with Q. boops and Q. acum inatus. Wüsthoff (1938) illustrated the structure of the aedeagus for Q. hauseri for the first time based on some material “aus Buchara” [from Buchara]. Next, the aedea gus for Q. hauseri was illustrated by Coiffait (1978), also based on some non-type material. dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 154 Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 154 Figure 22. Quedius hauseri, distribution and variability of the paramere (as an example specimens from one locality, indicated by black dot). Scale bar: 1 mm. Figure 22. Quedius hauseri, distribution and variability of the paramere (as an example specimens from one locality, indicated by black dot). Scale bar: 1 mm. which is rather common and widespread (Fig. 22), we place Q. peneckei in synonymy with Q. hauseri.f Diagnosis. Head and abdomen usually black, prono tum, elytra and appendages pale-brown to brown; scutel lum punctate (Figs 19A, 20A, 21A). Aedeagus (in lateral view) (Figs 19B, 21B): ventral contour of median lobe apically and basally from subapical tooth form one line, so that the tooth is protruding and median lobe apical ly from that does not look like an axe blade. Among all Middle Asian Raphirus, only Q. hauseri and Q. fulvicollis (see below) have the punctate scutellum. Quedius hauseri differs from Q. fulvicollis by the shape of the paramere (Figs 19C, 21C, 23C, respectively). Coiffait (1969) described Q. ouzbekiscus from Uzbeki stan based on the male holotype (Fig. 21) and 40 para types (4 males and 36 females). He considered it similar to the species from the boops-group and stated that Q. ouzbekiscus can be distinguished from other members of the group by the structure of aedeagus and proportions of the body. Also he noticed that Q. ouzbekiscus is es pecially similar to Q. fulvicollis. Our examination of the type material of Q. ouzbekiscus reveals that this species is conspecific with Q. hauseri and therefore we place the former in synonymy with the latter. Distribution. Quedius hauseri is common and widely distributed in Middle Asia where it occurs from south Distribution. Quedius hauseri is common and widely distributed in Middle Asia where it occurs from south 155 Dtsch. Entomol. Z. 65 (2) 2018, 117–159 eastern Kazakstan (southern border through Dzhungaskiy Alatau) to southern Tajikistan (Pamir Mountains, Schug nan) (Fig. Quedius (Raphirus) hauseri Bernhauer, 1918 Figs 19, 22 22). It was also recorded from Afganistan (Schülke and Smetana, 2015). Material examined. Quedius (Raphirus) scintillans Gravenhorst, 1806 Fig. 4A Quedius scintillans: Herman 2001, 3260 (summary of lit erature); Assing and Schülke 2012, 471, 473 (diagno sis, distribution and bionomics, aedeagus illustration) Figure 23. Quedius 'fulvicollis' (specimen from Uzbekistan). A, habitus. B, median lobe, lateral view; C, paramere, under side. Scale bars: A = 1 mm; B, C = 0.2 mm. dez.pensoft.net Maria Salnitska & Alexey Solodovnikov: Revision of the Quedius fauna of Middle Asia... 156 leg. (ZIN); 4 ♂, 1 ♀, Pyandj District, in hay, 28.IV.1988, S.V. Saluk leg. (cRyv); Uzbekistan or Tajikistan: 2 ♂, “Uzbekistan Buchara./ Staudinger. 823.” (ZMMU). leg. (ZIN); 4 ♂, 1 ♀, Pyandj District, in hay, 28.IV.1988, S.V. Saluk leg. (cRyv); Uzbekistan or Tajikistan: 2 ♂, “Uzbekistan Buchara./ Staudinger. 823.” (ZMMU). of Denmark, a much smaller, geographically uniform and flat area, contains 41 species (http://danbiller.dk) as opposed to 28 species recorded from Middle Asia. It is not expected that the Middle Asian Quedius fauna will significantly grow with more explorations. But some increase of this number is likely, due to widespread species to be found there and new species for science to be discovered, especially from the mountain areas of Middle Asia. We hope that our work will encourage further field exploration of this diverse re gion by using collecting techniques targeting Staphylinidae, especially sifting. Comments on taxonomy, distribution and bionom ics. Quedius scintillans is widely distributed in Europe, Western and Middle Asia, and its diagnostic characters, distribution and biology were recently summarized in Assing and Schülke (2012). In Middle Asia, from the newly examined material here, the species is recorded in southern and eastern Turkmenistan and southwestern Ta jikistan for the first time. From all Middle Asian Raphirus species it can be eas ily distinguished by the presence of two additional punc tures between anterior frontal punctures on the head. Acknowledgements Quedius scintillans prefers various wet ground-based debris mostly in lowland forests or open landscapes. In the mountains it can be found up to 1300 m elevation. We are very thankful to all curators and colleagues listed in the “Material and methods” section who provided the ma terial under their care for this paper. Our special thanks are due to Alexander Ryvkin (Moscow, Russia) for his valuable help in decoding and editing the label data and also to Bo ris Korotyaev, Igor Belousov, Ilya Kabak and Sergey Sinev (Saint-Petersburg, Russia) for their further help in georefer encing some of the labels. We are grateful to Lech Borow iec (Wrocław, Poland), Aslak Kappel Hansen (Copenha gen, Denmark), Andre Nel (Paris, France) all colleagues who provided or helped us to obtain some illustrations. We sincerely thank Michael Schülke for providing some dif ficult-to-obtain references, and Volker Assing (Hannover, Germany) for his critical and important comments on the content of the manuscript. And we are very grateful to both reviewers Aleš Smetana and Adam Brunke (Ottawa, Cana da) for their thoughtful comments and suggestions that sig nificantly improved the quality of this paper. This study was conducted using equipment of the Center for Molecular and Cell Technologies and the Resource Center ‘Chromas’ of the Research Park at St. Petersburg State University. Sup port for this research partly was provided by the Saint-Pe tersburg State University foundation (Saint-Petersburg, Russia) under the programm "Meropriyatie № 5". Discussion This revision is the first focused summary on Quedius of Middle Asia. It clarifies the taxonomy of many poorly or very poorly known species such as Q. (s. str.) subunicolor, Q. (M.) capitalis, Q. (M.) fusicornis, Q. (M.) solskyi and Q. (R.) cohaesus, and it records from Middle Asia a few widely distributed species such as Q. (s. str.) fuliginosus, Q. (s. str.) sundukovi and Q. (R.) pseudonigriceps for the first time. It shows how confusing and incomplete the tax onomy was of the species that constitute the core of this fauna. In the course of this revision (including Salnitska and Solodovnikov 2018) the rate of new species dicovery was negligible compared to the rate of revealed misidentifica tions and synonymies. Many “endemic” species described from various regions of Middle Asia, mainly by H. Coiffait, turned out to be synonyms of the species described from this region at the border between XIX and XX centuries (Table 1). These species, with the newly examined material, expectedly turned out to be more widespread than they were previously thought. Several species, especially in the subge nus Microsaurus, remain very poorly known (e.g., Q. (M.) bucharensis, Q. (M.) fusicornis, Q. (M.) solskyi, Q. (M.) koltzei and Q. (M.) tajikiscus). Here they are represented by fragmentary, poorly georeferenced type material (often females only) and, at most, a few additional specimens. For Q. (s. str.) subunicolor and Q. (s. str.) sundukovi and Q. (M.) koltzei, new distributional records from Middle Asia change our idea of their distribution patterns and calls for their more thorough exploration. In general this revision made it obvi ous that, apart from a handful of species such as Q. (s. str.) fuliginosus, Q. (s. str.) vicinus, Q. (M.) ochripennis, Q. (R.) hauseri, Q. (R.) imitator, Q. (R.) limbatus, Q. (R.) novus, Q. (R.) pseudonigriceps and Q. (R.) scintillans, well represent ed in the examined material, the Middle Asian species are known from very scarce sampling. Because Middle Asia is mainly a warm and arid region that is not well suited to such a distinctly temperate and mesophilous genus, the fauna of Quedius is relatively poor. For example, the Quedius fauna dez.pensoft.net References Stuttgarter Beiträge zur Naturkunde A, Neue Serie 8: 137–163. Boháč J, Matějíček J, Rous R (Eds) (2005) Staphylinidae (drabčíkovití). In: Farkač J, Král D, Škorpík M (Eds) Červený seznam ohrožených druhů České republiky. Bezobratlí. 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https://openalex.org/W2889061099	http://nrl.northumbria.ac.uk/id/eprint/43124/1/6842306.pdf	English	null	Scoping Review of the Driving Behaviour of and Driver Training Programs for People on the Autism Spectrum	Behavioural neurology	2,018	cc-by	14,695	This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/id/eprint/43124/ This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/id/eprint/43124/ Northumbria University has developed Northumbria Research Link (NRL) to enable users to access the University’s research output. Copyright © and moral rights for items on NRL are retained by the individual author(s) and/or other copyright owners. 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Published by: Hindawi URL: https://doi.org/10.1155/2018/6842306 <https://doi.org/10.1155/2018/684230 This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/id/eprint/43124/ This version was downloaded from Northumbria Research http://nrl.northumbria.ac.uk/id/eprint/43124/ Northumbria Research Link Citation: Wilson, Nathan J., Lee, Hoe C., Vaz, Sharmila, Vindin, Priscilla and Cordier, Reinie (2018) Scoping Review of the Driving Behaviour of and Driver Training Programs for People on the Autism Spectrum. Behavioural Neurology, 2018. pp. 1-17. ISSN 0953-4180 Correspondence should be addressed to Hoe C. Lee; hoe.lee@curtin.edu.au Correspondence should be addressed to Hoe C. Lee; hoe.lee@curtin.edu.au Received 28 February 2018; Revised 19 June 2018; Accepted 30 July 2018; Published 28 August 2018 Academic Editor: Veit Roessner Copyright © 2018 Nathan J. Wilson 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. Gaining a driver’s licence represents increased independence and can lead to improved quality of life for individuals and their families. Learning to drive a motor vehicle and maintaining safe on-road skills are often more diﬃcult for people on the autism spectrum. Many countries currently have no autism-speciﬁc licencing requirements for learner drivers, and there is a general lack of ASD-speciﬁc support and training packages for individuals, their families, and driving instructors. This review synthesises the peer-reviewed literature about the driving characteristics of drivers on the spectrum and driver training available for the cohort. The evidence in this review showed that individuals on the autism spectrum drive diﬀerently from their neurotypical counterparts. There are shortcomings in tactical skills of drivers on the autism spectrum, but the extent to which this aﬀects their own safety or the safety of other road users is unclear. Tactical skills can be improved through training programs. There are few autism spectrum-speciﬁc learner training programs available. Development of an eﬀective training program will beneﬁt individuals on the spectrum to learn to drive, be independent, and be safe on the road. Hindawi Behavioural Neurology Volume 2018, Article ID 6842306, 17 pages https://doi.org/10.1155/2018/6842306 Hindawi Behavioural Neurology Volume 2018, Article ID 6842306, 17 pages https://doi.org/10.1155/2018/6842306 Review Article Scoping Review of the Driving Behaviour of and Driver Training Programs for People on the Autism Spectrum Nathan J. Wilson,1 Hoe C. Lee ,2 Sharmila Vaz,2 Priscilla Vindin,2 and Reinie Cordier 2 1School of Nursing and Midwifery, Western Sydney University, Hawkesbury Campus, Locked Bag 3, Richmond, NSW 2753, Australia 2School of Occupational Therapy Social Work and Speech Pathology, Faculty of Health Sciences, Curtin University, GPO Box U1987, Perth, WA 6845, Australia 1. Introduction suggest that prevalences of people on the autism spectrum in Western countries range from 0.6% to 2.2% [5–7]; as far as we are aware, in the Australian context, there are currently no evidence-based autism-speciﬁc driving interventions available to people on the spectrum to help counter commu- nity mobility limitations that aﬀect social and community participation. This review synthesises the peer-reviewed liter- ature about the driving characteristics of drivers on the spec- trum and driver training available for the cohort. People on the autism spectrum experience problems with social and communication skills that are associated with executive function deﬁcits aﬀecting working memory, motor coordination, attention, planning, mental ﬂexibility, and visual perception [1, 2]. This means that learning to drive a motor vehicle and maintaining safe on-road skills are often more diﬃcult for people on the autism spectrum. Many peo- ple on the autism spectrum face barriers to community inclusion, which can create lifelong obstacles to experiencing individual independence. In most countries, gaining a driver’s licence represents increased independence and can lead to improved quality of life for individuals and their fam- ilies [3]. In fact, a recent retrospective longitudinal study showed that fewer people on the autism spectrum had a licence and generally, they obtained their licence later when compared to their peers [4]. Although recent estimates Driving plays a critical developmental role during the transition to adulthood and is associated with greater chances of securing paid employment, accessing education, training and services, and maintaining social relationships [8, 9]. Learner drivers on the autism spectrum demonstrate problems with driving skill acquisition and performance and typically require more, and longer, driving lessons and need to take the practical driving test more often when com- pared to their neurotypical peers [2, 3]. Further, research 2 Behavioural Neurology males with ASD), (2) had increased reaction times, (3) had more tactical driving diﬃculties, (4) reported more traﬃc crashes, citations, and intentional driving violations, and (5) had poorer situational awareness skills than drivers with- out autism. A major implication of this review was the need for strategic and systematic approaches to train people on the autism spectrum to drive. shows that people on the autism spectrum are also at greater risk of being involved in motor vehicle accidents, which poses risks not only for them but also for other road users in the community [10, 11]. 2. Methodology 2.1. Aim. The aim of this scoping review was to synthesise the current peer-reviewed research literature about the driving behaviours of individuals on the autism spectrum and explore the available training programs for this cohort with a view to suggest future research that promotes better train- ing outcomes for learner drivers on the autism spectrum. Three reviews of the literature covering various aspects of driving and use of public transportation in people on the autism spectrum have been published. The ﬁrst review by Classen and Monahan [15] presented an overview of evidence-based intervention and predictor studies of driving performance in young people with attention deﬁcit hyperac- tivity disorder (ADHD) or autism spectrum disorder (ASD). A total of ten papers, published between 1995 and 2011, met the criteria for inclusion in this review with nine of the ten papers focussing on driving in people with ADHD. Only one observational case-control study included ASD partici- pants and focussed on hazard recognition in young people with and without ASD, using a virtual reality video display interface [14]. Thus, this ﬁrst review highlighted the paucity of studies in the area and the urgent need for more empirical studies. The second review focussed on barriers and facilita- tors associated with driving and public transportation in people with ASD [9]. The review included 14 studies and nar- ratively summarised ﬁndings related to (1) diﬃculties faced in obtaining a driver’s licence (e.g., handling unexpected changes, sustaining attention for long drives, merging into traﬃc, and limited ability to read facial expressions and ges- tures), (2) driver conﬁdence (e.g., due to lower reaction time and anxiety), (3) driving behaviours (e.g., avoiding heavy traﬃc or highways or driving at night, adherence to speed regulation, and lane maintenance), and (4) strategies advo- cated to improve driving skills in ASD (e.g., direct communi- cation, minimal verbal correction, and-short duration lessons; introduction to driving in low noise density/quiet areas; and strategies to address anxiety). A third review focussed on the likely eﬀects that ASD has on driving abilities in young peo- ple [16]. The authors reported that drivers on the autism spectrum (1) were less likely to identify social hazards (i.e., 2.2. Design. A scoping review of contemporary peer-reviewed research articles published in English between 2000 and August 2017 was undertaken. Seven databases were searched: PubMed, Scopus, ProQuest, Embase, Medline, CINAHL, and TRID. 2.3. Search Strategy. 1. Introduction y Driving requires higher-order executive function mecha- nisms to work in a coordinated fashion to respond to, man- age, and cope with multiple demands and unexpected and unpredictable driving situations [10]. Research has shown that people on the autism spectrum may experience executive function deﬁcits [2, 9], which will directly impact on their ability to develop driving skills and drive safely. Research investigating the driving behaviour of people on the autism spectrum found that they notice driving hazards but they dis- played a delay in response to social hazards that required interactions with other road users, such as failing to give way to pedestrians who show clear intent to cross the road [12–14]. This is thought to be related to a combination of reduced processing of social stimuli and attention deﬁcits, which is very problematic when learning to drive a motor vehicle—a process which requires collaborative problem- solving skills [3]. These challenges are further confounded by research supporting the notion that people on the autism spectrum have atypical eye-gaze patterns, such as prolonged ﬁxation of the speedometer to check the car speed and scan- ning repeatedly on traﬃc-irrelevant roadside objects such as advertisement billboards [2]. p In summary, the body of knowledge on driving and ASD needs further consolidation. A comprehensive review of both empirical and grey literatures that focuses on bar- riers and enablers to obtaining a licence that are related to (a) people on the autism spectrum (skills, behaviours), (b) the learning environment (policy, driver-training prac- tice, instructor, and physical/built environments), and (c) within-the-person-environment context is needed to guide the design of a training intervention on the person with autism spectrum disorder, as well as their driving instructors [1, 9]. Many countries currently have no autism-speciﬁc licencing requirements for learner drivers and there is a gen- eral lack of ASD-speciﬁc support and training packages for individuals, their families, and driving instructors [3, 4, 17]. 3. Results All articles were grouped into four broad categories based on all author-agreed interpretation of each study’s ﬁndings. These categories included (1) on-road driving behaviours and transport statistics reports on drivers on the spectrum (n = 7), (2) performance in driving simulators (n = 9), (3) performance in virtual reality driving (n = 9), and (4) barriers to obtaining a licence and training of drivers on the spectrum (n = 3). Although driving simulators represent a virtual real- ity context, the simulator category diﬀers from the virtual reality driving category as the context of the latter was using devices such as a desktop computer, rather than a fully func- tional simulator. Between the two assessment regimes, the low-cost computer-based driving simulator has a better face validity in observing driving behaviours. Descriptive infor- mation was extracted from all articles and included the authors, year of publication, study design, objectives, popula- tion, and key ﬁndings. Study designs were described using the decision matrix recommended by the Centre for Evidence-Based Medicine [18]. This information is sum- marised in Table 1. Performing complex driving functions that required multitasking skills (e.g., merging and using roundabouts) were often more diﬃcult for the person on the autism spec- trum [23, 24]. Driving tasks, or situations, that were some- times diﬃcult for drivers on the autism spectrum included driving in heavy traﬃc, night driving, maintaining the cor- rect speed, lane maintenance, judging distance, and under- taking long journeys. In particular, drivers on the autism spectrum sometimes struggled to interpret the driving actions of other road users and found slight deviations from traﬃc rules of other drivers—a challenge and anxiety provok- ing [23]. 4.2. Performance in Driving Simulators. All of the studies in this category used a comparison group of drivers not on the autism spectrum, except for one study by Monahan et al. [19]. Bishop et al. [12] and Brooks et al. [1] reported that in comparing drivers on the spectrum and the drivers in the control groups, there were no between-group diﬀerences in reaction time to hazard perception and motor response time during predriving assessments. In Brooks et al.’s [1] driving simulator study, when compared with the neurotypical con- trols, participants on the autism spectrum required an addi- tional 30–35 minutes to complete 18 rounds of steering and pedal skill exercises. 2. Methodology The following search terms were used as appropriate, for each database: “Child development disorders, pervasive” OR “child developmental disorder∗” OR “pervasive developmental disorder∗” OR autism OR Asperger OR “autism spectrum disorder∗” OR “autistic disorder∗” OR “Develop- mental disabil∗” AND “Automobile Driving” OR driving or “driv∗training” OR “driver behaviour” OR “driver perfor- mance” OR “driver characteristic” OR “driv∗education” OR “driv∗testing” OR “driv∗procedures” OR “driving hazard∗” OR “car driv∗”OR “automobile driv∗” OR “driv∗packages”. 2.4. Study Selection. The search yielded 1389 results from all seven databases. After removing 609 duplicates, a total of 780 articles were screened at an abstract level for inclusion. Studies were excluded if they did not involve people with ASD and if they did not focus on an aspect of driver training or driving skill development. After two authors reviewed the abstracts, 56 articles were deemed to be suitable for review at full-text level. Of the 56 studies reviewed at full-text level, 28 were excluded based on the following exclusion criteria: (1) not peer-reviewed article, (2) no full text available, (3) focus on other disabilities (not ASD), and (4) focus on transporta- tion rather than independent driving. This resulted in a total 3 Behavioural Neurology of 28 studies for analysis; Figure 1 provides an illustration of the review process. of 28 studies for analysis; Figure 1 provides an illustration of the review process. interaction with other road users (e.g., being hesitant to merge into another lane when other drivers had already ges- tured and reduced speed to allow the manoeuvre to happen) [14, 23]. Chee et al. [23] also found that drivers on the autism spectrum performed better than neurotypical drivers in rule- following aspects of driving, such as using the indicator and checking for traﬃc when approaching an intersection. 3. Results There was not a wide distribution of empirical studies from around the globe, with over 70% of the studies from the USA (n = 20), followed by Australia (n = 3), the UK (n = 3), Canada (n = 1), and Sweden (n = 1). Research on the topic area was mainly conducted in high-income coun- tries. A total of 1481 participants on the autism spectrum were included across all 28 studies, with 79% of those partic- ipants being male. In terms of research designs, there were a total of 23 observational studies, two case studies, two review articles, and one driving simulator study with a quasi- experimental design. The published case study [19] used the same single male subject on the autism spectrum that was used in a comparison with a neurotypical male peer of the same age [20]. In the quasi-experimental study, the inter- vention group consisted of novice drivers on the autism spec- trum (n = 51) and the comparison group (n = 333) was experienced licenced drivers without autism aged over 25 years [21]. Other driving simulator studies reported no between- group diﬀerences in (1) errors of maintenance of lane posi- tion and speed, (2) adjustment to distractions and poorer right-sided visual acuity [10], (3) response time in braking and overall driving ability [2], and (4) interpreting the gap between the front car, the speed, and the traﬃc ﬂow of speciﬁc traﬃc scenarios [19]. Reimer et al. [25] also reported that participants on the autism spectrum showed diﬀerent eye-gaze patterns. When responding to added cognitive demands, they positioned their vertical gaze higher and toward distant objects with more visual diversion. This can reduce the detection of hazards on the peripheral visual ﬁeld of the individuals. The only quasi-experimental study included in the review used a driving simulator to training drivers on the spectrum [21]. The study showed that drivers on the autism spectrum had poorer baseline executive func- tion and underperformed in tactical skills during simulated scenarios of unanticipated traﬃc demands [21]. Participants were trained in stages using a driving simulator program dur- ing one-hour training sessions across 10–12 weeks, focusing on alternated training of executive functions and tactical driving skills in driving. Outcome measures from the study showed that the training program signiﬁcantly improved both executive function and driving skills. 4. Key Categories 4.1. On-Road Driving Behaviours and Transport Statistics Reports on Drivers on the Spectrum. Drivers on the autism spectrum were reported to have proportionally more traﬃc oﬀences than their neurotypical counterparts (p < 0 05) [22]. This study also reported that drivers on the autism spectrum obtained their licence later than other drivers and self-rated their driving skills on a 10-point Likert scale as lower. Cox et al. [21] reported on a greater proportion of traﬃc oﬀences in drivers on the autism spectrum. Other studies reported that drivers on the autism spectrum demon- strated decreased manoeuvring ability, particularly in left- and right-hand turns and an increased response time to traﬃc hazards, particularly in circumstances that required 4.3. Performance in Virtual Reality Driving. These studies all used some form of a desktop computer and screen as the Behavioural Neurology Records identiﬁed through: Pubmed \| Scopus \| ProQuest \| Embase \| Medline \| CINAHL \| TRID: 1389 Total number of studies included: 28 Number of abstracts excluded: 724 Number of full-text articles assesed for eligibility:56 Number of abstracts screened: 780 Afer 609 duplicates from 1389 records removed: 780 records Number of full-text articles excluded: 28 (i) Not peer reviewed (ii) Full text not available (iii) Other type of disabilities (not ASD) (iv) Public transport Identiﬁcation Screening Eligibility Included Figure 1: Flowchart of included studies. Afer 609 duplicates from 1389 records removed: 780 records Number of abstracts excluded: 724 Number of full-text articles assesed for eligibility:56 Number of full-text articles excluded: 28 (i) Not peer reviewed (ii) Full text not available (iii) Other type of disabilities (not ASD) (iv) Public transport Total number of studies included: 28 Figure 1: Flowchart of included studies. basis of the virtual reality driving experience with driving- styled consoles (e.g., Logitech™steering wheel) attached, to enable the participant to mimic driving. Six of the seven stud- ies were led by various members from the same US Electrical Engineering and Computer Science research team. Many were proof of concept studies to determine whether the var- ious data collection methods and tools can be augmented with a virtual reality program to detect between subject dif- ferences in areas, for example, such as eye gaze, EEG data, and physiological responses. 4. Key Categories (ii) Some preferred to use public transportation or walk; anxiety was a barrier to driving for some. (iii) 34% with ASD had a driver licence compared to 68% of typical developing. Diagnosis of ASD based on self-report Underrepresentation of nondrivers in the control group. Chee et al. 2017 (Australia) [23] Cross-sectional study (i) To assess and compare the driving performance of drivers with and without ASD in suburban traﬃc environment (ii) To explore how the symptomatology of ASD hinders or facilitates the on-road driving performance n = 37 16 with ASD (MA = 25.43 years; 86% male) 21 TD (MA = 27 years; 93% male) (i) Drivers with ASD underperformed in vehicle manoeuvring, especially at left turns, right turns (hesitant and slower), and pedestrian crossings. (ii) Drivers with ASD outperformed the TD group in rule-following aspects such as using the indicator at roundabouts and checking for cross-traﬃc when approaching intersections. Being small in sample size, the participants of this study may not be representative of the broader population and can impact on the generalisability of the results. Assessor bias—not blinded to diagnosis of participants or the purpose of the study No use of independent assessor or interrater reliability checks Impact of environmental factors, such as time of day and weather conditions on driving performance, cannot be disregarded. Driving route is developed by a researcher using the route of convenience for experimentation purposes; whether it mimics traﬃc demands on a normal road is questionable y Table 1 Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations On-road driving behaviours and transport statistics reports on drivers on the spectrum (n = 7) Chee et al. 2015 (Australia) [28] Cross-sectional study To understand people with ASD’s viewpoints on driving using Q- methodology (e.g., whether or not they are conﬁdent and prefer driving than other transportation). n = 100 50 with ASD (MA = 21.8 years; 82% male) 57 young adults without ASD (MA = 23.6 years; 72% male) (i) Some young adults with ASD perceived themselves as conﬁdent and independent drivers. (ii) Some preferred to use public transportation or walk; anxiety was a barrier to driving for some. (iii) 34% with ASD had a driver licence compared to 68% of typical developing. Diagnosis of ASD based on self-report Underrepresentation of nondrivers in the control group. Chee et al. 4. Key Categories 2015 (Australia) [28] Cross-sectional study To understand people with ASD’s viewpoints on driving using Q- methodology (e.g., whether or not they are conﬁdent and prefer driving than other transportation). n = 100 50 with ASD (MA = 21.8 years; 82% male) 57 young adults without ASD (MA = 23.6 years; 72% male) (i) Some young adults with ASD perceived themselves as conﬁdent and independent drivers. (ii) Some preferred to use public transportation or walk; anxiety was a barrier to driving for some. (iii) 34% with ASD had a driver licence compared to 68% of typical developing. Diagnosis of ASD based on self-report Underrepresentation of nondrivers in the control group. ll l h p yp developing. Chee et al. 2017 (Australia) [23] Cross-sectional study (i) To assess and compare the driving performance of drivers with and without ASD in suburban traﬃc environment (ii) To explore how the symptomatology of ASD hinders or facilitates the on-road driving performance n = 37 16 with ASD (MA = 25.43 years; 86% male) 21 TD (MA = 27 years; 93% male) (i) Drivers with ASD underperformed in vehicle manoeuvring, especially at left turns, right turns (hesitant and slower), and pedestrian crossings. (ii) Drivers with ASD outperformed the TD group in rule-following aspects such as using the indicator at roundabouts and checking for cross-traﬃc when approaching intersections. Being small in sample size, the participants of this study may not be representative of the broader population and can impact on the generalisability of the results. Assessor bias—not blinded to diagnosis of participants or the purpose of the study No use of independent assessor or interrater reliability checks Impact of environmental factors, such as time of day and weather conditions on driving performance, cannot be disregarded. Driving route is developed by a researcher using the route of convenience for experimentation purposes; whether it mimics traﬃc demands on a normal road is questionable. Cox et al. 4. Key Categories 2017 (Australia) [23] Cross-sectional study (i) To assess and compare the driving performance of drivers with and without ASD in suburban traﬃc environment (ii) To explore how the symptomatology of ASD hinders or facilitates the on-road driving performance n = 37 16 with ASD (MA = 25.43 years; 86% male) 21 TD (MA = 27 years; 93% male) (i) Drivers with ASD underperformed in vehicle manoeuvring, especially at left turns, right turns (hesitant and slower), and pedestrian crossings. (ii) Drivers with ASD outperformed the TD group in rule-following aspects such as using the indicator at roundabouts and checking for cross-traﬃc when approaching intersections. Being small in sample size, the participants of this study may not be representative of the broader population and can impact on the generalisability of the results. Assessor bias—not blinded to diagnosis of participants or the purpose of the study No use of independent assessor or interrater reliability checks Impact of environmental factors, such as time of day and weather conditions on driving performance, cannot be disregarded. Driving route is developed by a researcher using the route of convenience for experimentation purposes; whether it mimics traﬃc demands on a normal road is questionable. Cox et al. 2012 (USA) [24] Cross-sectional study An online survey to understand driving and ASD by surveying parents/caregivers of adolescents and young adults with ASD, who had driving licence or were in the process of learning to drive n = 123 parents/caregivers (mother 81%, father 13%, and caregiver 6%) The mean age of their child with ASD was 19 years, 73% male; 83% Asperger’s/high- function ASD, 9% PDD-NOS, 3% autistic disorder, and 5% others (i) Parents were active in the teaching process (mother 81%; father 62%). (ii) 48% had a driver’s licence, 29% possessed a learner’s permit, and 70% of the parents stated that ASD aﬀected driving ability. (iii) Particular challenges for people with ASD are complex driving demands (e.g., merging into traﬃc traﬃc awareness Diagnosis of ASD is not conﬁrmed through review of medical records Confounding impact of comorbidity is not accounted. Respondent bias (use of caregivers other than the person on the spectrum) limits validity. Use of a sample that was nonrepresentative; data collection is Table 1 Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations On-road driving behaviours and transport statistics reports on drivers on the spectrum (n = 7) Chee et al. 4. Key Categories 2012 (USA) [24] Cross-sectional study An online survey to understand driving and ASD by surveying parents/caregivers of adolescents and young adults with ASD, who had driving licence or were in the process of learning to drive n = 123 parents/caregivers (mother 81%, father 13%, and caregiver 6%) The mean age of their child with ASD was 19 years, 73% male; 83% Asperger’s/high- function ASD, 9% PDD-NOS, 3% autistic disorder, and 5% others (i) Parents were active in the teaching process (mother 81%; father 62%). (ii) 48% had a driver’s licence, 29% possessed a learner’s permit, and 70% of the parents stated that ASD aﬀected driving ability. (iii) Particular challenges for people with ASD are complex driving demands (e.g., merging into traﬃc, traﬃc awareness, multitasking, handling Diagnosis of ASD is not conﬁrmed through review of medical records Confounding impact of comorbidity is not accounted. Respondent bias (use of caregivers other than the person on the spectrum) limits validity. Use of a sample that was nonrepresentative; data collection is limited to internet survey. Cox et al. 2012 (USA) [24] Cross-sectional study An online survey to understand driving and ASD by surveying parents/caregivers of adolescents and young adults with ASD, who had driving licence or were in the process of learning to drive n = 123 parents/caregivers (mother 81%, father 13%, and caregiver 6%) The mean age of their child with ASD was 19 years, 73% male; 83% Asperger’s/high- function ASD, 9% PDD-NOS, 3% autistic disorder, and 5% others (i) Parents were active in the teaching process (mother 81%; father 62%). (ii) 48% had a driver’s licence, 29% possessed a learner’s permit, and 70% of the parents stated that ASD aﬀected driving ability. (iii) Particular challenges for people with ASD are complex driving demands (e.g., merging into traﬃc, traﬃc awareness, multitasking, handling Diagnosis of ASD is not conﬁrmed through review of medical records Confounding impact of comorbidity is not accounted. Respondent bias (use of caregivers other than the person on the spectrum) limits validity. Use of a sample that was nonrepresentative; data collection is limited to internet survey. 4. Key Categories The seventh study was from a team of UK researchers and reported that participants on the autism spectrum did not detect the time to arrival of two moving cars on a straight road as accurately as the com- parison group [26]. Although this study used a virtual reality driving program, none of the participants were actual drivers and so the ability to draw real-life conclusions is limited. practice was demanding [3]. Unexpected changes to the usual routine or driving norms—where other drivers did not obey the traﬃc rules—were problematic for people on the autism spectrum [3]. Other reported problems included a lack of conﬁdence, overconﬁdence, sensory overload, anxi- ety, poorer concentration, and greater distractibility. Although all the research studies in this category were either observational or literature reviews that focussed on barriers, there were a few suggestions of potential ways to overcome these barriers. These included strategies to assist driving instructors, such as using direct communication and concrete instructions, providing shorter driving lessons, to commence each lesson in a quiet neighbourhood, and only giving the minimum amount of required information at a time [9]. There was a broad suggestion to use strategies to address anxiety; however, these would need to be individua- lised to the person on the autism spectrum. In Tyler’s [17] four case studies with young adult males on the autism spectrum, some of these individualised strategies included using parent/learner/instructor communication logbooks to enhance consistency, structured language, tasks analysis, and breakdown, using a range of “what if” scenarios to increase insight into unpredictable driving and traﬃc situa- tions and using visual markers to aid with judging distance. 4.4. Barriers to Obtaining a Licence and Training of Drivers on the Spectrum. Both drivers with ASD (learners and with a licence) experienced greater problems with driving and require more lessons and more tests than neurotypical drivers. Learning to drive was sometimes more diﬃcult for the person on the autism spectrum as the process of reading, understanding, and converting driving theory into driving Behavioural Neurology 5 Chee et al. 2015 (Australia) [28] Cross-sectional study To understand people with ASD’s viewpoints on driving using Q- methodology (e.g., whether or not they are conﬁdent and prefer driving than other transportation). n = 100 50 with ASD (MA = 21.8 years; 82% male) 57 young adults without ASD (MA = 23.6 years; 72% male) p and independent drivers. questionable. 6 6 Behavioural Neurology Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations unexpected changes, and sustaining attention on a long drive) (iv) Other diﬃculties include interpreting the actions of other drivers (e.g. reading their nonverbal social cues when in ambiguous situations). Daly et al. 2014 (USA) [22] Cross-sectional study An online survey using a modiﬁed version of the Driver Behaviour Questionnaire to determine whether diﬀerences exist between adult drivers with ASD and non-ASD adult drivers in terms of (i) driving history and driving behaviours, (ii) the rates of reporting violations/mistakes/driving slips as deﬁned by DBQ, and (iii) the relative risk of such behaviours as quantiﬁed by the DBQ. n = 172 78 licenced drivers with ASD (MA = 32.9 years; 56% male, 98.7% autism/Asperger’s, and 1.3% PDD-NOS) 94 non-ASD drivers (MA = 35.5 years; 31% male) (i) Drivers with ASD obtained their driver’s licence approximately 2 years later that non-ASD drivers. (ii) Drivers with ASD drove less days per week and had signiﬁcantly lower ratings of their driving ability and a higher number of traﬃc violations. (iii) Drivers with ASD were also more likely to place voluntary restrictions on driving. The pilot study relied on anonymous self-report. Participants, due to poor insight or diﬃculty in comparing their own driving behaviour with other drivers, may underreport their symptoms and overrate their driving ability. Report of driving behaviours through the use of standardized self-report measures would have improved the study rigor. Only the use of internet outlets for data collection may limit sample representativeness. Impact of comorbidity cannot be disregarded. Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations unexpected changes, and sustaining attention on a long drive) (iv) Other diﬃculties include interpreting the actions of other drivers (e.g. reading their nonverbal social cues when in ambiguous situations). Daly et al. 2014 (USA) [22] Cross-sectional study An online survey using a modiﬁed version of the Driver Behaviour Questionnaire to determine whether diﬀerences exist between adult drivers with ASD and non-ASD adult drivers in terms of (i) driving history and driving behaviours, (ii) the rates of reporting violations/mistakes/driving slips as deﬁned by DBQ, and (iii) the relative risk of such behaviours as quantiﬁed by the DBQ. questionable. n = 172 78 licenced drivers with ASD (MA = 32.9 years; 56% male, 98.7% autism/Asperger’s, and 1.3% PDD-NOS) 94 non-ASD drivers (MA = 35.5 years; 31% male) (i) Drivers with ASD obtained their driver’s licence approximately 2 years later that non-ASD drivers. (ii) Drivers with ASD drove less days per week and had signiﬁcantly lower ratings of their driving ability and a higher number of traﬃc violations. (iii) Drivers with ASD were also more likely to place voluntary restrictions on driving. The pilot study relied on anonymous self-report. Participants, due to poor insight or diﬃculty in comparing their own driving behaviour with other drivers, may underreport their symptoms and overrate their driving ability. Report of driving behaviours through the use of standardized self-report measures would have improved the study rigor. Only the use of internet outlets for data collection may limit sample representativeness. Impact of comorbidity cannot be disregarded. ∗Deka et al. 2016 (USA) [31] Cross-sectional study An online survey to learn about the travel patterns, needs, and barriers people with ASD encounter regarding the use of diﬀerent transportation modes. n = 703 Male : female = 3 : 16 (i) Only 9.3% of the adults with ASD had a driver’s licence and many are using it as identity card rather than a licence to drive. (ii) 55.3% of the drivers with ASD (n = 47) had diﬃculty dealing with traﬃc, 34% mentioned diﬃculty caused by distractions near roads; 27.7% mentioned diﬃculty judging distance, and 27.7% had diﬃculty with parking. (iii) Because of these diﬃculties, 26.1% did not drive at all. Low representation of views of ASD adults who had a driving licence and that have driven a car regularly Huang et al. 2012 (USA) [32] Cross-sectional study An online survey to compare the characteristics of age-eligible driving and nondriving teens and explore the driving outcomes for teens with higher n = 235 (MA = 16.39 years; 83%; 24.7% autistic disorder, 14.5% PDD-NOS, 1.5% ASD, (i) 63% of teens currently drive or plan to drive. (ii) 29% who are eligible to drive currently drive. Respondent bias (use of caregivers other than the person on the spectrum) limits validity. Selection bias leading to ka 2016 A) Cross-sectional study An online survey to learn about the travel patterns, needs, and barriers people with ASD encounter regarding the use of diﬀerent transportation modes. questionable. n = 703 Male : female = 3 : 16 (i) Only 9.3% of the adults with ASD had a driver’s licence and many are using it as identity card rather than a licence to drive. (ii) 55.3% of the drivers with ASD (n = 47) had diﬃculty dealing with traﬃc, 34% mentioned diﬃculty caused by distractions near roads; 27.7% mentioned diﬃculty judging distance, and 27.7% had diﬃculty with parking. (iii) Because of these diﬃculties, 26.1% did not drive at all. Low representation of views of ASD adults who had a driving licence and that have driven a car regularly ng 2012 A) Cross-sectional study An online survey to compare the characteristics of age-eligible driving and nondriving teens and explore the driving outcomes for teens with higher n = 235 (MA = 16.39 years; 83%; 24.7% autistic disorder, 14.5% PDD-NOS, 1.5% ASD, (i) 63% of teens currently drive or plan to drive. (ii) 29% who are eligible to drive currently drive. Respondent bias (use of caregivers other than the person on the spectrum) limits validity. Selection bias leading to 7 Behavioural Neurology Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations functioning autism spectrum disorders 46.4% Asperger’s, and 4.7% others) (iii) Driving predictors included individualised education plans with driving goals, indicators of functional status, and parent experience with teaching teens to drive. (iv) 12% of teens received driving citations, and 12% of teens had been involved in a motor vehicle crash. nonrepresentative samples: families of teens who currently drive or plan to drive are more likely to participate in this study Sheppard et al. 2017 (UK) [33] Case control study Videos of 20 diﬀerent driving situations with inbuilt hazards to (i) compare the ability of identifying the driving hazards by stopping the video and pointing to the hazard between people with ASD and without ASD and (ii) deﬁne whether people with ASD have diﬃculty in responding to hazards or orienting to them. n = 35 18 ASD males (MA = 18.79 years) 17 TD males (MA = 18.19 years) (i) Although nonsigniﬁcant, participants with ASD showed a slower ﬁxation on the hazard source and a slower orientation to the hazards. (ii) Greater attentional capture in the time preceding the hazards’ onset was associated with lower verbal IQ. questionable. nonrepresentative samples: families of teens who currently drive or plan to drive are more likely to participate in this study Sheppard et al. 2017 (UK) [33] Case control study Videos of 20 diﬀerent driving situations with inbuilt hazards to (i) compare the ability of identifying the driving hazards by stopping the video and pointing to the hazard between people with ASD and without ASD and (ii) deﬁne whether people with ASD have diﬃculty in responding to hazards or orienting to them. n = 35 18 ASD males (MA = 18.79 years) 17 TD males (MA = 18.19 years) (i) Although nonsigniﬁcant, participants with ASD showed a slower ﬁxation on the hazard source and a slower orientation to the hazards. (ii) Greater attentional capture in the time preceding the hazards’ onset was associated with lower verbal IQ. (iii), Suggesting individuals with ASD may distribute or direct their attention diﬀerently when identifying driving hazards. The study is limited to young male ASD adults with no speciﬁc training or driving experience. Results cannot be generalised to ASD drivers with a licence or of diﬀerent age ranges or females with ASD. f d l ( ) mance in driving simulators (n = 9) 017 Cross-sectional study To investigate how drivers with ASD respond to social and nonsocial hazards in a driving simulator compared to typically developing drivers using a driving simulator n = 32 16 ASD drivers (MA = 23.88 years; 15% male) 16 TD drivers (MA = 22.94 years; 15% male) (i) There was no diﬀerence in reaction time to social versus nonsocial hazards for drivers with ASD. (ii) Typically developing drivers reacted more quickly to social hazards as compared to nonsocial hazards. Small sample size and underpowered Low scores on ASD symptomatology: ASD Social Responsive Scale. Unclear of any real-life translation of the simulated driving assessment 016 Cross-sectional study To investigate the utility of using a driving simulator and interactive exercises (steering and pedal tasks) to address the motor aspects of predriving skills of young adults with ASD n = 41 10 ASD (MA = 15.9 years; 10 male) 31 TD (MA = 16.7 years; males = 18) (i) Minimal performance diﬀerences were observed between the two groups in terms of the motor aspects of predriving skills. (ii) Participants with ASD needed more time (30–35 minutes) to complete the 18 interactive steering and pedal exercises. questionable. (iii), Suggesting individuals with ASD may distribute or direct their attention diﬀerently when identifying driving hazards. The study is limited to young male ASD adults with no speciﬁc training or driving experience. Results cannot be generalised to ASD drivers with a licence or of diﬀerent age ranges or females with ASD. Performance in driving simulators (n = 9) Bishop et al. 2017 (USA) [12] Cross-sectional study To investigate how drivers with ASD respond to social and nonsocial hazards in a driving simulator compared to typically developing drivers using a driving simulator n = 32 16 ASD drivers (MA = 23.88 years; 15% male) 16 TD drivers (MA = 22.94 years; 15% male) (i) There was no diﬀerence in reaction time to social versus nonsocial hazards for drivers with ASD. (ii) Typically developing drivers reacted more quickly to social hazards as compared to nonsocial hazards. Small sample size and underpowered Low scores on ASD symptomatology: ASD Social Responsive Scale. Unclear of any real-life translation of the simulated driving assessment Brooks et al. 2016 (USA) [1] Cross-sectional study To investigate the utility of using a driving simulator and interactive exercises (steering and pedal tasks) to address the motor aspects of predriving skills of young adults with ASD n = 41 10 ASD (MA = 15.9 years; 10 male) 31 TD (MA = 16.7 years; males = 18) (i) Minimal performance diﬀerences were observed between the two groups in terms of the motor aspects of predriving skills. (ii) Participants with ASD needed more time (30–35 minutes) to complete the 18 interactive steering and pedal exercises. (iii) The interactive exercises and the process used worked well to Small sample size and underpowered Females are excluded in the ASD group. Unclear of any real-life translation: interactive exercises do not include any road scenes that help to eliminate the sensory overload, anxiety, and stress that participants might experience in naturalistic driving environments Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations functioning autism spectrum disorders 46.4% Asperger’s, and 4.7% others) (iii) Driving predictors included individualised education plans with driving goals, indicators of functional status, and parent experience with teaching teens to drive. (iv) 12% of teens received driving citations, and 12% of teens had been involved in a motor vehicle crash. questionable. (iii) The interactive exercises and the process used worked well to Small sample size and underpowered Females are excluded in the ASD group. Unclear of any real-life translation: interactive exercises do not include any road scenes that help to eliminate the sensory overload, anxiety, and stress that participants might experience in naturalistic driving environments 8 Behavioural Neurology Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations address motor-related aspects of predriving skills in young adults with ASD. Classen and Monahan 2013 (USA) [15] Literature review To conduct an evidence-based review of intervention studies and predictor studies related to driving outcomes in teens with ADHD or ASD n = 10 intervention studies (2 RCT, 7 quasi-experiments, and 1 prospective observational study) Quasi-experimental studies: 7 on ADHD and 1 on ASD (i) The ASD group perceives driving hazards via video clips but has diﬃculty in perceiving hazards if the task also requires processing of social information. (ii) The ASD group responds slower to hazard perceptions compared to controls. (iii) An instrumented vehicle, simulator training, and parent involvement invention had positive eﬀect for speeding and hard braking for teens with ADHD. (iv) Male drivers with ADHD improved in hazard perception response times following training with hazard videos. Heterogeneity among the studies: variability in age Inclusion criteria restricted to articles published in English and male participants Using diﬀerent driving simulators, driving scenarios, outcome measures of assessment, failure to control for any prior rehabilitation, or clinical interventions lessened the methodological rigour of the studies. Grey literature is not included in the search. Classen et al. 2013 (USA) [8] Observational study Using a driving simulator to examine the demographic, clinical, and simulated driving (type and number of driving errors) diﬀerences between teens with ADHD-ASD, healthy controls (HCs). n = 44 22 ADHD-ASD (MA = 15.05 years; 17% male) 22 healthy controls (MA = 14.32 years; 59% male) (i) Teens with ADHD-ASD performed more poorly on right eye visual acuity, selective attention, visual motor integration, cognition, and motor performance. (ii) Teens with ADHD-ASD made more errors on the driving simulator regarding visual scanning, speed regulation, lane maintenance, adjustment to stimuli, and total number of driving errors. (iii) Compared with HC teens, teens with ADHD-ASD may have more predriving skill deﬁcits. Sample of convenience: a Caucasian sample is not representative of the population of interest. questionable. Small sample size: underpowered study Selection bias: more concerned parents and teens with better insight enrolled in the study Berkson’s bias: test taking and driving behaviours could be inﬂuenced by the evaluator’s sitting next to the client. Hawthorne bias: the test-taking and driving behaviours are inﬂuenced by the testing site and social conditions. Did not control for medication eﬀects on driving Simulator study: questionable of real-life driving equivalence with hazard videos. 13 Observational study Using a driving simulator to examine the demographic, clinical, and simulated driving (type and number of driving errors) diﬀerences between teens with ADHD-ASD, healthy controls (HCs). n = 44 22 ADHD-ASD (MA = 15.05 years; 17% male) 22 healthy controls (MA = 14.32 years; 59% male) (i) Teens with ADHD-ASD performed more poorly on right eye visual acuity, selective attention, visual motor integration, cognition, and motor performance. (ii) Teens with ADHD-ASD made more errors on the driving simulator regarding visual scanning, speed regulation, lane maintenance, adjustment to stimuli, and total number of driving errors. (iii) Compared with HC teens, teens with ADHD-ASD may have more predriving skill deﬁcits. Sample of convenience: a Caucasian sample is not representative of the population of interest. Small sample size: underpowered study Selection bias: more concerned parents and teens with better insight enrolled in the study Berkson’s bias: test taking and driving behaviours could be inﬂuenced by the evaluator’s sitting next to the client. Hawthorne bias: the test-taking and driving behaviours are inﬂuenced by the testing site and social conditions. Did not control for medication eﬀects on driving Simulator study: questionable of real-life driving equivalence Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations address motor-related aspects of predriving skills in young adults with ASD. Classen and Monahan 2013 (USA) [15] Literature review To conduct an evidence-based review of intervention studies and predictor studies related to driving outcomes in teens with ADHD or ASD n = 10 intervention studies (2 RCT, 7 quasi-experiments, and 1 prospective observational study) Quasi-experimental studies: 7 on ADHD and 1 on ASD (i) The ASD group perceives driving hazards via video clips but has diﬃculty in perceiving hazards if the task also requires processing of social information. (ii) The ASD group responds slower to hazard perceptions compared to controls. questionable. (iii) An instrumented vehicle, simulator training, and parent involvement invention had positive eﬀect for speeding and hard braking for teens with ADHD. (iv) Male drivers with ADHD improved in hazard perception response times following training with hazard videos. Heterogeneity among the studies: variability in age Inclusion criteria restricted to articles published in English and male participants Using diﬀerent driving simulators, driving scenarios, outcome measures of assessment, failure to control for any prior rehabilitation, or clinical interventions lessened the methodological rigour of the studies. Grey literature is not included in the search. Behavioural Neurology 9 Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations Cox et al. 2016 (USA) [2] Cross-sectional study To examine the relationship between driving performance and executive functioning for novice drivers, with and without ASD, using a driving simulator n = 44 males 17 ASD (MA = 18.28 years) 27 healthy controls (MA = 16.59 years) (i) ASD drivers had signiﬁcantly slower reaction times during steering but not braking. (ii) ASD drivers demonstrated impaired working memory functioning, such that adding working memory demands resulted in a signiﬁcant decrement in their driving performance relative to control drivers. (iii) ASD drivers demonstrated poorer overall driving ability than the control drivers. Simulator study—questionable real-life equivalence Diagnosis based on parent report: medical records/doctors not consulted Cognitive measure is not used to assess intellectual functioning. Equivalence of comparison groups questionable: the control group was younger licenced drivers, whereas the ASD group had learner’s permits. Previous driving experience was not accounted for in both groups. Cox et al. 2017 (USA) [21] Pre- and post-quasi- experimental studies Using a virtual reality driving simulator to investigate how novice drivers with ASD diﬀer from experienced drivers and whether virtual reality driving simulation training (VRDST) improves ASD driving performance 51 novice ASD drivers (MA = 17.96 years; 78% male) 23 in routine training (RT) 14 in standard VRDST 14 in automated VRDST 18 in eye-tracking VRDST only 333 normative licenced comparison group (MA = 40 years; 73% male) (i) ASD drivers showed worse baseline executive function (EF) and driving skills than experienced drivers. (ii) ASD drivers performed worse than normative drivers on general tactical driving (iii) Both standard and automated VRDST signiﬁcantly improved driving (e.g., better steering and speed control) and EF performance compared to RT. (iv) Eye-tracking VRDST did not signiﬁcantly improve tactical performance relative to RT. questionable. No random assignment of subjects to groups Small sample size: the study can be under powered Monahan et al. 2012 (USA) [19] Single subject case study To illustrate the predriving skills of a teen with ADHD/ASD using a driving simulator 1 male with ASD/ADHD, 15 years old (i) Demonstrated signiﬁcant impairments related to visual motor integration and attention shift (ii) Did not perform visual scanning for traﬃc at cross streets and during lane changes (iii) Approached all interactions with excessive speed (iv) Poor understanding of traﬃc ﬂow (v) Made gap acceptance error related to being overcautious Single case study design: inherently faces the lack of representativeness of general population 10 Behavioural Neurology Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations Monahan et al. 2013 (USA) [20] Cross-sectional study Using a driving simulator to compare the predriving skills of a teen with ADHD/ASD to an age- and gender- matched healthy control. 1 male with ADHD/ASD, 15 years old 1 youth without ADHD/ASD, 15 years old (i) Youth with ADHD/ASD demonstrated impairments in the ability to shift attention, perform simple sequential tasks, integrate visual-motor responses, and coordinate motor responses (ii) Youth without ADHD/ASD demonstrated intact skills in these abilities. (iii) Youth with ADHD/ASD made 44 driving error, while the youth without ADHD/ASD made 17 during the driving. (iv) Youth with ADHD/ASD had more lane maintenance, visual scanning, and speeding errors compared to the youth without ADHD/ASD. Single participant in each comparison group Reimer et al. 2013 (USA) [25] Cross-sectional study Using a driving simulator to explore driving behaviour and visual attention in young adult drivers with high- function (HF) ASD in comparison with a community sample of nonaﬀected individuals matched for age and sex n = 20 10 male with HF-ASD (MA = 20.2 years) 10 matched controls (MA = 20.7 years) (i) Youth with HF-ASD performed comparable to controls in terms of the frequency of simulated crashes and vehicle control. (ii) Youth with HF-ASD displayed a nominally higher and unvaried heart rate compared to controls. (iii) Youth with HF-ASD showed a gaze pattern suggestive of a diversion of visual attention away from high-stimulus areas of the roadway when responding to increased cognitive demands (cell phone task; continuous performance task). (iv) Youth with HF-ASD tended to position their vertical gaze higher and away from nearer objects and more toward the distance than controls. questionable. Unclear of any real-life translation of the driving simulator study. Fan et al. 2018 (USA) [35] Descriptive study To generate proof of concept data to determine if EEG data is useful in ASD driving interventions 20 ASD (MA = 15.29 years; 19 males and 1 female) (i) EEG-based group level classiﬁcation models are feasible for recognizing aﬀect and workload recognition in individuals with ASD in the context of using a desktop virtual reality driving program. (ii) Although promising, the applicability of this approach to collecting outcome data in other interventions requires further development. Small sample size of the neurotypical comparison group Limits with interpreting EEG data of virtual reality environments as in real-life contexts Sheppard et al. 2010 (UK) [14] Cross-sectional study To compare the ability of identifying 10 diﬀerent driving hazards by stopping the video and pointing to the hazard between people with ASD and without ASD n = 44 23 males with ASD (MA = 18.55 years; 30% ASD and 70% Asperger’s) 21 males without ASD (MA = 18.83 years) (i) Participants with ASD identiﬁed fewer social hazards (those that involved people like pedestrians, cyclists), but not nonsocial hazards (e.g., cars). (ii) Participants with ASD were slower to respond than those without ASD. Participants of the study are likely to have strategies prior to the experience to identify and respond to both types of hazard. Limited to only male participants Results cannot be generalised to ASD drivers with a licence or of diﬀerent age ranges or females with ASD. Sheppard et al. 2016 (UK) [26] Cross-sectional study To investigate whether individuals with ASD have diﬃculty judging the location of moving objects in a driving context using a time-to-arrival task on a desktop virtual reality program n = 44 23 males with ASD (MA = 18.55 years; 7 with Autism and 16 with Asperger’s) 21 males in comparison group (MA = 18.83 years) (i) Participants with ASD were less accurate at predicting which of the two cars in the virtual reality program would arrive ﬁrst at an intersection on a straight road. (ii) There were no diﬀerences between participants with ASD and the comparison group when the simulation was along a curved road. Participants were all nondrivers so generalisability to drivers is not possible. Unclear of any real-life translation Table 1: Continued. questionable. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations Performance in virtual reality driving (n = 9) Bian et al. 2016 (USA) [34] Study one: descriptive study Study two: case control experimental design, random allocation of members to comparison groups To assess the feasibility of outcome measures for and responsiveness of virtual reality driving package 4 teenagers with ASD—3 male and one female 2 in the performance-sensitive system (PS) group (degree of diﬃculty changed based on performance only) 2 in the engagement-sensitive system (ES) group (degree of diﬃculty changed based on performance and engagement) (i) All participants reported that they “enjoyed the game” and noticed the changes in task diﬃculty. (ii) The physiological data was successfully used to assess the user’s engagement level and performance enabling the dynamic adjustment of the driving diﬃculty level. Small sample size: underpowered study Equivalence of groups was questionable: eﬀects of neither age nor driving experience are suﬃciently controlled for the studies Study design does not take into account the possibility of comorbidity of ADHD in the sample group. Unclear of any real-life translation of the driving simulator study. Fan et al. 2018 (USA) [35] Descriptive study To generate proof of concept data to determine if EEG data is useful in ASD driving interventions 20 ASD (MA = 15.29 years; 19 males and 1 female) (i) EEG-based group level classiﬁcation models are feasible for recognizing aﬀect and workload recognition in individuals with ASD in the context of using a desktop virtual reality driving program. (ii) Although promising, the applicability of this approach to collecting outcome data in other interventions requires further development. Small sample size of the neurotypical comparison group Limits with interpreting EEG data of virtual reality environments as in real-life contexts n = 44 (i) Participants with ASD identiﬁed fewer social hazards (those that Participants of the study are likely to have strategies prior to the experience pard 2010 Cross-sectional study To compare the ability of identifying 10 diﬀerent driving hazards by stopping the video and pointing to the hazard between people with ASD and without ASD n = 44 23 males with ASD (MA = 18.55 years; 30% ASD and 70% Asperger’s) 21 males without ASD (MA = 18.83 years) (i) Participants with ASD identiﬁed fewer social hazards (those that involved people like pedestrians, cyclists), but not nonsocial hazards (e.g., cars). questionable. Sample of convenience—Caucasian comparison groups are not representative of the population of interest—limits generalisability of results. Small sample size: underpowered study Without screening on age and cognitive functions, a convenient sample from the community as the control group Assessment of intellectual functioning or ASD was not obtained. Degree to which outcome measures relate to other covariates such as driving frequency and exposure is not reported. Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations Monahan et al. 2013 (USA) [20] Cross-sectional study Using a driving simulator to compare the predriving skills of a teen with ADHD/ASD to an age- and gender- matched healthy control. 1 male with ADHD/ASD, 15 years old 1 youth without ADHD/ASD, 15 years old (i) Youth with ADHD/ASD demonstrated impairments in the ability to shift attention, perform simple sequential tasks, integrate visual-motor responses, and coordinate motor responses (ii) Youth without ADHD/ASD demonstrated intact skills in these abilities. (iii) Youth with ADHD/ASD made 44 driving error, while the youth without ADHD/ASD made 17 during the driving. (iv) Youth with ADHD/ASD had more lane maintenance, visual scanning, and speeding errors compared to the youth without ADHD/ASD. Single participant in each comparison group (i) Youth with HF-ASD performed comparable to controls in terms Behavioural Neurology 11 Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations Performance in virtual reality driving (n = 9) Bian et al. 2016 (USA) [34] Study one: descriptive study Study two: case control experimental design, random allocation of members to comparison groups To assess the feasibility of outcome measures for and responsiveness of virtual reality driving package 4 teenagers with ASD—3 male and one female 2 in the performance-sensitive system (PS) group (degree of diﬃculty changed based on performance only) 2 in the engagement-sensitive system (ES) group (degree of diﬃculty changed based on performance and engagement) (i) All participants reported that they “enjoyed the game” and noticed the changes in task diﬃculty. (ii) The physiological data was successfully used to assess the user’s engagement level and performance enabling the dynamic adjustment of the driving diﬃculty level. Small sample size: underpowered study Equivalence of groups was questionable: eﬀects of neither age nor driving experience are suﬃciently controlled for the studies Study design does not take into account the possibility of comorbidity of ADHD in the sample group. questionable. (ii) Participants with ASD were slower to respond than those without ASD. Participants of the study are likely to have strategies prior to the experience to identify and respond to both types of hazard. Limited to only male participants Results cannot be generalised to ASD drivers with a licence or of diﬀerent age ranges or females with ASD. pard 2016 Cross-sectional study To investigate whether individuals with ASD have diﬃculty judging the location of moving objects in a driving context using a time-to-arrival task on a desktop virtual reality program n = 44 23 males with ASD (MA = 18.55 years; 7 with Autism and 16 with Asperger’s) 21 males in comparison group (MA = 18.83 years) (i) Participants with ASD were less accurate at predicting which of the two cars in the virtual reality program would arrive ﬁrst at an intersection on a straight road. (ii) There were no diﬀerences between participants with ASD and the comparison group when the simulation was along a curved road. Participants were all nondrivers so generalisability to drivers is not possible. Unclear of any real-life translation Fan et al. 2018 (USA) [35] Descriptive study To generate proof of concept data to determine if EEG data is useful in ASD driving interventions 20 ASD (MA = 15.29 years; 19 males and 1 female) for recognizing aﬀect and workload recognition in individuals with ASD in the context of using a desktop virtual reality driving program. (ii) Although promising, the applicability of this approach to collecting outcome data in other interventions requires further development. Small sample size of the neurotypical comparison group Limits with interpreting EEG data of virtual reality environments as in real-life contexts Sheppard et al. 2010 (UK) [14] Cross-sectional study To compare the ability of identifying 10 diﬀerent driving hazards by stopping the video and pointing to the hazard between people with ASD and without ASD n = 44 23 males with ASD (MA = 18.55 years; 30% ASD and 70% Asperger’s) 21 males without ASD (MA = 18.83 years) (i) Participants with ASD identiﬁed fewer social hazards (those that involved people like pedestrians, cyclists), but not nonsocial hazards (e.g., cars). (ii) Participants with ASD were slower to respond than those without ASD. Participants of the study are likely to have strategies prior to the experience to identify and respond to both types of hazard. questionable. 2014 (USA) [36] Cross-sectional study To determine if a novel virtual reality driving environment can detect between-group diﬀerences n = 8 4 males with ASD (MA = 16.87 years) 4 TD controls (MA = 15.34 years; 3 males and 1 female) (i) The ASD group experiencing a higher number of simulated driving failures (ii) Participants with ASD had a vertically higher and horizontally right visual gaze. (iii) The ASD group had a signiﬁcantly higher skin conduction level and skin conductance response rate suggesting that participants with Small sample size: underpowered study Unclear of any real-life translation Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations Sheppard et al. 2017 (UK) [33] Case control study Videos of 20 diﬀerent driving situations with inbuilt hazards to (i) compare the ability of identifying the driving hazards by stopping the video and pointing to the hazard between people with ASD and without ASD and (ii) deﬁne whether people with ASD have diﬃculty in responding to hazards or orienting to them. n = 35 18 ASD males (MA = 18.79 years) 17 TD males (MA = 18.19 years) (i) Although nonsigniﬁcant, participants with ASD showed a slower ﬁxation on the hazard source and a slower orientation to the hazards. (ii) Greater attentional capture in the time preceding the hazards onset was associated with lower verbal IQ, suggesting that individuals with ASD may distribute or direct their attention diﬀerently when identifying driving hazards. Limited to male young adults with ASD, but with no speciﬁc training or driving experience Results cannot be generalised to ASD drivers or of diﬀerent age ranges or females with ASD. Wade et al. 2014 (USA) [36] Cross-sectional study To determine if a novel virtual reality driving environment can detect between-group diﬀerences n = 8 4 males with ASD (MA = 16.87 years) 4 TD controls (MA = 15.34 years; 3 males and 1 female) (i) The ASD group experiencing a higher number of simulated driving failures (ii) Participants with ASD had a vertically higher and horizontally right visual gaze. (iii) The ASD group had a signiﬁcantly higher skin conduction level and skin conductance response rate suggesting that participants with ASD may have greater anxiety. Small sample size: underpowered study Unclear of any real-life translation Wade et al. questionable. Limited to only male participants Results cannot be generalised to ASD drivers with a licence or of diﬀerent age ranges or females with ASD. Sheppard et al. 2016 (UK) [26] Cross-sectional study To investigate whether individuals with ASD have diﬃculty judging the location of moving objects in a driving context using a time-to-arrival task on a desktop virtual reality program n = 44 23 males with ASD (MA = 18.55 years; 7 with Autism and 16 with Asperger’s) 21 males in comparison group (MA = 18.83 years) (i) Participants with ASD were less accurate at predicting which of the two cars in the virtual reality program would arrive ﬁrst at an intersection on a straight road. (ii) There were no diﬀerences between participants with ASD and the comparison group when the simulation was along a curved road Participants were all nondrivers so generalisability to drivers is not possible. Unclear of any real-life translation 12 Behavioural Neurology Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations Sheppard et al. 2017 (UK) [33] Case control study Videos of 20 diﬀerent driving situations with inbuilt hazards to (i) compare the ability of identifying the driving hazards by stopping the video and pointing to the hazard between people with ASD and without ASD and (ii) deﬁne whether people with ASD have diﬃculty in responding to hazards or orienting to them. n = 35 18 ASD males (MA = 18.79 years) 17 TD males (MA = 18.19 years) (i) Although nonsigniﬁcant, participants with ASD showed a slower ﬁxation on the hazard source and a slower orientation to the hazards. (ii) Greater attentional capture in the time preceding the hazards onset was associated with lower verbal IQ, suggesting that individuals with ASD may distribute or direct their attention diﬀerently when identifying driving hazards. Limited to male young adults with ASD, but with no speciﬁc training or driving experience Results cannot be generalised to ASD drivers or of diﬀerent age ranges or females with ASD. Wade et al. questionable. 2015 (USA) [11] Cross-sectional study To compare the eﬀects on driving performance and gaze patterns using the gaze-contingent system and the gaze-insensitive, performance-based system n = 18 12 males with ASD in the gaze-contingent group (MA = 14.65 years) 6 in the performance-based group (MA = 15.93 years) (i) The performance-based group showed a signiﬁcantly higher mean vertical and right-sided gaze component when compared to the gaze-contingent group. (ii) The performance-based group showed a decrease in trial failures from pre- to post-post. Small sample size: underpowered study. Equivalence of groups was questionable. Zhang et al. 2015 (USA) [37] Descriptive study To evaluate the feasibility of combining psychophysiological data collection with performance based data using a virtual reality program 10 with ASD (age range = 13 to 17 years) (i) The best accuracy to assess cognitive load was by combining eye gaze and EEG data in a hybrid data analysis model. Small sample size: underpowered study Applicability to real-life situations was questionable. Zhang et al. 2014 (USA) [38] Descriptive study To determine if performance data and aﬀective data can be combined to predict the optimum driving diﬃculty level of a virtual reality program 7 with ASD (age range = 13 to 17 years) (i) Combining performance and aﬀective state data was better at predicting the diﬃculty level when compared to separating the data. (ii) The number of driving performance failures and the Small sample size: underpowered study 13 Behavioural Neurology Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations enjoyment level had a strong relationship with the diﬃculty level. Barriers to obtaining a licence and training on drivers of the spectrum (n = 3) Almberg et al. 2017 (Sweden) [3] Descriptive study Questionnaires with open and closed questions to explore the facilitators or barriers in driving education experienced by individuals with ASD or ADHD who obtained a learner’s permit, from the perspective of the learner drivers and their driving instructors n = 33 14 male ADHD 19 ASD (MA = 20.7 years; 16 males and 3 females) 9 driving instructors (4 with ADHD learner drivers and 5 with ASD learner drivers) (i) Participants with ASD required twice as many driving lessons and more on-road test than those with ADHD. (ii) Reading diﬃculties meant converting theory into practice was diﬃcult. questionable. (iii) Adjusting to on-road situations that were unfamiliar of deviated from the “textbook” script was challenging. (iv) Paying attention to other road users and road signs was reported to be a problem. (v) Hazard perception and the ability to regulate the driving according to the traﬃc conditions were diﬃcult for ASD participants. Small sample size: underpowered study Use of nonvalidated outcome measures Did not consider comorbidity as a confounder Did not consider impact of prior familiarity and experience in working with people with ASD and ADHD in measuring outcomes ∗Lindsay 2017 (Canada) [9] Systematic literature review To systematically review the literature on factors (e.g., barriers and facilitators) aﬀecting driving and motor vehicle transportation experiences of people with ASD 22 studies from eight databases with 2919 participants (mean age 17.3 years 364 individuals with ASD 2555 parents of youth with ASD 13 studies focused on factors aﬀecting driving including the following: (i) Challenges in obtaining a licence (e.g., handling unexpected changes, sustaining attention for long drives, merging into traﬃc, and limited ability to read facial expressions and gestures) (ii) Driving conﬁdence (not conﬁdent in reaction time, much resulted from experiences of anxiety) (iii) Driving behaviours (e.g., avoiding heavy traﬃc or highways, driving at night, adhering to speed regulation, and lane maintenance) (iv) Strategies to improving driving skills (e.g., direct communication, providing lessons in short intervals, starting oﬀin quiet Research conducted on March 2015 Inclusion restricted to articles published in English Grey literature not included 14 14 Behavioural Neurology Table 1: Continued. Authors, year (country) Type of study (study design) Objective Population Key ﬁndings Study limitations areas, not giving too much information or verbal correction, and address anxiety). Tyler 2013 (Australia) [17] Case study To examine the problems faced by supervisors and instructors during training and the strategies that can be implemented to decrease the risks associated for drivers with Asperger’s syndrome Four case studies on the following: (i) 20-year-old male with Asperger’s/ADHD (ii) 26-year-old male, with Asperger’s/anxiety/ depression (iii) 20-year-old male with Asperger’s/ADHD/ (iv) 18-year-old male with Asperger’s/dyspraxia (i) Problems included being easily distracted during driving, poor concentration, anxiety, and sensory overload (ii) Strategies included are as follows: (a) Communication book for parents/learners/instructors to reinforce the sequence of tasks using a consistent approach are provided. (b) Regular lessons were conducted. (c) The use of keywords and structured language in instructions for consistency reduced confusion and anxiety. 5. Discussion Individuals on the autism spectrum appear to have decreased ability to multitask and manoeuvre through com- plex traﬃc scenarios. To accomplish these tasks, drivers on the autism spectrum require training on the tactical level skills of the Michon’s model of driving [29]. Tactical behav- iour in driving involves striking a balance between the demands of driving and the driver’s ability to drive safely in accordance to the road rules [30]. Successful acquisition of the tactical skills may allow drivers on the autism spec- trum to reserve their cognitive capacity to cope with tasks with high number of executive functions and decision- making demands. There is inconsistent evidence on the driving ability of people on the autism spectrum. For example, some studies reported that there is no diﬀerence between drivers on the autism spectrum and neurotypical counterparts in the maintenance of lane position and speed [8], response time in braking, and overall driving ability [2]. These studies were conducted either in simulated driving or in virtual reality environments generated by computer desktops. Conversely, when the abil- ity of drivers was assessed in naturalistic on-road driving environments, drivers on the autism spectrum experience diﬃculty in complex traﬃc scenarios, especially driving tasks that required nonverbal communications with other drivers [23]. In addition, when compared with neurotypical controls, drivers on the autism spectrum had diﬃculty in multitasking and performing complex driving tasks and consistently maintaining a safe speed. They were also observed to have decreased manoeuvring ability and increased in response time to traﬃc hazards [23]. Unexpected changes to the usual driving routine or driving norms from other drivers dis- tracted the attention of drivers on the autism spectrum and induced performance anxiety. When responding to added cognitive demands, drivers on the autism spectrum had poorer baseline executive function and performed worse on general and tactical driving [23]. In identifying the training of the individuals on the spec- trum, there is no experimental study aimed at testing novel ways to train individuals on the autism spectrum to obtain their driver’s licence. In the current review, most observa- tional studies described the barrier individuals on the autism spectrum experience in getting a driver’s licence. None of them focussed on investigating the best strategies to help individuals on the spectrum to obtain their driver’s licence. 5.1. Future Research. 5. Discussion The current literature reviewed pro- vides a preliminary driving proﬁle of individuals on the autism spectrum in simulated contexts or during on-road assessments with predetermined routes. Given that individ- uals on the autism spectrum are susceptible to stress and anx- iety, their driving experience is very likely to deteriorate in a test environment. For this particular cohort, research into the individuals in naturalistic driving environments will generate a more comprehensive driver proﬁle. Driving is essential for individuals on the spectrum, but they showed a relatively low take-up rate of formal driving licence. There is limited research on how to eﬀectively train individuals on the autism spectrum to drive independently and safely. Future research should focus on identifying strategies and the best practice of training to support individuals on the autism spectrum to get their licences. g g [ ] Similarly, discrepancies were found by Cox et al. [21] who reported a greater proportion of traﬃc oﬀences in drivers on the autism spectrum, whereas Chee et al. [23] found that drivers on the autism spectrum performed better than neuro- typical drivers in rule-following aspects of driving. A possible explanation for these discrepancies is that the Cox et al. [21] study observed drivers on the autism spectrum using a driv- ing simulator assessment, which is an artiﬁcial context. Con- sequentially, the real-life behaviour changes of drivers on the autism spectrum in responding to high-demand traﬃc sce- narios were not known. This notion is supported in a validity study using driving simulators to assess driving behaviours of older drivers. Lee et al. [27] reported that only 67% of driving behaviours observed in a driving simulator was transferable to an on-road driving environment. Simulated driving envi- ronments might not be sensitive enough to detect changes of the driving performance. On-road driving is the gold stan- dard to assess and observe ability of drivers. Drivers on the autism spectrum underperform in some aspects of driving or drive diﬀerently in naturalistic environments, but there is insuﬃcient evidence to support the notion that drivers on the spectrum are more at risk of being involved in an accident than their neurotypical counterparts [28]. 5.2. Limitations. Most of the studies included in this review used to establish a proﬁle of drivers on the spectrum were based on observations in driving simulator and/or virtual reality settings. questionable. (d) Reduce discussions on topic of interest to increase focus on the driving task. (e) Break tasks down and working through smaller components in sequence to reduce anxiety. (f) Instructors used “what if” scenarios to broaden participant’s understanding of unpredictable drivers and pedestrians. (g) Instructors used visual markers for judgement of distance, crash avoidance space, and indicator distance rules. Interpretation of results of the study dependent on the sensitivity and integrity of the investigator Case study design: inherently faces the lack of representativeness of general population Eﬀorts undertaken to ensure trustworthiness, credibility, transferability, dependability, conﬁrmability, and rigour of the study were not reported. ∗Study included the collection of both driving and public transport data, with suﬃcient data about driving to be included in this review. Behavioural Neurology 15 5. Discussion This is a major limitation, given the low transferability of observations from a driving simulator envi- ronment to real-life on-road driving [27]. Findings from this scoping review should be interpreted with caution, as we only included studies published in English; grey literature, books, and theses were outside the scope of our review. Further, there are eight studies that employed self-report methodol- ogy to collect information from individuals on the autism spectrum on their ability to drive. Although there is no incen- tive for the participants to have falsely reported their driving ability, their reporting may have inﬂated their ability to drive and may have been inﬂuenced by recall bias. The observational studies [8, 26] using cross-sectional designs reported drivers on the autism spectrum to have dif- ﬁculty in interpreting the driving actions of other road users. The eye-gaze patterns of drivers on the autism spectrum demonstrated an increased response time in detecting haz- ards, especially those associated with ineﬀective interpreta- tion of the intentions of other drivers by means of body gesture and social clues. Insuﬃcient attention to the traﬃc events detected on the peripheral visual ﬁelds can be hazard- ous and unsafe on road [26]. References [1] J. Brooks, J. Kellett, J. 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Durbin, “Factors associated with driving in teens with autism spectrum disor- ders,” Journal of Developmental & Behavioral Pediatrics, vol. 33, no. 1, pp. 70–74, 2012. [33] E. Sheppard, E. van Loon, G. Underwood, and D. Ropar, “Attentional diﬀerences in a driving hazard perception task in adults with autism spectrum disorders,” Journal of Autism and Developmental Disorders, vol. 47, no. 2, pp. 405–414, 2017. [34] D. Bian, J. Wade, Z. Warren, and N. Sarkar, “Online engage- ment detection and task adaptation in a virtual reality based driving simulator for autism intervention,” in International Conference on Universal Access in Human-Computer Interac- tion, pp. 538–547, Toronto, ON, Canada, 2016, Springer. [35] J. Fan, J. W. Wade, A. P. Key, Z. E. Warren, and N. Sarkar, “EEG-based aﬀect and workload recognition in a virtual driv- ing environment for ASD intervention,” IEEE Transactions on Biomedical Engineering, vol. 65, no. 1, pp. 43–51, 2018. [36] J. Wade, D. Bian, L. Zhang et al., “Design of a virtual reality driving environment to assess performance of teenagers with ASD,” in International Conference on Universal Access in Human-Computer Interaction, pp. 466–474, Heraklion, Crete, Greece, 2014, Springer. References Stem Cells International Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 MEDIATORS INFLAMMATION of Endocrinology International Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Disease Markers Hindawi www.hindawi.com Volume 2018 BioMed Research International Oncology Journal of Hindawi www.hindawi.com Volume 2013 Hindawi www.hindawi.com Volume 2018 Oxidative Medicine and Cellular Longevity Hindawi www.hindawi.com Volume 2018 PPAR Research Hindawi Publishing Corporation http://www.hindawi.com Volume 2013 Hindawi www.hindawi.com The Scientific World Journal Volume 2018 Immunology Research Hindawi www.hindawi.com Volume 2018 Journal of Obesity Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Computational and Mathematical Methods in Medicine Hindawi www.hindawi.com Volume 2018 Behavioural Neurology Ophthalmology Journal of Hindawi www.hindawi.com Volume 2018 Diabetes Research Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Research and Treatment AIDS Hindawi www.hindawi.com Volume 2018 Gastroenterology Research and Practice Hindawi www.hindawi.com Volume 2018 Parkinson’s Disease Evidence-Based Complementary and Alternative Medicine Volume 2018 Hindawi www.hindawi.com Submit your manuscripts at www.hindawi.com Hindawi www.hindawi.com Volume 2018 MEDIATORS INFLAMMATION of Endocrinology International Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Disease Markers Hindawi Publishing Corporation http://www.hindawi.com Volume 2013 Hindawi www.hindawi.com The Scientific World Journal Volume 2018 Immunology Research Hindawi www.hindawi.com Volume 2018 Journal of Diabetes Research Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Gastroenterology Research and Practice Submit your manuscripts at www.hindawi.com The Scientific World Journal Hindawi Publishing Corporation Hindawi The Scientific World Journal Diabetes Research Journal of Hindawi Endocrinology International Journal of Hindawi www.hindawi.com Volume 2018 Submit your manuscripts at www.hindawi.com Hindawi www.hindawi.com Volume 2018 BioMed Research International Submit your manuscripts at www.hindawi.com Stem Cells International Hindawi www.hindawi.com Volume 2018 Oncology Journal of Hindawi www.hindawi.com Volume 2013 Hindawi www.hindawi.com Volume 2018 Oxidative Medicine and Cellular Longevity Obesity Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Computational and Mathematical Methods in Medicine Hindawi www.hindawi.com Volume 2018 Behavioural Neurology Ophthalmology Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Research and Treatment AIDS Hindawi www.hindawi.com Volume 2018 Parkinson’s Disease Evidence-Based Complementary and Alternative Medicine Volume 2018 Hindawi www.hindawi.com Stem Cells International Hindawi www.hindawi.com Volume 2018 Oncology Journal of Hindawi www.hindawi.com Volume 2013 Hindawi www.hindawi.com Volume 2018 Oxidative Medicine and Cellular Longevity Obesity Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Computational and Mathematical Methods in Medicine Hindawi www.hindawi.com Volume 2018 Behavioural Neurology Ophthalmology Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Research and Treatment AIDS Hindawi www.hindawi.com Volume 2018 Parkinson’s Disease Evidence-Based Complementary and Alternative Medicine Volume 2018 Hindawi www.hindawi.com Obesity Journal of Hindawi www.hindawi.com Volume 2018
https://openalex.org/W1586334646	https://online.unisc.br/seer/index.php/epidemiologia/article/download/3312/2645	Portuguese	null	Vigilância epidemiológica em Santa Cruz do Sul/RS A construção de um protocolo para atenção básica	Revista de Epidemiologia e Controle de Infecção	2,013	cc-by	1,841	PUBLICAÇÃO OFICIAL DO NÚCLEO HOSPITALAR DE EPIDEMIOLOGIA DO HOSPITAL SANTA CRUZ E PROGRAMA DE PÓS GRADUAÇÃO EM PROMOÇÃO DA SAÚDE - DEPARTAMENTO DE BIOLOGIA E FARMÁCIA DA PUBLICAÇÃO OFICIAL DO NÚCLEO HOSPITALAR DE EPIDEMIOLOGIA DO HOSPITAL SANTA CRUZ E PROGRAMA DE PÓS GRADUAÇÃO EM PROMOÇÃO DA SAÚDE - DEPARTAMENTO DE BIOLOGIA E FARMÁCIA DA DESCRITORES Vigilância Epidemiológica Atenção Primária à Saúde de risco para a comunidade, sempre se respeitando o direito de anonimato dos cidadãos. A Lei Orgânica da Saúde conceitua Vigilância Epidemiológica (VE) como: “conjunto de ações que proporciona o conhecimento, a detecção ou prevenção de qualquer mudança nos fatores deter- minantes e condicionantes da saúde individual ou coletiva, com a ﬁ nalidade de recomendar e adotar as medidas de prevenção e controle das doenças ou agravos”¹. O Ministério da Saúde através da Portaria nº 104, de 25 de janeiro de 2011, deﬁ niu as terminologias adotadas em legislação na- cional, conforme o disposto no Regulamento Sanitário Internacional 2005 (RSI 2005), a relação de doenças, agravos e eventos em saúde pública de notiﬁ cação compulsória em todo o território nacional e estabelece ﬂ uxo, critérios, responsabilidades e atribuições aos pro- ﬁ ssionais e serviços de saúde³. Podemos acrescentar que o desencadeamento do processo de vigilância tem inicio com a informação do problema de saúde que se destina à tomada de decisões e, por essa razão deﬁ ne-se a vigilância epidemiológica por meio da tríade informação – decisão – ação. Sendo que as doenças e eventos constantes devem ser notiﬁ - cados e registrados no Sistema de Informação de Agravos de Notiﬁ - cação - SINAN, obedecendo às normas e rotinas estabelecidas pela Secretaria de Vigilância em Saúde do Ministério da Saúde - SVS/MS. A VE constitui-se assim um importante instrumento de preven- ção e controle de doenças e agravos e fornece importantes subsídios para o planejamento, organização e operacionalização dos serviços de saúde, como também para a normatização de atividades técnicas correlatas². Para tanto a comunicação da ocorrência de determinada doença ou agravo à saúde, feita a autoridade sanitária por proﬁ ssionais e serviços de saúde ou qualquer cidadão, para ﬁ m de adoção de me- didas de intervenção pertinentes através das notiﬁ cações é essencial. A coleta de dados realizada pelos proﬁ ssionais de saúde da atenção básica deve ser realizado a partir da ocorrência de um evento sanitário de caso suspeito ou conﬁ rmado de doença ou agravo sob vigilância. A co- leta de dados ocorre em todos os níveis (municipal, estadual e federal) de atuação do sistema de saúde. A força e valor da informação (que é o dado analisado) dependem da qualidade e ﬁ dedignidade com que a mesma é gerada. Rev Epidemiol Control Infect. 2013;3(1):36-37 Kellen Nunes Rodrigues Gassen¹ Kellen Nunes Rodrigues Gassen¹ g 1Enfermeira – Especialista em Enfermagem do Trabalho (Unisc), Especialista em Educação Proﬁ ssional na Área da Saúde: Enfer- magem (FIOCRUZ/ENSP), Especialista em Saúde da Família (UNA-SUS/UFCPA). Mestranda em Educação (Unisc), bolsista CNPq. Coordenadora Técnica das Equipes de Saúde da Família de Santa Cruz do Sul/RS. g 1Enfermeira – Especialista em Enfermagem do Trabalho (Unisc), Especialista em Educação Proﬁ ssional na Área da Saúde: Enfer- magem (FIOCRUZ/ENSP), Especialista em Saúde da Família (UNA-SUS/UFCPA). Mestranda em Educação (Unisc), bolsista CNPq. Coordenadora Técnica das Equipes de Saúde da Família de Santa Cruz do Sul/RS. g 1Enfermeira – Especialista em Enfermagem do Trabalho (Unisc), Especialista em Educação Proﬁ ssional na Área da Saúde: Enfer- magem (FIOCRUZ/ENSP), Especialista em Saúde da Família (UNA-SUS/UFCPA). Mestranda em Educação (Unisc), bolsista CNPq. Coordenadora Técnica das Equipes de Saúde da Família de Santa Cruz do Sul/RS. Recebido em: 11/01/2013 Aceito em: 07/04/2013 enfkn@hotmail.com Recebido em: 11/01/2013 Aceito em: 07/04/2013 Recebido em: 11/01/2013 Aceito em: 07/04/2013 enfkn@hotmail.com ISSN 2238-3360 \| Ano III - Volume 3 - Número 1 - 2013 RELATO DE EXPERIÊNCIA RELATO DE EXPERIÊNCIA Kellen Nunes Rodrigues Gassen¹ 1Enfermeira – Especialista em Enfermagem do Trabalho (Unisc), Especialista em Educação Proﬁ ssional na Área da Saúde: Enfer- magem (FIOCRUZ/ENSP), Especialista em Saúde da Família (UNA-SUS/UFCPA). Mestranda em Educação (Unisc), bolsista CNPq. Coordenadora Técnica das Equipes de Saúde da Família de Santa Cruz do Sul/RS. Vigilância epidemiológica em Santa Cruz do Sul/RS A construção de um protocolo para atenção básica Epidemiological surveillance in Santa Cruz do Sul / RS Construction of a primary care protocol Vigilância epidemiológica em Santa Cruz do Sul/RS A construção de um protocolo para atenção básica Epidemiological surveillance in Santa Cruz do Sul / RS Construction of a primary care protocol RELATO atenção básica. Tais proﬁ ssionais foram escolhidos por serem muitas vezes os primeiros a realizarem o acolhimento dos usuários em suas queixas e demandas e por serem na grande maioria os coordenado- res locais dos serviços, sendo necessário, portanto terem um olhar mais aguçado a essas doenças e agravos e o importante papel de compartilhar as informações e o material com os demais proﬁ ssio- nais e acadêmicos de seus setores para uma vigilância mais efetiva e menos subnotiﬁ cada. Considerando a importância que os dados epidemiológicos gerados através das notiﬁ cações das doenças compulsórias no mu- nicípio de Santa Cruz do Sul têm para planejar as ações em saúde e visando o controle e monitoramento desses agravos foi elaborado o Protocolo de Vigilância Epidemiológica na Atenção Básica, que tem por objetivo, informar, padronizar e condensar as condutas e ﬂ uxos das notiﬁ cações das doenças Compulsórias na rede de Atenção Bá- sica. Dessa forma instrumentalizando os proﬁ ssionais de saúde das unidades de saúde em seu trabalho e possibilitando o acesso através da consulta no Protoloco do procedimento correto a ser tomado frente aos agravos notiﬁ cáveis. Assim cada serviço de unidade básica de saúde tem atualmen- te o Protocolo de Vigilância Epidemiológica de forma organizada, com a facilidade de consulta nesse material sempre que necessário em seus atendimentos, facilitando o acesso da informação, a agili- dade no atendimento e a correta conduta em casos de doenças e agravos notiﬁ cáveis. A metodologia utilizada foi organizada a partir das diﬁ culda- des de acesso a informações mais concisas das doenças e agravos notiﬁ cáveis e/ou da falta de uma sistematização dos ﬂ uxos e con- dutas do correto procedimento frente a uma doença ou agravo de notiﬁ cação compulsória. O Protocolo foi elaborado por proﬁ ssional enfermeira da Estratégia de Saúde da Família em parceira com a enfermeira responsável pelo setor de Vigilância epidemiológica do município, iniciando com uma revisão bibliográﬁ ca sobre as doenças de notiﬁ cação compulsória, utilizando-se do manual do Ministério da Saúde que aborda o Sistema de Informação de Agravos de No- tiﬁ cações - SINAN e da Lista de Doenças e Agravos do ano de dois mil e onze preconizados para o Estado do Rio Grande do Sul, esse constante na introdução do material produzido. VIGILÂNCIA EPIDEMIOLÓGICA EM SANTA CRUZ DO SUL/RS – A CONSTRUÇÃO DE UM PROTOCOLO PARA ATENÇÃO BÁSICA Kellen Nunes Rodrigues Gassen VIGILÂNCIA EPIDEMIOLÓGICA EM SANTA CRUZ DO SUL/RS – A CONSTRUÇÃO DE UM PROTOCOLO PARA ATENÇÃO BÁSICA Kellen Nunes Rodrigues Gassen RELATO Após construiu-se com base nas recomendações do MS uma padronização das con- dutas e ﬂ uxos para cada doença e agravo constante na lista, com forma e linguagem a facilitar o consulta pelo proﬁ ssional da rede nos atendimentos, incluindo nesse material o modelo de cada uma das ﬁ chas de notiﬁ cação a ser preenchido. CONSIDERAÇÕES FINAIS Em suma, o Protocolo foi construído baseado nas normas técnicas do Ministério da Saúde e nos ﬂ uxos organizados atualmente no município através da estrutura de serviços de saúde todos eles revisados pelo setor de Vigilância Epidemiológica do Município, mas sendo indispensável ser atualizado semestralmente ou sempre que necessário, conforme as alterações estipuladas pelo Ministério da Saúde, Secretaria Estadual da Saúde do Rio Grande do Sul e a organização dos serviços de saúde do município. Com isso, que o Protocolo de Vigilância Epidemiológica tem se mostrado um instrumento facilitador do processo de trabalho nessa área, contribuindo para melhorar a qualidade das informações epidemiológicas das unidades de saúde e o planejamento das ações em saúde em Santa Cruz do Sul. Nessa fase foram realizados encontros, reuniões e contatos breves para estudos, debates e informações com outros proﬁ ssio- nais de setores por onde ocorrem os ﬂ uxos e que estão envolvidos diretamente nesse processo, tais como a Unidade Municipal de Referência em Saúde do Trabalhador (UMREST), Centro Municipal de Atendimento à Sorologia DST/AIDS (CEMAS), Ambulatório de Tuberculose e Hanseníase, setor de Ginecologia entre outros. Enﬁ m, é possível aﬁ rmar que a produção desse material se efetiva continuamente em seu objetivo quando passa a ser utilizado de fato nos serviços das unidades básicas de saúde do município. Rev Epidemiol Control Infect. 2013;3(1):36-37 DESCRITORES Para isso, faz- se necessário que os responsáveis pelo atendimento estejam bem preparados para diagnosticar corretamente o caso, bem como realizar uma boa investigação epidemiológica, com anotações claras e conﬁ áveis. Segundo as diretrizes Nacionais da VE, deve ser notiﬁ cada a simples suspeita da doença, sem aguardar a conﬁ rmação do caso, que pode signiﬁ car perda de oportunidade de adoção das medidas de prevenção e controle indicadas, essa tem que ser sigilosa, só podendo ser divulgada fora do âmbito médico sanitário em caso Páginas 01 de 02 não para ﬁ ns de citação Páginas 01 de 02 não para ﬁ ns de citação REFERÊNCIAS 1. BRASIL. Lei 8080 de 19/09/1990 – que dispõe sobre as condições para a promoção, proteção e recuperação da saúde. Disponível em http://www.planalto.gov.br/ccivil_03/leis/l8080.htm (Acesso em 12/09/2011). 1. BRASIL. Lei 8080 de 19/09/1990 – que dispõe sobre as condições para a promoção, proteção e recuperação da saúde. Disponível em http://www.planalto.gov.br/ccivil_03/leis/l8080.htm (Acesso em 12/09/2011). Após a conclusão do material passou-se a fase de imple- mentação do Protocolo de Vigilância Epidemiológica nas unidades básicas de saúde do município de Santa Cruz do Sul, contando com o apoio da Universidade de Santa Cruz do Sul, através do departamen- to de enfermagem, coordenação da Estratégia de Saúde da Família e Coordenação das Unidades Básicas de Saúde do município, o ma- terial e os formulários de notiﬁ cação foram organizados em pastas a ﬁ m de facilitar o acesso das informações nas unidades de saúde. 2. BRASIL. Ministério da Saúde. Secretaria de Vigilância em Saúde. Departamento de Vigilância Epidemiológica. Guia de vigilância epidemiológica / Ministério da Saúde, Secretaria de Vigilância em Saúde, Departamento de Vigilância Epidemiológica. – 7. ed. – Brasília: Ministério da Saúde, 2009. 816 p. (Série A. Normas e Manuais Técnicos). 3. BRASIL. PORTARIA Nº 104, DE 25 DE JANEIRO DE 2011. Disponível A seguir uma capacitação foi realizada com todos proﬁ ssio- nais técnicos de enfermagem e enfermeiros da rede, com o objetivo de sensibiliza-los para a importância da notiﬁ cação frente a esses agravos, além de apresentar o novo instrumento de trabalho da BRASIL. PORTARIA Nº 104, DE 25 DE JANEIRO DE 2011. Disponível em: http://bvsms.saude.gov.br/bvs/saudelegis/gm/2011/ prt0104_25_01_2011.html (Acesso em 02/08/2012). prt0104_25_01_2011.html (Acesso em 02/08/2012). Rev Epidemiol Control Infect. 2013;3(1):36-37 Páginas 02 de 02 não para ﬁ ns de citação Páginas 02 de 02 não para ﬁ ns de citação
https://openalex.org/W3015362892	https://hrcak.srce.hr/file/316891	Latin	null	Multifunctional Product Marketing Using Social Media Based on the Variable-Scale Clustering	Tehnički vjesnik/Tehnički vjesnik	2,019	cc-by	5,838	1 INTRODUCTION electronic word of mouth, and thus positively influence customers' overall satisfaction. Geetha et al., [15] confirm the consistency between the online ratings and actual customer feelings across two different categories of hotels. The research also suggests that managers of budget hotels should pay more attention to staff performance, compared with other premium hotels. Political factors have been increasing the uncertainty of our fragile global economy recently, and unpredictable trade risks might last even longer [1, 2]. Enterprises, especially multinational firms, must focus on the continuous improvement of their product quality and service level to survive [3]. Thus, the key issue depends on whether the enterprise is able to understand and respond to the customer needs. p As for the product-oriented enterprises, the study related to direct the product planning through mining customer-generated topics gains more popularity. Jeong et al., [4] propose an opportunity mining approach based on social media modelling for product planning. According to the topic discovery and sentiment analysis method, the research evaluates the satisfaction level of each product topic and devises the development strategies referring to the primary keywords of topics. Jan [16] presents an automatic opinion mining method of customers' textual online reviews. The algorithm provides the word cloud of each product, which could enlighten enterprises on the production operation in the next stage. However, customer demands have gradually become more dynamic and complicated due to the functional diversity and lifecycle reduction of products [4]. That pushes enterprises urgently to mine the real-time needs or opinions of different customers through appropriate techniques and methods. Although the traditional customer satisfaction investigation (usually based on questionnaires) is able to get the reliable customer attitude，relatively long data acquisition time and high communication cost make it hard to meet today’s business requirements [5, 6]. In a word, researches above mostly apply the social media analysis to improve the enterprise competitiveness of the core business and then affect customers' feelings or experience. However, incentive strategies and marketing activities could directly promote customer satisfaction if they can arouse customers' interest accurately. Therefore, this paper studies the problem of customer satisfaction identification and improvement based on social media mining methods, which enriches the intention of the current customer relationship management (CRM). Contributing to the rapid development of information network technology, social media comes into being and shows excellent advantages on the social interaction and information exchange [7]. ISSN 1330-3651 (Print), ISSN 1848-6339 (Online) ISSN 1330-3651 (Print), ISSN 1848-6339 (Online) https://doi.org/10.17559/TV-20181120082714 Original scientific paper Multifunctional Product Marketing Using Social Media Based on the Variable-Scale Clustering Ai WANG, Xuedong GAO Abstract: Customers' demands have become more dynamic and complicated owing to the functional diversity and lifecycle reduction of products which pushes enterprises to identify the real-time needs of distinct customers in a superior way. Meanwhile, social media turned as an emerging channel where customers often spontaneously can express their perceptions and thoughts about products promptly. This paper examines the customer satisfaction identification and improvement problem based on social media mining. First, we proposed the public opinion sensitivity index (POSI) to uncover target customers from extensive short-textual reviews. Subsequently, we presented a customer segmentation approach based on the sentiment analysis and the variable-scale clustering (VSC). The approach is able to get several customer clusters with the same satisfaction level where customers belonging to each cluster have similar interests. Finally, customer-centered marketing strategies and customer difference marketing campaigns are planned under the shadow of customer segmentation results. The experiments illustrate that our proposed method can support marketing decision marketing in practice that enriches the intention of the current customer relationship management. Keywords: customer satisfaction; scale transformation; short text clustering; social media mining Tehnički vjesnik 26, 1(2019), 193-200 1 INTRODUCTION Since the convenience and freedom of social media, it turns into an emerging channel where customers often spontaneously express their feelings and ideas about products [8,9]. Hence, social media platforms unconsciously provide an open data source, which stores plenty of real-time customer opinions on various products of different companies even from their competitors [4, 10-12]. The main contributions are as follows. First, we propose the public opinion sensitivity index (POSI) to measure the importance and popularity of a customer review. The POSI could be utilized to find target customers from extensive initial textual reviews. Second, we present a customer segmentation approach based on the sentiment analysis and the variable-scale clustering (VSC). The approach is able to get several customer clusters with the same satisfaction level, and also customers belonging to each cluster have similar interests or concerns. Third, specific customer-centered strategies are provided to Several researchers have noticed the potential value of social media, and previous works are organized mainly from two perspectives. As for the service-oriented enterprises, the study related to improving the business performance through mining customer-concerned factors gain more popularity. Zhao et al., [13] and Guo et al., [14] study the customer satisfaction influence factors gathering from online reviews to improve the management performance of hotels. One important conclusion illustrates that online reviews positively influence the Tehnički vjesnik 26, 1(2019), 193-200 193 Ai WANG, Xuedong GAO: Multifunctional Product Marketing Using Social Media Based on the Variable-Scale Clustering support the enterprise marketing decision following the results of our customer segmentation approach. establishing a satisfaction function. They figure out that the strength of customer satisfaction is slightly increased when the actual feeling surpasses a certain value (expectation), while it is significantly reduced when the actual feeling falls below expectation. Let Tui represent the actual feeling (score) of customer Cu after purchasing product Pi or receiving service Si offered by an enterprise, and Texp represents the expectation of customer Cu, the customer satisfaction function Cu is defined as [22]: The rest of the paper is organized as follows. Section 2 presents previous works related to this research, including the customer satisfaction analysis and social media mining. Section 3 describes our main methodology together with related data analysis tools in detail. We present our experiment analysis that verifies the effectiveness of the proposed approach in Section 4. And the paper is concluded in Section 5. 1 INTRODUCTION ( 1) exp ui ui ui exp exp ui ui ui T T T C T T T T T δ  ≥  =  − + ≤  (1) (1) 2 LITERATURE REVIEW 2.1 Customer Satisfaction Analysis Customer satisfaction refers to the difference between customers’ expected effect and their actual feeling after purchasing the product or receiving the service [16]. Facing the fierce market competition, high customer satisfaction will promote enterprises to win more customer loyalty and earn more customer value. On the contrary, low customer satisfaction will cause much customer loss and make enterprises hard to maintain their competitiveness. Therefore, customer satisfaction is one of the best indicators reflecting the future profitability of enterprises. where the parameter δ ∈ N+ reflects the customer’s tolerance to low-quality Pi or Si. Although customer satisfaction can be directly calculated via Eq. (1), enterprises still encounter the challenge of missing value, since most of the customers ignore the evaluation process due to their carelessness or unwillingness. On the other hand, customers would like to post online reviews to express their attitudes towards some products or services on public social media platforms, which implies their satisfaction degree. The discussions usually consist of textual words, emoticons, and even pictures, etc. Jeong et al., [4] gather the online reviewing topics of a mobile phone product and then calculate the sentiment stock of every topic based on deep learning methods. The sentiment polarity of a topic is determined by its keyword weight (frequency) and the sentiment intensity of each keyword. Besides, they transform the sentiment stock of product topics to a scale of 0-10, that is satisfaction level, which successfully supports the subsequent application. Hence, this research selects the social network mining method to study the customer satisfaction identification of multifunctional products. Since different enterprises operate characteristic products and service, they have to establish their own customer satisfaction indices (CSI). For example, Helia et al., [17] calculate the CSI for a health referral centre from five dimensions that is reliability, responsiveness, assurance, empathy, and tangibility. Masound [18] builds a five-layered CSI system for e-commerce enterprises, consisting of the product layer, service layer, information technology, and network systems layer, payment system layer, and finally the loyalty and bonus program layer. Take the product layer as an example, and it is divided into six aspects, i.e., product customization, product price, product information, product scope, product quality, and product warranty indices. Besides, several countries have developed their standard CSI, such as American (ACSI), Swedish (SCSI), and Korean (KCSI) [19-21]. 3 RESEARCH METHOD Customers with higher POSI either have larger frequency or higher importance, both represent that they are exactly the active and important target audience of enterprise's marketing campaigns. In this section, we present a customer satisfaction estimation method using social media data. There are three significant issues that need to be solved. First, filter and exclude irrelevant and worthless reviews or metadata collected from social media platforms, such as advertisements, news, etc. Second, identify the essential and active customers regarding the numerous textual reviews, who are more probably to become the target audience of enterprises' marketing campaigns. Third, divide the customer base and reveal their demographics, perceptions, and concerns, to better support marketing decision making. Relative solutions are proposed in the following three phases. 2.2 Social Media Mining According to the CSI, enterprises attempt various ways to gather the basic information. Common approaches depend on establishing the information feedback and consultation system, as well as conducting the satisfaction questionnaire survey [16]. The customer complaint system facilitates the direct dialogue with customers via the telephone contact or on-site visit, which enables enterprises to know the satisfaction degree of different customers. Similarly, the statistical analysis on a large number of satisfaction questionnaires also successfully quantifies the customer satisfaction degree. However, these approaches have the same disadvantages, i.e. the extended data acquisition time and high communication cost. Social media mining aims to discover the inherent information and knowledge using the online data of social media platforms, which requires high data processing techniques including social media data gathering, pre- processing, modelling and analysis (see Fig. 1). Web crawling is the most common technique to gather numerous online reviews in the text or picture format on social media [24]. Although crawlers can scrape the target dataset conveniently and continuously from any websites, they consume resources of visited systems and will cause the load and schedule issue [25]. To improve the data extraction efficiency, several crawling tools could be installed and utilized directly, such as Scrapy (scrapy.org), python-requests (docs.python-requests.org), and bazhuayu (www.bazhuayu.com). In this regard, the network information technique provides new methods for customer satisfaction identification. On the one hand, a great many online platforms, like e-commerce websites, have already opened the rating function, which invites customers to score the product or service they offered, and the customer satisfaction degree could be derived from the evaluation scores. Ding et al., [22, 23] estimate the customer satisfaction of the personalized cloud service by Different from other websites, the web crawling results of social media platforms always exist in the short content, big noise, low normativity and high sparsity feature, which challenges the traditional text mining techniques. [26]. Certain data preparing and modelling methods should be designed especially for social media mining. Traditional data pre-processing work of text mining mainly contains the word segmentation and stop words Technical Gazette 26, 1(2019), 193-200 194 Ai WANG, Xuedong GAO: Multifunctional Product Marketing Using Social Media Based on the Variable-Scale Clustering 3.1 Data Gathering and Preprocessing removal. If we implement the word segmentation directly on the initial short-textual data of social media, it is bound to drive a high dimensional sparse model with massive noise in the next text modelling stage. Thus, appropriate text filtering work is of great significance utilizing the extra descriptive statistics information provided by social media platforms. For instance, Howells et al., [27] indicates that online users usually express their support of a post on Facebook by a click of "like," which enables us to filter valuable short texts according to the number of "likes" that a post gets. In the beginning, we decide a target product and collect all the related online data from the social media platform via an open source crawling tool. The retrieval content not only contains all the textual reviews that at least mentioned the target product once but also includes the descriptive information of every review (e.g., number of forwarding, number of comments, number of likes), as well as the necessary information of every social media user. According to the characteristic of the social network, the more significant a review is, the more it spreads, and vice versa [39]. We assume that social media users only interact with valuable and attractive reviews. Therefore, the social media data cleaning is achieved by setting coefficients on the descriptive information of the whole reviews. p g There are two classic approaches of the text representation, that is the vector space model (VSM) and word embedding [28]. The VSM maps a document to a great many content-related words or phrases, which successfully translates the textual document calculation to the vector calculation [29]. Word embedding is widely utilized in the natural language processing (NLP) that contains the one-hot representation and distributed representation [32]. 3.2 Target Customers Identification After data pre-processing, we start the formal word segmentation and stop words removal, where multiple reviews from the same user are merged for the completeness. Although marketers are eager to know the personality of consumers as much as possible (that is not limited to their past experiences, brand loyalty, perception of brands) when planning a marketing campaign, what they care about most is their response to campaigns, which might rapidly form the public opinion of an enterprise in a short term. Thus, we propose the public opinion sensitivity index (POSI) to measure the influence of online review(s) by the same customer to the enterprise’s current public opinion. After solving the data pre-processing and modelling problem for the unstructured short texts of social media, various common text mining applications could be utilized directly to the social media analysis, e.g., social media topic modelling, sentiment analysis, short text classification, etc. Notably, the sentiment analysis that focuses on discovering people's polarity of sentiment implied in textual words [38] could form the basis of our customer segmentation method by estimating the satisfaction degree of every online customer. Figure 1 The research framework of social media mining Given the keywords of the enterprise’s current public opinion on the target product, the public opinion sensitivity index (POSI) is expressed as: ( ) 1 lg N i ik k k tf H µ = = ∗ ∑ (2) (2) where N is the total number of keywords in the public opinion, Hk ∈ (0,1] is the importance of keyword wk and 1 1 N k k H = = ∑ , tfik is the term frequency of word wk in the reviews of customer Ci. Figure 1 The research framework of social media mining 3.3 Customer Segmentation Although the POSI is able to distinguish target customers in terms of their short-textual reviews, it is still unable to satisfy marketers when creating deep insight into the consuming markets. Therefore, a customer segmentation algorithm based on the VSC is proposed to provide rich customer information for marketers in detail (see Fig. 2). The algorithm consists of three steps as (1) Classify customer opinions through the sentiment analysis. At this point, customers have been classified into different groups following their sentiment attitudes (i.e., positive, neutral, Tehnički vjesnik 26, 1(2019), 193-200 195 Ai WANG, Xuedong GAO: Multifunctional Product Marketing Using Social Media Based on the Variable-Scale Clustering 𝐶𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞 and negative), which greatly contributes to developing marketing strategies. (2) Segment customer bases via the VSC. Since multiple reviews of one customer have been merged during data preprocessing, customers with similar interests or concerns are divided into the same customer base according to the short text clustering results. (3) Identify customer characteristics by the VSC. Identify the key characteristic of every customer base to support enterprises planning specific marketing campaigns. The VSC overcomes the problem that the characteristics of clusters are less prominent especially handling the high dimensional dataset, of the traditional clustering algorithms. The time complexity of VSC is 𝑂𝑂(𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛) , whereas the number of instances, 𝑚𝑚is the number of attributes, 𝑘𝑘 is the number of clusters, and 𝑡𝑡 is the number of iterations. Figure 2 The process of the customer segmentation approach 4 RESULTS AND DISCUSSIONS Figure 2 The process of the customer segmentation approach Wechat, QQ, and Weibo are the three most popular social media networks in China and their usage from Dec 2017 to Jun 2018 is shown in Fig. 3. Currently, the usage rate of Wechat and QQ remains relatively stable, while Weibo gains a substantial increase due to the prevalence of short videos and multi-channel network (MCN) institutions [39]. Hence, this paper takes Weibo as the data source of social media, and the data gathering scheme is shown in Tab. 1. As is known that people naturally are able to analyze and decide a problem from different perspectives, hierarchies, and dimensions spontaneously, that is referred to as scale transformation (ST) [42]. According to the basic definitions (i.e., the concept space (CS), the scale transformation rate (STR), and the granular deviation (GrD)) in [41], we propose a variable-scale clustering (VSC) method to solve the customer segmentation problem. Given a dataset D, the CS of attributes in D, the scale transformation threshold S0, and the initial clustering parameter k, the pseudo code of the VSC is shown in Algorithm 1. 𝑆 Figure 3 The usage rate of major social media in China from Dec 2017 to Jun 2018 Figure 3 The usage rate of major social media in China from Dec 2017 to Jun 2018 Algorithm 1 AttributeScaleUpClustering(D, CS, 𝑆𝑆0,k) 1: C=D.initialCluster(k) 2: 𝑅𝑅0 = max (𝐺𝐺𝐺𝐺𝐺𝐺(𝐶𝐶𝑖𝑖. 𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞)) 3: D. delete (𝐶𝐶𝑖𝑖. 𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞) 4: for𝐷𝐷≠∅do 5: for all 𝐴𝐴𝑗𝑗∈𝐷𝐷do 6: ifSTR൫𝐴𝐴𝑗𝑗, 𝐶𝐶𝐶𝐶(𝐴𝐴𝑗𝑗)൯< 𝑆𝑆0 then 7: D. update (𝐴𝐴𝑗𝑗, 𝐶𝐶𝐶𝐶(𝐴𝐴𝑗𝑗)) 8: break 9: end if 10: end for 11: 𝑅𝑅0 = max (𝑅𝑅0, 𝐺𝐺𝐺𝐺𝐺𝐺(𝐶𝐶𝑖𝑖. c𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙)) Figure 3 The usage rate of major social media in China from Dec 2 2018 We apply the professional web crawling tool to collect approved online reviews on Weibo, an experiments are performed in OS X (10.11.3) env on a machine with 8GB RAM. Figure 4 The POSI of qualified social media users Algorithm 1 AttributeScaleUpClustering(D, CS, 𝑆𝑆0,k) 1: C=D.initialCluster(k) 2: 𝑅𝑅0 = max (𝐺𝐺𝐺𝐺𝐺𝐺(𝐶𝐶𝑖𝑖. 𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞)) 3: D. delete (𝐶𝐶𝑖𝑖. 𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞) 4: for𝐷𝐷≠∅do 5: for all 𝐴𝐴𝑗𝑗∈𝐷𝐷do 6: ifSTR൫𝐴𝐴𝑗𝑗, 𝐶𝐶𝐶𝐶(𝐴𝐴𝑗𝑗)൯< 𝑆𝑆0 then 7: D. update (𝐴𝐴𝑗𝑗, 𝐶𝐶𝐶𝐶(𝐴𝐴𝑗𝑗)) 8: break 9: end if 10: end for 11: 𝑅𝑅0 = max (𝑅𝑅0, 𝐺𝐺𝐺𝐺𝐺𝐺(𝐶𝐶𝑖𝑖. c𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙)) Algorithm 1 AttributeScaleUpClustering(D, CS, 𝑆𝑆0,k) 1: C=D.initialCluster(k) 2: 𝑅𝑅0 = max (𝐺𝐺𝐺𝐺𝐺𝐺(𝐶𝐶𝑖𝑖. 𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞)) 3: D. delete (𝐶𝐶𝑖𝑖. 4.1 Experimental Results Analysis public opinion of iPhone X, we finally choose the top 634 users as our target customers. Following the requirements of data gathering in Tab.1, there are 15165 original microblogs related to iPhone X during the valid period, while identity authorized online users posted only 4350 of them. Hence, we take 4350 microblogs as the initial social media dataset of our experiments. Fig. 5 presents the customer segmentation results based on the proposed method VSC. Different colour represents different customer satisfaction (sentiment), i.e., positive, neutral, and negative, and each rectangle represents a customer base distinguishing the gender factor. We find out that (1) the distribution of the customer structure is extremely unbalanced, and the first two customer bases own over 54% customers. (2) there are nine customer bases in total, and five in positive, two in negative, one in neutral, which means most of the customers are currently satisfied with iPhone X. (3) The male proportion of customer base 2-9 is quite high (over 90%) and two of them (that is customer base 3 and 9) even reach 100%. However, the largest customer base has only the female. The descriptive information is applied to the data preprocessing, where we set the minimum likes number as 3, the minimum comments number as 2, the minimum forwarding number as 1. Consequently, we gain 1607 qualified microblogs in total. It can be seen that all the irrelevant and worthless microblogs are excluded, which is consistent with the investigation result that over 40% reviews on the social media are advertisements, news, or other spam messages [40]. We merge multiple microblogs posted by the same user, and finish the word segmentation and stop words removal through professional tools in Section 2. What's more, Tab. 2 also provides further insight into the customer segmentation results, including customer satisfaction, gender proportion, average age, and customer characteristics (interests or concerns) of every customer base. The number in brackets of the attribute Avg. Age represents the age missing rate of each customer base and floats about 30%. And most importantly, the VSC discovers the various customer characteristics known as interests or concerns being hidden in customers' reviews. Then, we begin to identify the target customers from qualified social media users via the POSI (see Fig. 4). It can be seen that the POSI curve stays in a rapid decline trend in general, while part of it is relatively gentle. 4 RESULTS AND DISCUSSIONS 𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞) 4: for𝐷𝐷≠∅do 5: for all 𝐴𝐴𝑗𝑗∈𝐷𝐷do 6: ifSTR൫𝐴𝐴𝑗𝑗, 𝐶𝐶𝐶𝐶(𝐴𝐴𝑗𝑗)൯< 𝑆𝑆0 then 7: D. update (𝐴𝐴𝑗𝑗, 𝐶𝐶𝐶𝐶(𝐴𝐴𝑗𝑗)) 8: break 9: end if 10: end for 11: 𝑅𝑅0 = max (𝑅𝑅0, 𝐺𝐺𝐺𝐺𝐺𝐺(𝐶𝐶𝑖𝑖. c𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙)) Algorithm 1 AttributeScaleUpClustering(D, CS, 𝑆𝑆0,k) 𝑅𝐺𝐺𝐺𝐶𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞 Figure 3 The usage rate of major social media in China from Dec 2017 to Jun 2018 2018 We apply the professional web crawling tool, Scrapy, to collect approved online reviews on Weibo, and all the experiments are performed in OS X (10.11.3) environment on a machine with 8GB RAM. 11: 𝑅𝑅0 = max (𝑅𝑅0, 𝐺𝐺𝐺𝐺𝐺𝐺(𝐶𝐶𝑖𝑖. c𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙)) Figure 4 The POSI of qualified social media users Technical Gazette 26, 1(2019), 193-200 196 Ai WANG, Xuedong GAO: Multifunctional Product Marketing Using Social Media Based on the Variable-Scale Clustering Ai WANG, Xuedong GAO: Multifunctional Product Marketing Using Social Media Based on the Variable-Scale Clustering Ai WANG, Xuedong GAO: Multifunctional Product Marketing Using Social Media Based on the Variable Scale Clustering Figure 5 The customer segmentation results based on the VSC Table 1 The data gathering scheme Social Media Weibo Target Product iPhone X Time Period 2018-06-18 00:00to 2018-06-24 24:00 Retrieval Conditions (1) Original microblogs (2) Social media users have passed the personal identity authentication Data Structure Object Descriptive Information Customer Information Customer reviews Number of forwarding, Number of comments, Number of likes, Release time Gender, Birthday, Country, Education, Affiliation, Occupation, Position Table 2 The statistic results of the VSC No. Customer Satisfaction Proportion Avg. Age Interests / Concerns 1 Positive 0,0000 22,8(0,3046) After-sales service, promo 2 Negative 0,9448 24,9 (0,3379) Video apps 3 Neutral 1,0000 24,7 (0,3034) Sales, consumer 4 Positive 0,9032 23,6 (0,2742) Running 5 Positive 0,9149 26,1 (0,3404) Movie 6 Positive 0,8250 26,3 (0,3750) Discount, product, gifts 7 Positive 0,9750 23,8 (0,4000) Celebrity 8 Positive 0,9500 24,6 (0,2500) Photograph 9 Negative 1,0000 24,1 (0,3671) Member, World Cup Figure 5 The customer segmentation results based on the VSC Figure 5 The customer segmentation results based on the VSC Figure 5 The customer segmentation results based on the VSC Tehnički vjesnik 26, 1(2019), 193-200 https://doi.org/10.1016/j.ijhm.2018.04.009 [6] Radojevic, T., Stanisic, N., Stanic, N. & Davidson, R. (2018). The effects of traveling for business on customer satisfaction with hotel services. Tourism Management, 67(1), 326-341. https://doi.org/10.1016/j.tourman.2018.02.007 4.2 Customer-Centered Marketing Strategies This section illustrates how to make marketing decisions using the results of the VSC. Firstly, decide customer-centered marketing strategies through customer satisfaction. For example, there are totally three different satisfaction levels in Tab. 2, i.e., positive, neutral, and negative. We could offer the precise benefits to positive customers, improve the stickiness of neutral customers, and increase the feedback collection of negative customers respectively. Secondly, plan customer personalization marketing campaigns following the proposed marketing strategies. As for the precise benefits strategy, customer bases 1, 5, 8 most probably respond to the exclusive benefits of their interested software or apps. Customer base 7 might look forward to the intangible spiritual enjoyment (like meeting favourite celebrities), while customer base 6 might expect the tangible material reward (like valuable or saleable gifts). And termly pushing the running related information, such as distinctive or available marathon races to customer base 1 could improve their satisfaction. Referring to these, various marketing campaigns for positive customers could be designed. As for the customer stickiness and feedback collection improvement strategy, marketers could make full use of the known customer concerns to set up a more effective conversation and gain better solutions. However, excessive spam messages on the social media provide challenges for our proposed methods. The identity authentication also limits the amount of the available online data. Therefore, future study will focus on the data gathering phase to better fit the data environment of social media. And more contrast experiments will also be implemented on other popular social media (like WeChat and QQ) to further verify the effectiveness of our proposed methods. Acknowledgments The research is supported by the national natural science fund of China (71272161). 4.1 Experimental Results Analysis In order to identify the online users that truly affect the current Tehnički vjesnik 26, 1(2019), 193-200 197 Ai WANG, Xuedong GAO: Multifunctional Product Marketing Using Social Media Based on the Variable-Scale Clustering perspective of social media mining. The public opinion sensitivity index (POSI) was presented to measure the importance and popularity of a customer review. We also proposed a customer segmentation approach based on the sentiment analysis and our improved algorithm, the variable-scale clustering (VSC) algorithm. The functionality of the method was verified herein using the iPhone X short-textual data crawling from Weibo, one of the major social media in China, between 18 Jun 2018 and 24 Jun 2018. Customer-centered marketing strategies and customer personalization marketing campaigns were designed through this case study. Moreover, an evaluation measurement of marketing plans was also established to support marketers decide the final candidate plans under funding constraints. Our proposed methods enrich the intention of the current customer relationship management with the help of the popular social media, which could not only develop the scale transformation thought of clustering algorithms in theory, but also save enterprises' time and cost of the marketing decision process in practice. These characteristics not only represent the similarity of customers but also are at the lowest (detailed) conceptual hierarchy, which further verifies the effectiveness and feasibility of the VSC in practice. These characteristics not only represent the similarity of customers but also are at the lowest (detailed) conceptual hierarchy, which further verifies the effectiveness and feasibility of the VSC in practice. Finally, marketers could make reasonable marketing strategies on behalf of the estimated customer satisfaction and characteristics (see Section 4.2). 6 REFERENCES Last but not least, evaluate all the possible marketing plans and make the final decision. Since experience marketers are likely to brainstorm lots of marketing plans towards different customer base, it is necessary to establish an evaluation measurement of marketing plans. Let Pij represents the jth marketing plan of customer base CBi, the evaluation indicator of Pij as: [1] Antoine, G. (2018). 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Therefore, this paper focuses on the problem of customer satisfaction identification and improvement on the Technical Gazette 26, 1(2019), 193-200 198 Ai WANG, Xuedong GAO: Multifunctional Product Marketing Using Social Media Based on the Variable-Scale Clustering https://doi.org/10.1016/j.inffus.2017.11.002 [22] Ding S., Wang Z., Wu D., & David L. O. (2017). Utilizing customer satisfaction in ranking prediction for personalized cloud service selection. Decis. Support Syst, 93(1), 1-10. https://doi.org/10.1016/j.dss.2016.09.001 [41] Gao, X. & Wang, A. (2018). Variable-scale clustering, 2017 Int. Conf. Logist. INFORMATICS Serv (LISS’ 2018). In press. [23] Ding S., Yang S., Zhang Y., Liang C., & Xia C. (2014). Combining QoS prediction and customer satisfaction estimation to solve cloud service trustworthiness evaluation problems. Knowledge-Based Syst., 56(1), 216-225. https://doi.org/10.1016/j.knosys.2013.11.014 [42] Zhang, Q., Wang, G., & Hu, J. (2013). Multi-granularity knowledge acquisition and uncertainty measurement. Beijing: Science Press, 1(1), 16-25. [24] Qian, X., Li, M., Ren, Y., & Jiang, S. (2018). Social media based event summarization by user-text-image co-clustering. Knowledge-Based Syst. 199 Tehnički vjesnik 26, 1(2019), 193-200 Ai WANG, Xuedong GAO: Multifunctional Product Marketing Using Social Media Based on the Variable-Scale Clustering Contact information: Ai WANG, PhD student (Corresponding author) University of Science and Technology Beijing No. 30 Xueyuan Road, Haidian District, Beijing, China wangai22222@126.com Xuedong GAO, Professor University of Science and Technology Beijing No. 30 Xueyuan Road, Haidian District, Beijing, China gaoxuedong@manage.ustb.edu.cn 200 Technical Gazette 26, 1(2019), 193-200
https://openalex.org/W3022736574	https://europepmc.org/articles/pmc7200679?pdf=render	English	null	Physical presence of spouse enhances brain-to-brain synchrony in co-parenting couples	Scientific reports	2,020	cc-by	10,532	Physical presence of spouse enhances brain-to-brain synchrony in co-parenting couples OPEN Atiqah Azhari1, Mengyu Lim1, Andrea Bizzego2, Giulio Gabrieli 1, Marc H. Bornstein3,4,5 & Gianluca Esposito 1,2,6 ✉ Co-parenting spouses who live together remain in close physical proximity to each other and regularly engage in reciprocal social interactions in joint endeavors to coordinate their caregiving. Although bi-parental rearing is a common occurrence in humans, the influence of the physical presence of a co- parenting spouse on parental brain responses remains largely unknown. Synchrony is conceptualized as the matching of behavioral and physiological signals between two individuals. In this study, we examined how the presence of a co-parenting spouse influences brain-to-brain synchrony when attending to salient infant and adult vocalizations. We hypothesized that brain-to-brain synchrony would be greater in the presence of a spousal partner. Functional Near-infrared Spectroscopy (fNIRS) was used on 24 mother-father dyads (N = 48) to measure prefrontal cortical (PFC) activities while they listened to infant and adult vocalizations in two conditions, together (in the same room at the same time) and separately (in different rooms at different times). Couples showed greater synchrony in the together condition; when comparing fNIRS data between true couples and randomly matched controls, this synchronous effect was only seen in true couples, indicating a unique effect of spousal co-regulation toward salient stimuli. Our results indicate that the physical presence of the spouse might establish synchrony in attentional regulation mechanisms toward socially relevant stimuli. This finding holds implications for the role of the co-parenting spouse in influencing social and parental brain mechanisms. Parturition in humans initiates collaborative efforts between spouses toward effective co-parenting1. Although an evolutionary perspective asserts specific parenting roles of mothers and fathers2, the transition to parenthood in bi-parental species is accompanied by parallel modifications in mothers’ and fathers’ brains, notably in regions implicated in attention, cognition, and affect regulation2–4. Thus, the emergence of co-parenting in humans is likely supported by biobehavioural synchrony in couples3,5, which entails the entrained temporal coordination of physiological and behavioural signals between two individuals in an affiliative bond6. For co-parenting couples, constant interaction with each other presents partners with abundant opportunities to engage in daily rhythms of reciprocal exchanges that establish synchrony across numerous biological systems7. For example, couples have been shown to exhibit synchronised patterns of gaze and affect along with coordinated changes in electrodermal activity8, respiratory sinus arrhythmia9, diurnal cortisol patterns10, and brain activation patterns11,12. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2020) 10:7569 \| https://doi.org/10.1038/s41598-020-63596-2 Physical presence of spouse enhances brain-to-brain synchrony in co-parenting couples OPEN Among studies that investigated physiological synchrony in couples, some have evinced the importance of physical presence of a spouse in initiating synchrony, whereas others have determined that synchrony is still observed in the absence of a spouse. A study that measured cortisol and mood patterns of spousal partners21 revealed that the extent to which biological rhythms of spousal partners synchronise hinges greatly on the amount of time the two spent in close proximity. When cortisol levels were compared at times when partners were together and apart, synchrony in cortisol level was evident only when couples were together. Conversely, another study22 found that synchrony in cortisol level was not depend- ent on whether the spousal partner was present, but was instead determined by the amount of time spousal partners spent with each other throughout the duration of the experiment. Taken together, these contradictory findings underscore an important gap in our knowledge regarding whether the physical presence of a spousal partner affects dyadic synchrony. Understanding this association may aid in uncovering how spousal proximity in day-to-day life facilitates efficient co-parenting responses. y yfi p g p To fill this research gap, the present study employed functional Near-infrared Spectroscopy (fNIRS) to measure the brain signals of co-parents when they were exposed to salient infant and adult vocalisations either together (in the same room at the same time) or separately (in different rooms at different times). We decided to focus on the prefrontal cortical (PFC) region of the brain due to its integral role in both attentional regulation and social cognition, making it a likely region to be implicated when partners jointly attend to salient emotive vocal- isations23–26. First, we aimed to investigate whether synchrony was significantly higher when couples were in the physical presence of one another compared to when they listened to vocalisations separately. To do so, we derived a synchrony index. As the co-parents in our sample lived in the same household, we hypothesised that, compared to when they listened to salient vocalisations in the absence of their partner (separate-condition; SEP), cou- ples would exhibit greater brain-to-brain synchrony in the physical presence of each other (together-condition; TOG)21. Physical presence of spouse enhances brain-to-brain synchrony in co-parenting couples OPEN Because syn- chrony is associated with enhanced mutual attunement to emotional states13 and facilitates behavioral and phys- iological coordination between partners14,15, theories have been advanced that such synchrony may constitute a central pathway to achieving emotional stability16,17. When partners are “in-sync” in their subjective emotional experiences, they respond more optimally to one another’s needs and bolster support for one another14. Partner support buffers stress evoked by the novel demands of parenting and forms a critical ingredient in adaptation to parenthood18,19.h p The pursuit of a joint goal to protect the altricial human infant provides fertile ground for adaptive and syn- chronous functioning of mothers’ and fathers’ brains20. To our knowledge, only one study has so far investigated co-parenting and brain-to-brain synchrony: Using fMRI imaging techniques, the authors examined co-parents’ brain responses to viewing own infant play videos in comparison with standard infant play videos. Results showed 1Psychology Program, School of Social Sciences, Nanyang Technological University, Singapore, Singapore. 2Division of Psychology, Department of Psychology and Cognitive Science, University of Trento, Trento, Italy. 3National Institute of Child Health and Human Development, Bethesda, USA. 4Institute for Fiscal Studies, London, United Kingdom. 5UNICEF, New York USA. 6Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore. ✉e-mail: gianluca.esposito@ntu.edu.sg Scientific Reports \| (2020) 10:7569 \| https://doi.org/10.1038/s41598-020-63596-2 www.nature.com/scientificreports/ Channel Area SEP TOG p (uncorrected) p d Mean SD N Mean SD N 3 IFG 0.007 0.025 141 0.031 0.083 138 0.00221 0.01471 0.4 7 MFG 0.009 0.025 135 0.024 0.045 132 0.00041 0.00409 0.4 11 aPFC 0.013 0.064 135 0.062 0.119 138 0.00003 0.00062 0.5 13 aPFC 0.026 0.109 135 0.043 0.090 138 0.00484 0.02421 0.2 Table 1. Mother-father synchrony indexes in true dyads. Comparison between separate-condition (SEP) and together-condition (TOG) for the significant synchrony indexes. Note: IFG = inferior frontal gyrus, MFG = middle frontal gyrus, aPFC = anterior prefrontal cortex. Table 1. Mother-father synchrony indexes in true dyads. Comparison between separate-condition (SEP) and together-condition (TOG) for the significant synchrony indexes. Note: IFG = inferior frontal gyrus, MFG = middle frontal gyrus, aPFC = anterior prefrontal cortex. that synchrony in neural circuits involved in empathy and social cognition emerged in co-parents exposed to infant distress cues3. This finding suggests that mothers and fathers attune their brain responses to each other and that brain-to-brain synchrony may stem from unique couple coordination. However, the circumstance under which this coordination emerges requires further verification. Physical presence of spouse enhances brain-to-brain synchrony in co-parenting couples OPEN Second, to ascertain whether the co-presence effect was a result of the physical presence of a co-parent, or due to the unique pairing of couples who had existing relationships with each other, we compared the syn- chrony index of true couples to that of control couples (a synchrony index generated from randomly paired brain signals of a mother and father who were not spouses) in both the SEP and TOG conditions. We hypothesized that synchrony would only be observed between true couples but not in control couples. Third, we aimed to investigate whether stimuli- and parent-related factors play a role in driving the enhanced synchronous response observed in TOG compared to SEP. Thus, in channels where, compared to SEP, synchrony was found to be higher in TOG, we would examine if synchrony was influenced by emotional valence of acoustic stimuli (i.e. positive or negative vocalisations) and parents’ characteristics, namely, (i) the ratio of mother to father taking the lead in attending to the child, (ii) primiparous or multiparous status of parents, and (iii) age of parents. As little is known regarding the effects of these variables on brain-to-brain synchrony, we did not have any specific hypotheses for this third exploratory aim. Resultshi The first aim of the study was to verify that, compared to the separate-condition (SEP), synchrony was higher in the together-condition (TOG) condition for true dyads. Analysis of Variance (ANOVA) revealed that the differ- ence in synchrony index between SEP and TOG was significant in four fNIRS channels, namely channel 3 (CH3), CH7, CH11 and CH13 (see Table 1), which were mapped to the left inferior frontal gyrus (IFG), left middle fron- tal gyrus (MFG) and left as well as right bilateral anterior PFC (aPFC), respectively. In all significant channels, the synchrony index in TOG was higher than in SEP (see Table 1).hf y y g The second aim of the study was to prove that the co-presence effect was due to the unique physical presence of a co-parenting spouse. The same ANOVA analysis was conducted on control dyads but the differences in syn- chrony index between TOG and SEP were never significant in any channel (see Supplementary Table 1 for results for all channels). Taken together, significant differences between TOG and SEP in only true dyads, but not control dyads, confirmed that the unique presence of a spousal partner increases couple’s brain-to-brain synchrony.hf yi q p p p p y y The third aim of this study was to test whether the co-presence effect also depended on the type of acoustic stimuli and parent-related characteristics: (i) ratio of mother to father taking the lead in attending to the child which was measured by the Average Co-parenting Ratio Score, (ii) primiparous or multiparous parents, and (iii) age of parents. Scientific Reports \| (2020) 10:7569 \| https://doi.org/10.1038/s41598-020-63596-2 www.nature.com/scientificreports/ www.nature.com/scientificreports re.com/scientificreports/ Figure 1. Comparison of the MCC2 measures between the SEP (red) and TOG (blue) conditions for the six stimuli in the four channels for which a significant effect of physical presence was found. Outliers are not shown. Figure 1. Comparison of the MCC2 measures between the SEP (red) and TOG (blue) conditions for the six stimuli in the four channels for which a significant effect of physical presence was found. Outliers are not sho Synchrony and type of acoustic stimulus. Comparing the difference in synchrony indexes between SEP and TOG in the four significant channels, for each acoustic stimulus, we observed that infant laughter, adult laughter, and static sound induced greater synchrony in TOG compared to SEP (see Fig. 1). Resultshi Except for CH13 (right aPFC), for which no statistical difference across acoustic stimuli was found, infant laughter significantly increased synchrony index in CH3 (left IFG), CH7 (left MFG) and CH11 (left aPFC), while adult laughter and static only increased synchrony in CH7 (left MFG) and CH11(left aPFC). Notably, differences for Infant Cry, both high- and low-pitched, as well as Adult Female Cry were never significant.iff g p y gi No other channel showed significant differences between SEP and TOG across the different acoustic stimuli. g p y gi No other channel showed significant differences between SEP and TOG across the different acoustic stimuli. Synchrony and parent characteristics. First, synchrony was positively correlated with the Average Co-parenting Ratio Score, where a high ratio is associated with the predominance of the mother (rather than the father) in responding to the child (see Supplementary Table 2): ρ = 0.18, p = 0.00003; significant correlations between synchrony index and Average Co-parenting Ratio Score emerged for CH7 (MFG, ρ = 0.29, p = 0.004) and CH11 (left aPFC, ρ = 0.23, p = 0.015).i t Second, significantly (p = 0.00066) higher synchrony was found for primiparous (Mean = 0.06, SD = 0.117) compared to multiparous parents (Mean = 0.028, SD = 0.065) when considering all channels together; and for Channel 7 (p = 0.036; Primiparous: Mean = 0.041, SD = 0.059; Multiparous: Mean = 0.014, SD = 0.031) when considering each channel separately (see Supplementary Table 4).h g p y ( pp y ) Third, parent age was negatively correlated with synchrony: ρ = −0.11, when considering the average age of parents, and ρ = −0.10, p = 0.01, and ρ = −0.14, p = 0.001, when considering mothers’ and fathers’ ages respec- tively (see Supplementary Table 3). Notably, none of the tests was significant on randomly paired, unrelated control dyads.if y Additional analyses of parental characteristics in channels which were not significantly different in TOG SEP conditions could be found in the Supplementary Materials. www.nature.com/scientificreports/ Adopting this perspective, synchrony that emerged in the attentional and cognitive control networks of the brain might reflect couples’ tendency to similarly perceive and process auditory and affective information so as to coordinate joint impending behaviours, when together. g The lack of brain-to-brain synchrony in response to negatively valenced sounds when partners are together might be adaptive for both spousal and co-parenting relationships. Although synchrony is commonly represented as a positive characteristic in the extant literature, synchrony is not necessarily always beneficial for the couple or child. For example, biobehavioural synchrony has also been theorized to indicate poor emotional adjustment10,40. In these studies, biophysiological measures, such as cortisol levels10 and heart rate variability40, exhibited higher synchronous activity in couples who experienced greater conflict and higher levels of stress. There it was theo- rised that synchrony may be better construed as a tendency for these couples to be more easily affected by each other during stressful periods, therefore reflexively reacting to their partner’s physiological arousal. Likewise, in the context of co-parenting, synchrony in attentional and regulatory mechanisms to stressful cries might indicate maladaptive emotional adjustment between partners that subsequently undermines effective co-parenting. In the real world, cognitive and emotional control allows a parent to attend to a crying child, avoid distractions, and manage impulses and emotions. These capacities support flexible parenting41 and are recruited when parents plan and change their behaviours to meet the everyday demands of caregiving41,42. If co-parents are prone to being affected by the stress experienced by their partner when their infant is crying, they may not be able to organise effective caregiving behaviours to optimally respond to their child’s needs. Although it is unproven if synchrony is a marker of positive or negative qualities in a spousal relationship, in light of the results from this study we pro- pose that synchronous attentional regulatory mechanisms may generally be adaptive but still become maladaptive in stressful situations. Parent characteristics, such as frequency of each parent’s response to their child, parental multiparous or primiparous status, and the ages of the parents, were found to moderate the co-presence effect. In general, these variables were only statistically significant in the together condition, which may mean that the presence of the co-parent adds a social dimension to the co-regulation of synchronous responses to external stimuli. First, primiparous parents experienced higher synchrony compared to multiparous parents. www.nature.com/scientificreports/ mother, rather than the father, took the lead in responding to the child. We also found that synchrony was higher in primiparous compared to multiparous couples. Finally, synchrony was also noted to be greater in older parents. Taken together, co-parents showed enhanced brain-to-brain synchrony in the physical proximity of their spousal partner, although this effect was moderated by several stimuli and social factors.hi p gf y The central finding from this study is that the presence of a spousal partner is associated with greater syn- chrony in attentional and cognitive control mechanisms. The dorsal and ventral fronto-parietal pathways con- figure the network that supports attentional regulation functions27. Salient human vocalisations have been found to activate these dual attentional pathways28. The dorsal stream governs “top-down” (i.e., endogenous) voluntary resources to attend to features or location, whereas the ventral stream oversees resources allocated to “bottom-up” unattended (i.e., exogenous) stimuli that cue attentional shifts. Despite these specialised functions, the two pathways dynamically interact and exert flexible attentional control29,30. The MFG acts as a “circuit-breaker” connecting the dorsal and ventral attentional streams31–33. In this model, the MFG controls both ventral and dorsal pathways and directs a flexible switch between the “top-down” and “bottom-up” attentional streams31 and is involved in involuntary attention shifting34. In our study, by displaying a fixation cross “+” that trig- gered “top-down” attention, followed by its disappearance along with the broadcast of vocalisations that incited “bottom-up” attention, our experimental design specifically targeted at partners’ attentional switching capacities. In this line of reasoning, synchronous activation of the MFG indicates coupled attentional regulation between co-parenting spouses. Moreover, we observed synchronous activation in the left MFG and IFG, areas in the brain that preferentially regulate attention during processing of affective emotional information25,26. Besides the MFG and IFG, synchrony also emerged in the bilateral aPFC, which constitutes a component of the frontoparietal control network24,35,36. This network is postulated to underpin executive control processes37 that promote deci- sion making38. In the context of our study, the presence of a spousal partner might facilitate matched executive control processes that could help to organise couples’ upcoming joint behaviours. Our findings are supported by previous studies which have observed co-regulation in couples (e.g.21,39). Scientific Reports \| (2020) 10:7569 \| https://doi.org/10.1038/s41598-020-63596-2 Discussion Bi-parental rearing intuitively conjures the notion of a coordinated set of responses between co-parenting spouses. The present study investigated whether physical proximity influences brain-to-brain synchrony of co-parents and whether synchrony is being influenced by stimuli and parents’ characteristics. Our first aim was to show that there was greater synchrony when partners were in each other’s physical presence (together-condition; TOG) compared to when they listened to salient vocalisations individually (separate-condition; SEP). We found that synchrony was indeed higher in TOG than SEP in the left inferior frontal gyrus (IFG), left middle frontal gyrus (MFG) and bilateral anterior PFC (aPFC) regions of the brain, all of which fall within the purview of an extensive attentional regulation and cognitive control network23,24. The second aim of the study was to prove that the co-presence effect was unique to co-parenting spouses. Our results showed that physical proximity only enhanced synchrony in true couples, but not in control couples, which suggests that synchrony emerged due to the unique presence of one’s spousal partner. From the channels found to be significant, our third aim was to investigate whether acoustic stimuli and parents’ characteristics influenced synchrony. The sounds which induced greater synchrony in TOG than SEP conditions were infant laughter, adult laughter, and static. The fact that pos- itive (i.e. infant and adult laughter) and neutral stimuli (i.e. static), but not negative stimuli (i.e. infant and adult cry), elicited greater synchrony suggests that synchrony depended on the emotional valence of the sound. With regard to parents’ characteristics, synchrony between couples was higher in the left MFG and aPFC when the Scientific Reports \| (2020) 10:7569 \| https://doi.org/10.1038/s41598-020-63596-2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ be sufficient to definitively conclude the precise direction and magnitude of influence of these social factor spousal synchrony.f Although the focus of the paper centres on the effect of co-presence of spouses in facilitating synchrony, we have also conducted additional analyses to examine the roles of stimuli- and parent-related factors in channels which did not show a significant difference in synchrony between TOG and SEP. In regard to acoustic stimuli, there was no significant effect of stimuli-related factors in other channels besides the ones which significantly differed in TOG and SEP. These findings suggest a unique role of positively- and neutrally-valenced sounds (i.e. infant laughter, adult laughter, static), but not negatively-valenced sounds (i.e. infant cry, adult cry) in enhancing synchrony in couple’s brain responses in the co-presence of each other. In regard to parent characteristics, higher average co-parenting ratio, primiparous parental status and younger age of parents were all found to be signifi- cantly associated with greater synchrony in the TOG condition, even in channels which did not significantly differ between TOG and SEP. These results point to the general effect that parent-related variables have in enhancing synchrony between spouses which might not underpin the difference specifically observed due to the co-presence of a spouse. Like all research studies, this study carries several limitations. First, we implemented standard infant vocal- isations that might not have elicited mother-father brain synchrony as distinctly as own-child vocalisations. Comparing standard to own-child infant vocalisations would have provided a measure of whether synchrony was specific to the child that the couple co-parented. On this limitation, our results may underestimate couple synchrony. Second, we did not include data on parental efficacy and competence. These variables might explain further variance observed in mother-father brain synchrony as related to parenting experience. For example, as children develop, parents could gain a greater sense of competence as individuals or as a couple, which may have implications for couple brain synchrony. Third, our study employed a cross-sectional design, which does not allow us to observe changes in the co-parental brain over time. Different co-parenting partners might exhibit unique changes in the pace and trajectory of brain-to-brain synchrony that might be better captured in a longitu- dinal design. www.nature.com/scientificreports/ Caring for a child is an intensely demanding task that is qualitatively different from other life experiences43,44. Primiparous and multip- arous mothers follow different parental adjustment trajectories45. Primiparous parents may experience a greater need to co-regulate responses than multiparous parents who have had much more experience in caring for their children together. Primiparous parents have greater physiological responses to infant cries than multiparous or even non-parents46, suggesting that making decisions relating to child-related cues are indeed of great priority for new parents. Second, a higher frequency of mother (compared to father) taking the lead in responding to the child was related to higher synchrony. Consonant with a family systems perspective, this gender difference may reflect the role of the mother as the primary mediator in the family during stressful situations47. As mothers are more likely to employ mediation strategies compared to fathers, they may adapt their responses according to the emotional signals of their partner when co-parenting an infant, and therefore be crucial in determining the level of synchrony in a couple. Last, parental age showed a negative correlation with synchrony, where older couples (or couples with older mothers or older fathers) experienced lower levels of synchrony compared to younger cou- ples. With age comes a greater sense of maturity, competence, and stability48–52 suggested that older parents tend to be more secure in their role as parents and have parenting strengths that are consistent with their higher level of maturity. Thus, a lower level of synchrony between older couples may reflect a diminished need to respond similarly to each other as they experience greater security in their own roles as parents. These results point to the malleability of the adult brain during the parenting process and in different phases of life. Thus far, studies have mostly focused on the plasticity of the maternal brain in the context of parenting53,54, but it is plausible to deduce that similar brain malleability may be observed in fathers. Unfortunately, the methodology of this study may not Scientific Reports \| (2020) 10:7569 \| https://doi.org/10.1038/s41598-020-63596-2 www.nature.com/scientificreports/ Stimulus Gender Mean S.D. Infant cry (low- pitched) Mothers 2.21 0.885 Fathers 1.83 0.874 Combined 1.96 0.876 Infant cry (high- pitched) Mothers 2.88 1.09 Fathers 2.29 1.09 Combined 2.58 1.08 Table 2. Distress Rating of Low- and High-Pitched Infant Cry. Table 2. Distress Rating of Low- and High-Pitched Infant Cry. Table 2. Distress Rating of Low- and High-Pitched Infant Cry. www.nature.com/scientificreports/ Fourth, while our sample sample size achieved adequate power to compare between SEP and TOG, future studies ought to employ larger sample sizes that will allow for extensive analyses of the moderating roles of acoustic and social variables. Fifth, we limited the scope of our investigation to the prefrontal cortex. Other brain areas are involved in parental behaviours and responses, including subcortical regions, such as the amygdala3. These brain areas may also evince significant couple-specific responses not captured in our study. Sixth, although we included a control static sound, we did not include a baseline reading of brain activation without exposure to any stimuli which might have provided resting state readings of brain synchrony. Finally, our sample consisted of parents with children at heterogenous stages of development, which may be a cause of different responses towards infant vocalisations. For instance, parents with infants may find infant crying and laughter more relevant to their current parenting experience, whereas such vocalisations may not be relevant to the immediate caregiving con- text of parents with older children. Future studies may opt to disambiguate between sub-populations of parents. In humans, spousal partners naturally co-parent their infant. While the parental brain has been extensively investigated in regard to infant rearing, research on the influence of co-parenting on caregiving mechanisms is still lacking. Findings from our study suggest that, in the physical presence of a spousal partner, couples exhibit greater brain-to-brain synchrony in regions implicated in attentional regulation and cognitive control. Synchrony is also augmented in response to auditory vocalisations that are positive and neutral, rather than negative. Results from this study can be extrapolated to the real-world context when considering the nature of parenting and spousal relationships. As attentional regulation and cognitive control are especially vital to evoking soothing responses during stressful situations, such as attending to a crying infant55–57, matching the brain signals of a stressed spousal partner may be detrimental to co-parenting responses. Conversely, synchrony during favourable situations, such as that invoked by infant laughter, may enhance the spousal relationship. This study presents persuasive evidence that the parental brain may be shaped by the presence of a co-parenting spousal partner, although future studies should investigate how synchrony during positive and negative emotional situations directly affects coordinated caregiving behaviours. Materials & Methods y p p p y Data were collected from 27 couples (N = 54, Mean age = 33.6, SD = 5.65) consisting of 27 mothers (N = 27, Mean age = 32.8, SD = 5.65) and 27 fathers (N = 27, Mean age = 34.4, SD = 5.67) in two separate experimental conditions: together and separate (see Experimental Procedure), with their youngest child aged 48 months (4 years) and below (N = 27, 15 males and 12 females; Mean age = 17.28 months, SD = 9.43, ranging from 2 to 39 months). We also noted whether the couples were primiparous (one child only) or multiparous (more than one child) as we wanted to investigate whether previous parenting experience affects levels of brain-brain synchrony. Data of 3 couples (n = 6) were excluded from the analysis of the together condition because their children were present in the room (n = 4) or their children cried during the experiment (n = 2), disrupting their attention. The final sample consisted of 24 pairs of parents, of which 10 were primiparous and 14 were multiparous. Data, scripts and materials for this study are available here: https://doi.org/10.21979/N9/KF1JOG and https://gitlab. com/abp-san-public/MF-nirs. p p A power analysis (G*Power61, version 3.1.9.4, Windows 64 bit) was performed to ensure that the number of couples and stimuli were adequate to detect the effect of being in the TOG condition on the single channel. With an α = 0.0025 (corresponding to an α = 0.05 with 20 independent tests, i.e. the number of channels), and an aver- age of 260 samples (NSEP = 130, NTOG = 130), we are able to detect both strong (d = 0.7) and moderate (d = 0.5) effect sizes with high power (0.99 and 0.83 respectively). Questionnaire. Couples completed a questionnaire that recorded their demographic information, parental status (i.e., whether the couple had one or more children), and the age of their youngest child. Each partner was also asked to provide a parenting response ratio; that is, the typical ratio to which the mother:father takes the lead in responding to their child: When you and your partner are together and you hear your child(ren) cry, what is the ratio of Mother:Father taking the lead in attending to the child(ren)? Parents were asked to choose from one of the following options: 0:5, 1:4, 2:3, 3:2, 4:1 and 5:0. Materials & Methods Participant recruitment. All experimental protocols were approved by the ethics committee of the Psychology Programme of Nanyang Technological University, Singapore. All methods in this study were con- ducted in accordance with the guidelines and regulations stipulated by the ethical committee. Participants needed to be at least 21 years old to be eligible for this study. 27 pairs of heterosexual couples were recruited through poster and online advertisements on social media platforms, namely Facebook parenting groups and parenting forums. Participants were then screened for their eligibility, with the inclusion criteria as follows: (i) couples who Scientific Reports \| (2020) 10:7569 \| https://doi.org/10.1038/s41598-020-63596-2 www.nature.com/scientificreports/ S/No. Stimulus Frequency (Hz) 1 Adult female laughter 348.9 2 Infant laughter 331.2 3 Infant cry (low-pitched) 354.3 4 Infant cry (high-pitched) 554.3 5 Adult female cry 318.2 6 Static Not Applicable Table 3. Audio Frequency of Experimental Stimuli. Table 3. Audio Frequency of Experimental Stimuli. live together; (ii) couples with a young child, aged 4 years or younger; (iii) couples who do not have any known psychological and mental health conditions; and (iv) couples who do not have known medical conditions that affect the oxygen-carrying capacity of their blood. These criteria were selected for their relevance to the exper- imental procedure. First, couples must live together to afford the opportunity to co-regulate their physiological signals when caring for their child. Second, as experimental stimuli consisted of infant vocalizations (see Table 2), parents with a young child were chosen for their likelihood of being more attentive to these vocalisations58. Third, couples should not possess any mental health condition as the study investigates healthy parental populations. Fourth, as fNIRS collects data based on the level of oxygenated and deoxygenated blood within each region of the brain59, medical conditions (such as G6PD deficiency60) that may cause the data to reflect higher or lower oxygen levels than normal were excluded. Informed consent was obtained from all participants prior to the start of the study. Participants were reimbursed a total of SGD 50 per couple for their time at the end of the study. Scientific Reports \| (2020) 10:7569 \| https://doi.org/10.1038/s41598-020-63596-2 Materials & Methods The answers were coded into the parent ratio score, from 0 (mother never takes the lead, father always takes the lead) to 1 (mother always takes the lead, father never takes the lead). The scores of the two parents were then averaged to obtain the Average Parent Ratio score. Audio Stimuli. Audio stimuli (see Table 3) were selected from online public databases of sound files62 to reflect both negative valence sounds, such as crying63, and positive valence sounds, such as laughing64. A control sound, static noise (Stimulus 6), and adult vocalizations (Stimuli 1 and 5) were included for comparisons to adult and infant sounds.ht The audio stimuli were analysed for their frequency with Praat software65–67 (version 6.1.08, Macintosh 64 bit) to fall within the range of 300 Hz to 400 Hz, with the exception of Stimulus 4, which was modified from Stimulus 3 in terms of frequency to be 200 Hz higher. Stimulus 4, at higher frequency, was intended to induce an enhanced perception of distress68, thus providing a means of comparison between relatively less and more distressing infant cries. Table 2 shows the mean and SD of participants’ reported distress on a 5-point scale (1 = Not distressing at all to 5 = Extremely distressing) in response to low- and high-pitched infant cries. Compared to the low-pitched cry, significantly greater distress was reported to the high-pitched cry (t(95) = −4.09, p < 0.05, d = 0.561). All audio stimuli were then digitally equated at 15 s. Stimuli 2, 3, 5, and 6 have been used previously69. g y q p y NIRStim (NIRx Medical Technologies LLC, 2000, Version 4.0, Windows 64 bit) was used to present all audi- tory stimuli in a randomised manner. Each stimulus was presented three times (i.e. three trials for each sound) for a duration of 15-s each. A 10-s inter-stimulus interval (ISI) which displayed a white fixation cross “+” against a black background was present between every two stimuli. During the presentation of auditory stimuli, the fix- ation cross disappeared, and participants were only shown a black screen. The stimuli were screened on a 38-cm Acer Laptop, and participants sat approximately 40 cm in front of the laptop. In the separate-condition, partici- pants wore headphones with volume set at 27.5 dB; in the together-condition, sound stimuli were played aloud Scientific Reports \| (2020) 10:7569 \| https://doi.org/10.1038/s41598-020-63596-2 www.nature.com/scientificreports/ Figure 2. Materials & Methods Experimental set-up in together (left) and separated (right) conditions. Figure illustrated by Farouq Azizan. Figure 2. Experimental set-up in together (left) and separated (right) conditions. Figure illustrated by Farouq Azizan. Figure 3. The adopted NIRS montage consisting of 8 sources (gray dots) and 7 detectors (black dots) to form 20 source-detector channels (bold lines). Colors indicate brain areas: Superior Frontal Gyrus (SFG), Middle Frontal Gyrus (MFG), Inferior Frontal Gyrus (IFG) and anterior Pre-Frontal Cortex (aPFC). Figure 3. The adopted NIRS montage consisting of 8 sources (gray dots) and 7 detectors (black dots) to form 20 source-detector channels (bold lines). Colors indicate brain areas: Superior Frontal Gyrus (SFG), Middle Frontal Gyrus (MFG), Inferior Frontal Gyrus (IFG) and anterior Pre-Frontal Cortex (aPFC). with volume set at 44 dB. Earphones were used in the separate-condition to emphasise listening to the sounds in isolation; sounds were played aloud in the together-condition to simulate a shared experience between spousal partners. Scientific Reports \| (2020) 10:7569 \| https://doi.org/10.1038/s41598-020-63596-2 Experimental procedure h l l The first aim of this study was to show that, compared to individually listening to vocalisations (i.e. SEP condition), the presence of a co-parenting spouse (i.e. TOG condition) would be associated with greater brain-to-brain synchrony between partners. To test this hypothesis, we statistically compared the distributions of TOG and SEP synchrony measures of all stimuli. As we had no prior assumptions about which brain areas should be more affected by the co-presence effect, we tested all 20 channels and corrected the p-values using the False Discovery Rate (FDR) correction with the algorithm proposed by Benjamini and Hochberg88 (alpha = 0.05, 20 independent tests, i.e. the number of channels). The two-sided Mann-Whitney test was used to account for the different number of couples in the two conditions.if f Our second aim was to confirm that the co-presence effect was unique to the presence of a spouse (i.e. true couples). Thus, we randomly paired the brain signals of mothers and fathers who were not couples and generated synchrony indexes for the control dyads. We then compared the distributions of the TOG and SEP synchrony measures for control dyads. Our third aim was more exploratory in nature, namely to investigate whether the emotional valence of sound stimuli (i.e. positive vs negative vocalisations) and parents’ characteristics, (i) ratio of mother to father taking the lead in attending to infant, (ii) primiparous compared to multiparous parents and (iii) parents’ age, influ- enced the extent of synchrony. For each channel in which the co-presence of a spouse significantly led to greater synchrony, we compared (Mann-Whitney test, one-sided) the synchrony index for each sound stimulus in both SEP and TOG conditions, and corrected the p-values (Benjamini-Hochberg FDR88, alpha = 0.05, 6 independent tests, i.e. the number of stimuli). As for the social variables, we compared primiparous and multiparous couples (Mann-Whitney test, two-sided) and calculated Spearman correlations to investigate the influence of the Average Parent Ratio (i.e. ratio of mother to father taking the lead in responding to the child) and age of parents. Each sta- tistical test was first performed on the data of all channels together then separately on each of the channels which resulted significant in the SEP v. TOG analysis. P-values of the tests on the separate channels were corrected for multiple hypotheses (Benjamini-Hochberg FDR88, alpha = 0.05, 4 independent tests, i.e. four channels). Experimental procedure h l l fNIRS data were first pre-processed using NIRSlab software (NIRx Medical Technologies LLC, v2016.01, Windows 64 bit).fi For each subject, channels that presented a gain greater than 8 and coefficient of variation greater than 7.5 were excluded from the analysis, as these characteristics are associated with high signal noise76–78. As a conse- quence of the automatic rejection of channels with high signal noise, a different number of couples was available for analyzing each channel and condition. Spike artifacts, which are signal components with an abnormal change in amplitude, normally produced by head movements, were replaced with the nearest surrounding signals79, and discontinuities in the signals, if any, were corrected using the remove_discontinuities function on NIRSlab. Finally, a band-pass filter of 0.01–0.2 Hz was applied to eliminate baseline shift variations, and hemoglobin concentra- tions were determined using the modified Beer-Lambert law. Finally, the signal was visually inspected by two independent experts for validation. NIRS time-series of oxygenated haemoglobin (oxy-Hb) for each subject, condition, stimulus, and channel were exported from NIRSlab to be analysed. Synchrony measures. To obtain an index of synchrony between the brain activities of spousal partners, we computed the similarity in partners’ oxy-Hb concentration levels over time. Cross Correlation, which measures the extent to which two time-series signals co-vary, was used as a time-series similarity metric80–82. To account for minimal anticipations or delays of brain activation in one parent with respect to the other, we computed the cross correlation with shifted copies of one signal, between −2 s to 2 s with increments of 0.125 s. We refer to this metric as the Maximum Cross Correlation within a delay of 2 s (MCC2). The same metric has been adopted in the literature to quantify synchrony in different contexts and with different types of physiological signals80,83–85. The maximum delay was set to 2 s to account for the temporal characteristics of the brain response signals according to the hemodynamic response function86. For each stimulus, we computed the MCC2 for each trial and then averaged MCC2 data across the three trials as a similarity metric. As a control, we also computed MCC2 between the brain signals of randomly paired moth- ers and fathers. The random pairing was done for each sound, channel, and condition for all control couples. The computation of synchrony was performed using a custom code based on pyphysio87 and physynch packages85. Analytic plan. Experimental procedure h l l Experimental procedure. Couples were invited to attend an experimental session lasting 1.5 hours at the Nanyang Technological University (NTU) Lee Kong Chian School of Medicine (LKCSoM) Campus. Each cou- ple participated in two experimental conditions (see Fig. 2) and order was counterbalanced across couples: (i) together-condition (TOG), in which partners were presented with auditory stimuli at the same time in the same room, and (ii) separate-condition (SEP), in which partners listened to the auditory stimuli in separate rooms and at different times.ht f The sitting arrangement in the TOG condition was standardised so that the male was always seated on the left and female on the right. Spouses were instructed to refrain from physical contact during the together-condition, as physical touch may result in potentially confounding synchrony70. The presence of the fixation cross on the screen assured that participants would not make eye contact with each other during the course of the experiment. Although verbal communication was not restricted during the session, participants were instructed to pay atten- tion to the sounds. The auditory nature of the experimental stimuli enjoined spouses from conversing with each other. fNIRS data acquisition. Brain activity of the prefrontal-cortical regions was acquired using the non-invasive fNIRS neuroimaging system (NIRSport, NIRx Medical Technologies LLC), using a sampling rate of 7.81 Hz with light wavelengths at 760 nm and 850 nm71. fNIRS allows the quantification of oxygenated and de-oxygenated hemoglobin in different brain areas: brain areas exhibiting higher concentrations of oxygenated haemoglobin (oxy-Hb) indicate localised cerebral activation.h y The fNIRS cap placed on mothers and fathers utilised a 20-channel system with 8 sources and 7 detectors (Fig. 3). This channel configuration is similar to the international 10–20 system employed in EEG recordings, and Scientific Reports \| (2020) 10:7569 \| https://doi.org/10.1038/s41598-020-63596-2 www.nature.com/scientificreports/ analogous brain regions recorded by fNIRS were identified using this system72. 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Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-63596-2. Correspondence and requests for materials should be addressed to G.E. Correspondence and requests for materials should be addressed to G.E. Correspondence and requests for materials should be addressed to G.E. 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 institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W3116880962	https://europepmc.org/articles/pmc7823742?pdf=render	English	null	NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma	Pharmaceuticals	2,020	cc-by	14,963	Citation: Zhai, Z.; Vaddi, P.K.; Samson, J.M.; Takegami, T.; Fujita, M. NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma. Pharmaceuticals 2021, 14, 23. https://doi.org/ 10.3390/ph14010023 Citation: Zhai, Z.; Vaddi, P.K.; Samson, J.M.; Takegami, T.; Fujita, M. NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma. Pharmaceuticals 2021, 14, 23. https://doi.org/ 10.3390/ph14010023 Received: 26 November 2020 Accepted: 26 December 2020 Published: 30 December 2020 Keywords: melanoma; NLRP1; ATF4; resistance; targeted therapy Keywords: melanoma; NLRP1; ATF4; resistance; targeted therapy Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- ms in published maps and institutio- nal afﬁliations. Zili Zhai 1, Prasanna K. Vaddi 1, Jenny Mae Samson 1, Tomoya Takegami 1 and Mayumi Fujita 1 Department of Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; zili.zhai@cuanschutz.edu (Z.Z.); prasanna.vaddi@cuanschutz.edu (P.K.V.); jenny.samson@cuanschutz.edu (J.M.S.); takegami.tomoya.45z@kyoto-u.jp (T.T.) 1 Department of Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; zili.zhai@cuanschutz.edu (Z.Z.); prasanna.vaddi@cuanschutz.edu (P.K.V.); jenny.samson@cuanschutz.edu (J.M.S.); takegami.tomoya.45z@kyoto-u.jp (T.T.) 1 Department of Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; zili.zhai@cuanschutz.edu (Z.Z.); prasanna.vaddi@cuanschutz.edu (P.K.V.); jenny.samson@cuanschutz.edu (J.M.S.); takegami.tomoya.45z@kyoto-u.jp (T.T.) 2 2 Department of Veterans Affairs Medical Center, VA Eastern Colorado Health Care System, Aurora, CO 80045, USA 3 Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA , * Correspondence: mayumi.fujita@cuanschutz.edu; Tel.: +1-303-724-4045; Fax: +1-303-724-4048 , * Correspondence: mayumi.fujita@cuanschutz.edu; Tel.: +1-303-724-4045; Fax: +1-303-724-4048 Abstract: The BRAF V600E mutation leads to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and its downstream effector responses. Uncovering the hidden downstream effectors can aid in understanding melanoma biology and improve targeted therapy efﬁcacy. The inﬂammasome sensor, NACHT, LRR, and PYD domains-containing protein 1 (NLRP1), is responsible for IL-1β maturation and itself is a melanoma tumor promoter. Here, we report that NLRP1 is a downstream effector of MAPK/ERK signaling through the activating transcription factor 4 (ATF4), creating regulation in metastatic melanoma cells. We conﬁrmed that the NLRP1 gene is a target of ATF4. Interestingly, ATF4/NLRP1 regulation by the MAPK/ERK pathway uses distinct mechanisms in melanoma cells before and after the acquired resistance to targeted therapy. In parental cells, ATF4/NLRP1 is regulated by the MAPK/ERK pathway through the ribosomal S6 kinase 2 (RSK2). However, vemurafenib (VEM) and trametinib (TRA)-resistant cells lose the signaling via RSK2 and activate the cAMP/protein kinase A (PKA) pathway to redirect ATF4/NLRP1. Therefore, NLRP1 expression and IL-1β secretion were downregulated in response to VEM and TRA in parental cells but enhanced in drug-resistant cells. Lastly, silencing NLRP1 in drug-resistant cells reduced their cell growth and inhibited colony formation. In summary, we demonstrated that NLRP1 functions downstream of the MAPK/ERK signaling via ATF4 and is a player of targeted therapy resistance in melanoma. Targeting NLRP1 may improve the therapeutic efﬁcacy of targeted therapy in melanoma. Keywords: melanoma; NLRP1; ATF4; resistance; targeted therapy NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma 1, Prasanna K. Vaddi 1, Jenny Mae Samson 1, Tomoya Takegami 1 and Mayumi Fujita 1,2,3,* pharmaceuticals pharmaceuticals pharmaceuticals 1. Introduction Melanoma is a highly aggressive cancer causing the majority of skin cancer deaths. Approximately 50% of melanoma patients harbor a BRAF, particularly V600E [1], mutation in tumors, which leads to increased expression of downstream effectors, favoring tumor sur- vival [2]. The application of targeted therapy, including BRAF inhibitors (e.g., vemurafenib (VEM) and debrafenib) and mitogen-activated protein kinase kinase (MEK) inhibitors (e.g., trametinib (TRA) and cobimetinib), was regarded as a therapeutic breakthrough when used in patients with BRAF-mutated melanoma [3]. However, the downstream effectors that are suppressed by targeted therapy can be rewired during the development of adaptive or acquired resistance [2]. Therefore, uncovering the hidden downstream effectors of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway can provide clues to understand melanoma biology and improve the therapeutic efﬁcacy of targeted therapy. Copyright: © 2020 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, 23. https://doi.org/10.3390/ph14010023 2 of 15 Pharmaceuticals 2021, 14, 23 It has been suggested that the inﬂammatory tumor microenvironment, driven by tumor-intrinsic signaling pathways, is a major checkpoint to therapeutic efﬁcacy [4]. Tumor- promoting stromal cells, such as tumor-associated macrophages, dendritic cells, and ﬁbrob- lasts, sustain tumor cell survival, immunosuppression, and drug resistance by producing diverse inﬂammatory mediators. We and others have demonstrated that, in addition to stromal cells, metastatic melanoma cells spontaneously release inﬂammatory interleukin (IL)-1β, which in turn induces stromal cells to secrete more IL-1β, thus augmenting in- ﬂammatory signaling [5,6]. As an early response to VEM, IL-1β secretion from melanoma and stromal cells is reduced [7,8]. However, following VEM treatment, IL-1β-associated signaling from inﬂammatory niches is enhanced and confers drug tolerance [9], suggesting that melanoma-derived IL-1β is a master regulator for drug-resistant cells. g g The mechanism behind the autocrine production of IL-1β of melanoma cells is that NACHT, LRR, and PYD domains-containing protein (NLRP) inﬂammasomes, the cellular machinery responsible for IL-1β maturation, are constitutively activated [5]. Among a dozen NLRP proteins, NLRP1 promotes melanoma by increasing IL-1β secretion and suppresses apoptosis by inhibiting CARD-containing caspase activity [10]. NLRP1 gain-of- function mutations have been demonstrated to contribute to constitutive inﬂammasome activation and IL-1β signaling in skin inﬂammation and cancer development [11]. 1. Introduction There- fore, we hypothesized that NLRP1 contributes positively to the development of drug resistance in human melanoma. Recently, we demonstrated the involvement of NLRP1 in the acquired resistance of metastatic melanoma cells to the chemotherapy temozolomide (TMZ) by amplifying IL-1β signaling and activating nuclear factor κB (NF-κB) activity [12]. In the present study, we further demonstrate, for the ﬁrst time, that NLRP1 functions downstream of the MAPK/ERK signaling and contributes to acquired targeted therapy resistance in melanoma. Our ﬁndings may help develop resistance mechanism-targeted inhibitors as a strategy to improve the efﬁcacy of current therapeutics. 2. Results 2.1. The MAPK/ERK Pathway Regulates NLRP1 Expression and IL-1β Secretion through Activating Transcription Factor 4 (ATF4) in BRAFV600E-Mutant Human Metastatic Melanoma Cells Investigating the early response to the MAPK/ERK pathway inhibitors can help understand and thwart drug resistance [13]. Therefore, we ﬁrst determined the mechanism of NLRP1 regulation by the MAPK/ERK pathway using drug-sensitive melanoma cells. g y p y g g In metastatic melanoma cells, IL-1β is released due to the constitutive activation of both NF-κB and NLRP inﬂammasomes [5,14,15]. While NF-κB transcriptionally regulates pro-IL-1β production, NLRP inﬂammasomes are involved in the maturation and secre- tion of this pleiotropic cytokine. To test the effects of VEM and TRA on IL-1β secretion, we used 1205Lu cells, which carry BRAFV600E mutation and have a relatively high IL1B mRNA expression, as well as IL-1β secretion among human metastatic melanoma cells (Figure S1) [5]. As shown in Figure 1a, in contrast to the effect of TMZ that signiﬁcantly enhanced IL-1β secretion [12], VEM and TRA displayed an opposite effect and abrogated IL-1β secretion. Similar effects were observed in BRAFV600E-mutant A375 cells treated with TMZ, the BRAF inhibitor PLX-4720, and the MEK inhibitor CI-1040 (Figure 1b). Western blot data show that a single dose of VEM and TRA suppressed ERK phosphorylation and NLRP1 protein expression, whereas TMZ increased NLRP1 protein expression [12] (Figure 1c and Figure S2). These data suggest that the MAPK/ERK pathway regulates NLRP1 protein expression and IL-1β secretion. 3 of 15 4 of 16 Pharmaceuticals 2021, 14, 23 Pharmaceuticals 2021, 14, Figure 1. Regulation of NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). (a) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. (b) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI-1040 for 24 h. (c) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expres- sion in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). 2. Results (d–f) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. (d) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. (e) NLRP1 mRNA expression. (f) NLRP3 mRNA expression. (g–i) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction followed by qRT-PCR analysis gure 1. Regulation of NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) by the mitogen-activated protein nase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). ) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), 0.5 µM trametinib (TRA) for 24 h. (b) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 05 µM CI-1040 for 24 h. (c) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The and densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin ght panel). (d–f) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for nother 18 h. (d) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, spectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control -actin. (e) NLRP1 mRNA expression. (f) NLRP3 mRNA expression. (g–i) ATF4 binds to the NLRP1 gene promoter in Figure 1. Regulation of NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). (a) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. 2. Results (b) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI-1040 for 24 h. (c) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expres- sion in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). (d–f) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. (d) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. (e) NLRP1 mRNA expression. (f) NLRP3 mRNA expression. (g–i) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter (g). (h) ATF3 was used as a positive control of ATF4 genomic targets. (i) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, p < 0.01, and * p < 0.001. Figure 1. Regulation of NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). (a) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. (b) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI-1040 for 24 h. (c) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. 2. Results The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). (d–f) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. (d) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. (e) NLRP1 mRNA expression. (f) NLRP3 mRNA expression. (g–i) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter (g). (h) ATF3 was used as a positive control of ATF4 genomic targets. (i) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, p < 0.01, and * p < 0.001. Figure 1. Regulation of NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). (a) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. (b) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI-1040 for 24 h. (c) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expres- sion in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). (d–f) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. (d) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. 2. Results The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. (e) NLRP1 mRNA expression. (f) NLRP3 mRNA expression. (g–i) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter (g). (h) ATF3 was used as a positive control of ATF4 genomic targets. (i) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, p < 0.01, and * p < 0.001. Figure 1. Regulation of NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through activating transcription factor 4 (ATF4). (a) ELISA assay of IL-1β secretion from 1205Lu cells treated with 100 µM temozolomide (TMZ), 1 µM vemurafenib (VEM), or 0.5 µM trametinib (TRA) for 24 h. (b) IL-1β secretion from A375 cells treated with 200 µM TMZ, 0.1 µM PLX-4720, or 0.05 µM CI-1040 for 24 h. (c) Western blot analysis of ERK and RSK2 phosphorylation as well as ATF4 and NLRP1 expression in 1205Lu cells treated with dimethyl sulfoxide (DMSO, control), 100 µM TMZ, 5 µM VEM, or 0.5 µM TRA for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). (d–f) 1205Lu (top panels) and SK-MEL-28 (bottom panels) cells were transfected with 50 nM control siRNA or ATF4 siRNA overnight and treated with 1 µM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, for another 18 h. (d) ATF4 mRNA (left panel) and protein expression (right panel) analyzed by qRT-PCR and Western blot, respectively. The band densities of ATF4 were quantitated and normalized to those of the corresponding loading control β-actin. (e) NLRP1 mRNA expression. (f) NLRP3 mRNA expression. (g–i) ATF4 binds to the NLRP1 gene promoter in metastatic melanoma SK-MEL-28 cells. SK-MEL-28 cells were treated with DMSO or 1 µM TG for 18 h, and chromatin immunoprecipitation (ChIP) assay was performed to evaluate the protein-DNA interaction, followed by qRT-PCR analysis of ATF4 occupancy at the NLRP1 promoter (g). 2. Results (h) ATF3 was used as a positive control of ATF4 genomic targets. (i) The region 2 kb away from the putative binding site was used as a negative control. Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05, p < 0.01, and * p < 0.001. Figure 1. Regulation of NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) by the mitogen- Figure 1. Regulation of NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) by the mitogen-a 4 of 15 Pharmaceuticals 2021, 14, 23 To understand how the MAPK/ERK pathway controls the NLRP1/IL-1β, we inves- tigated the ribosomal S6 kinase (RSK)/activating transcription factor 4 (ATF4) axis. RSK family proteins are known to regulate multiple cellular functions. RSK2, activated by ERK1/2, translocates to the nucleus and phosphorylates several transcriptional factors, in- cluding ATF4 [16]. Western blot data show that a single dose of VEM and TRA suppressed RSK2 phosphorylation and ATF4 protein expression (Figure 1c and Figure S2). In contrast, TMZ had no such effect. Interestingly, the promoter region of human NLRP1 has a binding motif for ATF4 (Figure S3). These data indicate a role for the RSK2/ATF4 as a link between the MAPK/ERK pathway and NLRP1/IL-1β. / p y / β To understand the relationship between the MAPK/ERK pathway and NLRP1, we tested whether ATF4 works upstream of NLRP1 expression in metastatic melanoma cells. As a transcriptional factor, ATF4 regulates downstream gene expression in response to endoplasmic reticulum (ER) stress [17]. D’Osualdo et al. reported that NLRP1 is upregu- lated in human cervical cancer HeLa cells under ER stress conditions [18]. Therefore, we evaluated the regulation of NLRP1 by ATF4 using 1205Lu and another BRAFV600E-mutant cell line, SK-MEL-28 (Figure 1d–f). We silenced ATF4 using siRNA in cells and treated them with an ER stress inducer, thapsigargin (TG), to induce ATF4 expression (Figure 1d and Figure S4). In the presence of TG, both ATF4 and NLRP1 were upregulated at mRNA levels (Figure 1d,e, respectively), suggesting that NLRP1 expression responds to ER stress activation in melanoma cells. However, ATF4 knockdown inhibited TG-induced NLRP1 gene transcription (Figure 1e). As a nontarget gene control, NLRP3 gene expression was evaluated and found to be unchanged by ATF4 knockdown (Figure 1f). These results suggest that NLRP1 lies downstream of the ER stress signaling cascade in melanoma cells, possibly through ATF4 regulation. 2. Results Next, to evaluate whether this enhanced ATF4 expression by TG has a direct regulatory effect on NLRP1, we tested the direct binding of ATF4 to the promoter region of NLRP1 gene by protein–DNA interaction chromatin immunoprecipitation (ChIP) assay. We found increased recruitment of ATF4 to the NLRP1 promoter upon TG treatment, while no NLRP1 DNA enrichment was found in the negative IgG control group (Figure 1g). ATF3, a positive control of ATF4 target genes, was found to interact with ATF4 protein under the same conditions (Figure 1h). The region 2 kb away from the putative binding site on the NLRP1 promoter was used as a negative control and showed no interaction with ATF4 protein (Figure 1i). These data demonstrate that ATF4 is an activator of the NLRP1 gene in metastatic melanoma cells, especially under ER stress conditions. 2.2. The MAPK/ERK Pathway Shows an RSK2-Dependent Regulation of NLRP1 Gene Promoter Activity and Protein Expression in Metastatic Melanoma Cells 2.2. The MAPK/ERK Pathway Shows an RSK2-Dependent Regulation of NLRP1 Gene Promoter Activity and Protein Expression in Metastatic Melanoma Cells Next, we tested whether the MAPK/ERK pathway regulates the NLRP1 gene by measuring NLRP1 promoter activity in SK-MEL-28 cells treated with a single dose of VEM or TRA. We found that both inhibitors signiﬁcantly reduced NLRP1 promoter activity (Figure 2a). In agreement with these ﬁndings, Western blot analysis shows that NLRP1 protein expression was also signiﬁcantly inhibited by VEM and TRA (Figure 2b and Figure S2). In contrast, the drug, TMZ, had no such effects on NLRP1 promoter activity and its protein expression. p p To test whether NLRP1 is regulated by the MAPK/ERK pathway through RSK2, we used the RSK inhibitor, BI-D1870, to inhibit RSK2 activity (Figure 2c). Luciferase reporter activity shows that the RSK inhibitor signiﬁcantly reduced NLRP1 promoter activity and had a synergistic effect with VEM (Figure 2d). Western blot analysis conﬁrmed the synergistic inhibition of NLRP1 expression by the RSK inhibitor and VEM (Figure 2e and Figure S5). This synergistic inhibitory effect on NLRP1 protein expression was also conﬁrmed in A375 and 1205Lu cells (Figure S6). These data indicate an RSK-dependent regulation of ATF4/NLRP1 by the MAPK/ERK signaling pathway in drug-sensitive cells. 5 of 15 Pharmaceuticals 2021, 14, 23 y g g p y g Figure 2. Effects of VEM, TRA, and the RSK inhibitor on NLRP1 gene promoter activity and pro- tein expression in SK-MEL-28 cells. 2. Results (a) Around ~ 1000 bp NLRP1 promoter region was cloned into a luciferase reporter vector and transfected into cells overnight, and cells were treated with 100 µM TMZ, 1 µM VEM, or 0.1 µM TRA for 24 h before measuring luciferase activity. (b) Western blot analysis of ERK activation, p-RSK2, ATF4, and NLRP1 expression in cells treated with inhibi- tors for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). (c) Hypothesized regulation of NLRP1 by the ERK pathway through RSK2. Arrows indicate the direction of signal transduction; bar stands for inhibition; and “p” in green oval represents phosphorylation. (d) NLRP1 promoter luciferase activity in cells treated with 1 µM VEM and/or 10 µM BI-D1870, an RSK inhibitor (RSKi), for 24 h. (e) Western blot analysis of NLRP1 expression in cells treated with 10 µM BI-D1870 and/or 1 µM VEM for 24 h. The band densities of NLRP1 were quantitated and normalized to those of the cor- responding loading control β-actin. Representative images are shown and data expressed as the mean ± SEM, n = 4. * p < 0.001. Figure 2. Effects of VEM, TRA, and the RSK inhibitor on NLRP1 gene promoter activity and protein expression in SK-MEL-28 cells. (a) Around ~1000 bp NLRP1 promoter region was cloned into a luciferase reporter vector and transfected into cells overnight, and cells were treated with 100 µM TMZ, 1 µM VEM, or 0.1 µM TRA for 24 h before measuring luciferase activity. (b) Western blot analysis of ERK activation, p-RSK2, ATF4, and NLRP1 expression in cells treated with inhibitors for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). (c) Hypothesized regulation of NLRP1 by the ERK pathway through RSK2. Arrows indicate the direction of signal transduction; bar stands for inhibition; and “p” in green oval represents phosphorylation. (d) NLRP1 promoter luciferase activity in cells treated with 1 µM VEM and/or 10 µM BI-D1870, an RSK inhibitor (RSKi), for 24 h. (e) Western blot analysis of NLRP1 expression in cells treated with 10 µM BI-D1870 and/or 1 µM VEM for 24 h. The band densities of NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin. 2. Results Representative images are shown and data expressed as the mean ± SEM, n = 4. * p < 0.001. Figure 2. Effects of VEM, TRA, and the RSK inhibitor on NLRP1 gene promoter activity and pro- tein expression in SK-MEL-28 cells. (a) Around ~ 1000 bp NLRP1 promoter region was cloned into a luciferase reporter vector and transfected into cells overnight, and cells were treated with 100 µM TMZ, 1 µM VEM, or 0.1 µM TRA for 24 h before measuring luciferase activity. (b) Western blot analysis of ERK activation, p-RSK2, ATF4, and NLRP1 expression in cells treated with inhibi- tors for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). (c) Hypothesized regulation of NLRP1 by the ERK pathway through RSK2. Arrows indicate the direction of signal transduction; bar stands for inhibition; and “p” in green oval represents phosphorylation. (d) NLRP1 promoter luciferase activity in cells treated with 1 µM VEM and/or 10 µM BI-D1870, an RSK inhibitor (RSKi), for 24 h. (e) Western blot analysis of NLRP1 expression in cells treated with 10 µM BI-D1870 and/or 1 µM VEM for 24 h. The band densities of NLRP1 were quantitated and normalized to those of the cor- responding loading control β-actin. Representative images are shown and data expressed as the mean ± SEM, n = 4. * p < 0.001. Figure 2. Effects of VEM, TRA, and the RSK inhibitor on NLRP1 gene promoter activity and protein expression in SK-MEL-28 cells. (a) Around ~1000 bp NLRP1 promoter region was cloned into a luciferase reporter vector and transfected into cells overnight, and cells were treated with 100 µM TMZ, 1 µM VEM, or 0.1 µM TRA for 24 h before measuring luciferase activity. (b) Western blot analysis of ERK activation, p-RSK2, ATF4, and NLRP1 expression in cells treated with inhibitors for 24 h. The band densities of ATF4 and NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin (right panel). (c) Hypothesized regulation of NLRP1 by the ERK pathway through RSK2. Arrows indicate the direction of signal transduction; bar stands for inhibition; and “p” in green oval represents phosphorylation. (d) NLRP1 promoter luciferase activity in cells treated with 1 µM VEM and/or 10 µM BI-D1870, an RSK inhibitor (RSKi), for 24 h. 2. Results (a) Generation of VEM-resistant (VEM-R; left panel) and TRA-resistant (TRA-R; right panel) cells by growing cells in the drug-containing medium for 2 months. An increase in 50% inhibitory concentrations (IC50; dashed line) indicates drug resistance. Par, parental cells. (b) Recovered ERK1/2 activation in VEM-R cells treated with 0.1 or 1 µM VEM for 24 h. CyPA, cyclophilin A as a loading control. (c) Western blot analysis of RSK2 phosphorylation in VEM-R and TRA-R cells treated with 1 µM VEM and 0.5 µM TRA, respectively, for 24 h. (d) ELISA assay of IL-1β secretion from parental and resistant cells. (e) Western blot analysis of intracellular localization of ATF4 in parental and resistant cells. Cytoplasmic and nuclear fractions of cells were isolated and assayed for ATF4 expression with CyPA and Lamin B used as markers for cytoplasmic and nuclear proteins, respectively. (f–h) qRT-PCR analysis of ATF4, NLRP1, and NLRP3 expression, respectively, in parental and resistant cells transfected with 50 nM control siRNA or ATF4 siRNA overnight. (i) The ratios of MITF/AXL gene expression in parental and MAPK inhibitor-resistant SK-MEL-28 cells. In (b,c,e), the band densities of p-ERK1/2, p-RSK2, and ATF4 were quantitated and normalized to those of the corresponding total ERK1/2 (b), loading controls β-actin (c), CyPA or Lamin B (e). Representative images are shown and data expressed as the mean ± SEM, n = 3. p < 0.01 and * p < 0.001. Figure 3. Increased IL-1β secretion, upregulated gene expression of ATF4 and NLRP1, and decreased MITF/AXL Figure 3. Increased IL-1β secretion, upregulated gene expression of ATF4 and NLRP1, and decreased M Increased IL-1β secretion, upregulated gene expression of ATF4 and NLRP1, and decreased MITF/AXL ratio in sed IL-1β secretion, upregulated gene expression of ATF4 and NLRP1, and decreased MITF/AXL ratio p R; right panel) cells by growing cells in the drug-containing medium for 2 months. An increase in 50% inhibitory concen- trations (IC50; dashed line) indicates drug resistance. Par, parental cells. (b) Recovered ERK1/2 activation in VEM-R cells treated with 0.1 or 1 µM VEM for 24 h. CyPA, cyclophilin A as a loading control. (c) Western blot analysis of RSK2 phos- phorylation in VEM-R and TRA-R cells treated with 1 µM VEM and 0.5 µM TRA, respectively, for 24 h. (d) ELISA assay of IL-1β secretion from parental and resistant cells. (e) Western blot analysis of intracellular localization of ATF4 in paren- tal and resistant cells. 2. Results Cytoplasmic and nuclear fractions of cells were isolated and assayed for ATF4 expression with CyPA and Lamin B used as markers for cytoplasmic and nuclear proteins, respectively. (f–h) qRT-PCR analysis of ATF4, NLRP1, and NLRP3 expression, respectively, in parental and resistant cells transfected with 50 nM control siRNA or ATF4 siRNA overnight. (i) The ratios of MITF/AXL gene expression in parental and MAPK inhibitor-resistant SK-MEL-28 cells. In (b), (c), and (e), the band densities of p-ERK1/2, p-RSK2, and ATF4 were quantitated and normalized to those of the corre- sponding total ERK1/2 (b), loading controls β-actin (c), CyPA or Lamin B (e). Representative images are shown and data expressed as the mean ± SEM, n = 3. p < 0.01 and * p < 0.001. 2.4. The cAMP/PKA Pathway is a Regulator of ATF4/NLRP1 in Resistant Melanoma Cells Next, we evaluated whether the upregulated NLPR1 in resistant cells is through RSK2. Compared to parental cells, both VEM- and TRA-resistant SK-MEL-28 cells mani- in VEM and TRA resistant SK MEL 28 cells. (a) Generation of VEM resistant (VEM R; left panel) and TRA resistant (TRA-R; right panel) cells by growing cells in the drug-containing medium for 2 months. An increase in 50% inhibitory concentrations (IC50; dashed line) indicates drug resistance. Par, parental cells. (b) Recovered ERK1/2 activation in VEM-R cells treated with 0.1 or 1 µM VEM for 24 h. CyPA, cyclophilin A as a loading control. (c) Western blot analysis of RSK2 phosphorylation in VEM-R and TRA-R cells treated with 1 µM VEM and 0.5 µM TRA, respectively, for 24 h. (d) ELISA assay of IL-1β secretion from parental and resistant cells. (e) Western blot analysis of intracellular localization of ATF4 in parental and resistant cells. Cytoplasmic and nuclear fractions of cells were isolated and assayed for ATF4 expression with CyPA and Lamin B used as markers for cytoplasmic and nuclear proteins, respectively. (f–h) qRT-PCR analysis of ATF4, NLRP1, and NLRP3 expression, respectively, in parental and resistant cells transfected with 50 nM control siRNA or ATF4 siRNA overnight. (i) The ratios of MITF/AXL gene expression in parental and MAPK inhibitor-resistant SK-MEL-28 cells. In (b,c,e), the band densities of p-ERK1/2, p-RSK2, and ATF4 were quantitated and normalized to those of the corresponding total ERK1/2 (b), loading controls β-actin (c), CyPA or Lamin B (e). Representative images are shown and data expressed as the mean ± SEM, n = 3. 2. Results Interestingly, when we examined the relationship between MITF/AXL ratio and IL1B using 18 human melanoma cells without drug treatment, we found a strong inverse correlation between the MITF/AXL ratio and IL1B gene expression (Figure S11). Together, these data suggest that the MAPK/ERK pathway inhibitor-resistant SK-MEL-28 cells present with an increase in ATF4, NLRP1, and IL-1β secretion and a decrease in MITF/AXL ratio. R PEER REVIEW 7 of 16 Figure 3. Increased IL-1β secretion, upregulated gene expression of ATF4 and NLRP1, and decreased MITF/AXL ratio in VEM- and TRA-resistant SK-MEL-28 cells. (a) Generation of VEM-resistant (VEM-R; left panel) and TRA-resistant (TRA- R; right panel) cells by growing cells in the drug-containing medium for 2 months. An increase in 50% inhibitory concen- trations (IC50; dashed line) indicates drug resistance. Par, parental cells. (b) Recovered ERK1/2 activation in VEM-R cells treated with 0.1 or 1 µM VEM for 24 h. CyPA, cyclophilin A as a loading control. (c) Western blot analysis of RSK2 phos- phorylation in VEM-R and TRA-R cells treated with 1 µM VEM and 0.5 µM TRA, respectively, for 24 h. (d) ELISA assay of IL-1β secretion from parental and resistant cells. (e) Western blot analysis of intracellular localization of ATF4 in paren- tal and resistant cells. Cytoplasmic and nuclear fractions of cells were isolated and assayed for ATF4 expression with CyPA and Lamin B used as markers for cytoplasmic and nuclear proteins, respectively. (f–h) qRT-PCR analysis of ATF4, NLRP1, and NLRP3 expression, respectively, in parental and resistant cells transfected with 50 nM control siRNA or ATF4 siRNA overnight. (i) The ratios of MITF/AXL gene expression in parental and MAPK inhibitor-resistant SK-MEL-28 cells. In (b), (c), and (e), the band densities of p-ERK1/2, p-RSK2, and ATF4 were quantitated and normalized to those of the corre- sponding total ERK1/2 (b), loading controls β-actin (c), CyPA or Lamin B (e). Representative images are shown and data expressed as the mean ± SEM, n = 3. p < 0.01 and * p < 0.001. 2.4. The cAMP/PKA Pathway is a Regulator of ATF4/NLRP1 in Resistant Melanoma Cells Next, we evaluated whether the upregulated NLPR1 in resistant cells is through RSK2. Compared to parental cells, both VEM- and TRA-resistant SK-MEL-28 cells mani- Figure 3. Increased IL-1β secretion, upregulated gene expression of ATF4 and NLRP1, and decreased MITF/AXL ratio in VEM- and TRA-resistant SK-MEL-28 cells. 2. Results (e) Western blot analysis of NLRP1 expression in cells treated with 10 µM BI-D1870 and/or 1 µM VEM for 24 h. The band densities of NLRP1 were quantitated and normalized to those of the corresponding loading control β-actin. Representative images are shown and data expressed as the mean ± SEM, n = 4. * p < 0.001. 2.3. VEM- and TRA-Resistant Melanoma Cells Show Increased IL-1β Secretion, Upregulation of ATF4 and NLRP1 Gene Expression, and Downregulation of MITF/AXL Ratio To understand the role of NLRP1 in targeted therapy resistance, we generated SK- MEL-28 cells resistant to VEM and TRA, indicated by increased IC50 values (Figure 3a), and verified reactivation of ERK (Figure 3b and Figure S7) and RSK2 (Figure 3c and Figure S8) in these cells. We examined how IL-1β signaling and ATF4/NLRP1 pathways were altered in these drug-resistant cells. Compared to other metastatic melanoma cells, SK-MEL-28 cells secrete extremely low to undetectable levels (<1.9 pg/mL) of IL-1β; however, both VEM- and TRA-resistant SK-MEL-28 cells displayed significantly increased IL-1β secretion (Figure 3d). The elevated levels of IL-1β secreted from these SK-MEL-28 cells indicate that NLRP inflammasomes are activated in resistant cells, leading to cellular IL-1β processing and release into the extracellular space. NLRP1 and NLRP3 are two key NLRP family members for IL-1β secretion in melanoma [12,19]. Since increased nuclear ATF4 protein (Figure 3e and Figure S9) and ATF4 gene (Figure 3f) expression were seen in drug-resistant cells, we determined ATF4-regulated NLRP1 gene expression. NLRP1 expression was significantly upregulated at mRNA levels in drug-resistant cells but suppressed by knockdown of ATF4 (Figure 3g). By comparison, NLRP3 gene expression was upregulated in drug-resistant cells but remained unchanged with ATF4 knockdown (Figure 3h), confirming that NLRP1 but not NLRP3 is regulated by ATF4, though both NLRP1 and NLRP3 were upregulated in drug-resistant cells. It has been reported that MAPK inhibitor-resistant SK-MEL-28 cells have a decreased expression ratio of melanocyte-inducing transcription factor (MITF) to AXL receptor tyrosine kinase, a marker of targeted therapy resistance in melanoma [20], which is possibly driven by increased ATF4 expression [21]. We confirmed the low MITF/AXL ratio Pharmaceuticals 2021, 14, 23 6 of 15 by analyzing mRNA levels in drug-resistant cells (Figure 3i and Figure S10), which seems to correlate with IL-1β secretion inversely (Figure 3d). 2. Results p < 0.01 and * p < 0.001. fested significantly increased NLRP1 promoter activity. However, unlike parental cells, increased NLRP1 promoter activity in resistant cells remained unaffected following RSK 2.4. The cAMP/PKA Pathway Is a Regulator of ATF4/NLRP1 in Resistant Melanoma Cells inhibition (Figure 4a), suggesting the loss of the ATF4/NLRP1 regulation by RSK2 and a newly acquired regulation by other signaling pathways. In fact, in addition to the ER stress and MAPK/ERK pathways, other pathways, in- cluding cAMP/protein kinase A (PKA) and MAPK/c-Jun N-terminal kinase (JNK) path- ways, regulate ATF4 expression and activation [22,23]. Therefore, we investigated their effects on NLRP1 promoter activity and found that the PKA inhibitor significantly re- Next, we evaluated whether the upregulated NLPR1 in resistant cells is through RSK2. Compared to parental cells, both VEM- and TRA-resistant SK-MEL-28 cells manifested signiﬁcantly increased NLRP1 promoter activity. However, unlike parental cells, increased NLRP1 promoter activity in resistant cells remained unaffected following RSK inhibition (Figure 4a), suggesting the loss of the ATF4/NLRP1 regulation by RSK2 and a newly acquired regulation by other signaling pathways. 7 of 15 Pharmaceuticals 2021, 14, 23 phoryl was fu Figure 4. Effects of inhibiting RSK and protein kinase A (PKA) on NLRP1 gene promoter activity in drug-resistant cells. (a) NLRP1 promoter luciferase activity in parental, VEM-resistant (VEM-R), and TRA-resistant (TRA-R) SK-MEL-28 cells treated with DMSO or 10 µM BI-D1870 (RSKi) for 24 h. (b) NLRP1 promoter luciferase activity in parental and resistant SK-MEL-28 cells treated with DMSO, 1 µM GSK2606414 (inhibitor of the stressor protein kinase R-like ER kinase, PERKi), 5 µM H 89 2HCl (inhibitor of PKA, PKAi), or 10 µM SP600125 (inhibitor of c-Jun N-terminal kinase, JNKi) for 24 h. (c) Western blot analysis of PKA IIβ regulatory subunit (PKA IIβ reg) phosphoryla- tion in VEM-R and TRA-R SK-MEL-28 cells treated with VEM or TRA for 24 h as described in Fig- ure 3c. (d) VEM-R 1205Lu cells were generated, indicated by reactivation of ERK1/2 phosphoryla- tion. (e) Western blot analysis of PKA IIβ reg phosphorylation in parental 1205Lu cells treated with different drugs for 24 h as described in Figure 1c. (f) Western blot analysis of PKA IIβ reg phosphorylation in parental, TMZ-resistant (TMZ-R), and VEM-R 1205Lu cells treated with DMSO as control, 100 µM TMZ, or 5 µM VEM for 24 h. In (c–f), the band densities of p-PKA IIβ reg and p-ERK1/2 were quantitated and normalized to those of the corresponding loading control β actin (c e and f) or total ERK1/2 (d) Representative images are shown and data expressed as Figure 4. fested significantly increased NLRP1 promoter activity. However, unlike parental cells, increased NLRP1 promoter activity in resistant cells remained unaffected following RSK 2.4. The cAMP/PKA Pathway Is a Regulator of ATF4/NLRP1 in Resistant Melanoma Cells Effects of inhibiting RSK and protein kinase A (PKA) on NLRP1 gene promoter activity in drug-resistant cells. (a) NLRP1 promoter luciferase activity in parental, VEM-resistant (VEM-R), and TRA-resistant (TRA-R) SK-MEL-28 cells treated with DMSO or 10 µM BI-D1870 (RSKi) for 24 h. (b) NLRP1 promoter luciferase activity in parental and resistant SK- MEL-28 cells treated with DMSO, 1 µM GSK2606414 (inhibitor of the stressor protein kinase R-like ER kinase, PERKi), 5 µM H 89 2HCl (inhibitor of PKA, PKAi), or 10 µM SP600125 (inhibitor of c-Jun N-terminal kinase, JNKi) for 24 h. (c) Western blot analysis of PKA IIβ regulatory subunit (PKA IIβ reg) phosphorylation in VEM-R and TRA-R SK-MEL-28 cells treated with VEM or TRA for 24 h as described in Figure 3c. (d) VEM-R 1205Lu cells were generated, indicated by reactivation of ERK1/2 phosphorylation. (e) Western blot analysis of PKA IIβ reg phosphorylation in parental 1205Lu cells treated with different drugs for 24 h as described in Figure 1c. (f) Western blot analysis of PKA IIβ reg phosphorylation in parental, TMZ-resistant (TMZ-R), and VEM-R 1205Lu cells treated with DMSO as control, 100 µM TMZ, or 5 µM VEM for 24 h. In (c–f), the band densities of p-PKA IIβ reg and p-ERK1/2 were quantitated and normalized to those of the corresponding loading control β-actin (c,e,f) or total ERK1/2 (d). Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05 and ** p < 0.01. ure 4. Effects of inhibiting RSK and protein kinase A (PKA) on NLRP1 gene promoter activity of inhibiting RSK and protein kinase A (PKA) on NLRP1 gene promoter activity in drug-resistant cells. Figure 4. Effects of inhibiting RSK and protein kinase A (PKA) on NLRP1 gene promoter act Figure 4. Effects of inhibiting RSK and protein kinase A (PKA) on NLRP1 gene promoter activity in dru Figure 4. Effects of inhibiting RSK and protein kinase A (PKA) on NLRP1 gene promoter activity in drug-resistant cells. (a) NLRP1 promoter luciferase activity in parental, VEM-resistant (VEM-R), and TRA-resistant (TRA-R) SK-MEL-28 cells treated with DMSO or 10 µM BI-D1870 (RSKi) for 24 h. fested significantly increased NLRP1 promoter activity. However, unlike parental cells, increased NLRP1 promoter activity in resistant cells remained unaffected following RSK 2.4. The cAMP/PKA Pathway Is a Regulator of ATF4/NLRP1 in Resistant Melanoma Cells (b) NLRP1 promoter luciferase activity in parental and resistant SK-MEL-28 cells treated with DMSO, 1 µM GSK2606414 (inhibitor of the stressor protein kinase R-like ER kinase, PERKi), 5 µM H 89 2HCl (inhibitor of PKA, PKAi), or 10 µM SP600125 (inhibitor of c-Jun N-terminal kinase, JNKi) for 24 h. (c) Western blot analysis of PKA IIβ regulatory subunit (PKA IIβ reg) phosphoryla- tion in VEM-R and TRA-R SK-MEL-28 cells treated with VEM or TRA for 24 h as described in Fig- ure 3c. (d) VEM-R 1205Lu cells were generated, indicated by reactivation of ERK1/2 phosphoryla- tion. (e) Western blot analysis of PKA IIβ reg phosphorylation in parental 1205Lu cells treated with different drugs for 24 h as described in Figure 1c. (f) Western blot analysis of PKA IIβ reg phosphorylation in parental, TMZ-resistant (TMZ-R), and VEM-R 1205Lu cells treated with DMSO as control, 100 µM TMZ, or 5 µM VEM for 24 h. In (c–f), the band densities of p-PKA IIβ reg and p-ERK1/2 were quantitated and normalized to those of the corresponding loading control β actin (c e and f) or total ERK1/2 (d) Representative images are shown and data expressed as g g p ( ) g p y g (a) NLRP1 promoter luciferase activity in parental, VEM-resistant (VEM-R), and TRA-resistant (TRA-R) SK-MEL-28 cells treated with DMSO or 10 µM BI-D1870 (RSKi) for 24 h. (b) NLRP1 promoter luciferase activity in parental and resistant SK- MEL-28 cells treated with DMSO, 1 µM GSK2606414 (inhibitor of the stressor protein kinase R-like ER kinase, PERKi), 5 µM H 89 2HCl (inhibitor of PKA, PKAi), or 10 µM SP600125 (inhibitor of c-Jun N-terminal kinase, JNKi) for 24 h. (c) Western blot analysis of PKA IIβ regulatory subunit (PKA IIβ reg) phosphorylation in VEM-R and TRA-R SK-MEL-28 cells treated with VEM or TRA for 24 h as described in Figure 3c. (d) VEM-R 1205Lu cells were generated, indicated by reactivation of ERK1/2 phosphorylation. (e) Western blot analysis of PKA IIβ reg phosphorylation in parental 1205Lu cells treated with different drugs for 24 h as described in Figure 1c. (f) Western blot analysis of PKA IIβ reg phosphorylation in parental, TMZ-resistant (TMZ-R), and VEM-R 1205Lu cells treated with DMSO as control, 100 µM TMZ, or 5 µM VEM for 24 h. 2.5. NLRP1 Is Required for Cell Growth and Colony Formation of Targeted Therapy-Resistant Melanoma Cells Melanoma Cells Lastly, we determined the role of NLRP1 in cell growth and colony formation o Lastly, we determined the role of NLRP1 in cell growth and colony formation of MAPK inhibitor-resistant melanoma cells. Whereas VEM alone had no inhibitory effect on VEM-resistant SK-MEL-28 cell proliferation, silencing NLRP1 decreased the proliferation of resistant cells (Figure 5a), which was conﬁrmed using VEM-resistant 1205Lu cells (Figure 5b). The colony formation assay shows a slower growth of TRA-resistant cells compared to parental cells. However, when they were treated with TRA, the treatment left no visible colonies of parental SK-MEL-28 cells, whereas TRA-resistant cells enhanced their colony-forming ability in the presence of TRA (Figure 5c). When NLRP1 was silenced, the knockdown inhibited the colony formation in not only parental cells without treatment, but also TRA-resistant SK-MEL-28 cells with TRA treatment (Figure 5c), indicating an essential role of NLRP1 for survival and cell growth of parental and resistant cells. MAPK inhibitor-resistant melanoma cells. Whereas VEM alone had no inhibitory effect on VEM-resistant SK-MEL-28 cell proliferation, silencing NLRP1 decreased the prolifera- tion of resistant cells (Figure 5a), which was confirmed using VEM-resistant 1205Lu cells (Figure 5b). The colony formation assay shows a slower growth of TRA-resistant cells compared to parental cells. However, when they were treated with TRA, the treatment left no visible colonies of parental SK-MEL-28 cells, whereas TRA-resistant cells enhanced their colony-forming ability in the presence of TRA (Figure 5c). When NLRP1 was si- lenced, the knockdown inhibited the colony formation in not only parental cells without treatment, but also TRA-resistant SK-MEL-28 cells with TRA treatment (Figure 5c), indi- cating an essential role of NLRP1 for survival and cell growth of parental and resistant cells. Figure 5. Silencing NLRP1 slows down resistant cell growth and colony formation. (a) Cell viabil- ity assay of VEM-resistant (VEM-R) SK-MEL-28 cells transfected with 50 nM control or NLRP1 siRNA overnight and treated with 1 µM VEM for 48 h. (b) Similarly, cell viability assay of NLRP1 siRNA transfected VEM-R 1205Lu cells treated with 5 µM VEM for 48 h. (c) NLRP1 siRNA trans- fected parental or TRA-resistant (TRA-R) SK-MEL-28 cells treated with DMSO or 0.5 µM TRA for 7 days, assessed for colony formation by crystal violet staining (left) and quantitated (right). Rep- resentative images are shown and data expressed as the mean ± SEM, n = 4. * p < 0.05 and ** p < 0.01. Figure 5. Silencing NLRP1 slows down resistant cell growth and colony formation. 2.5. NLRP1 Is Required for Cell Growth and Colony Formation of Targeted Therapy-Resistant Melanoma Cells Melanoma Cells Lastly, we determined the role of NLRP1 in cell growth and colony formation o (c) NLRP1 siRNA trans- fected parental or TRA-resistant (TRA-R) SK-MEL-28 cells treated with DMSO or 0.5 µM TRA for 7 days, assessed for colony formation by crystal violet staining (left) and quantitated (right). Rep- resentative images are shown and data expressed as the mean ± SEM, n = 4. * p < 0.05 and ** p < 0 01 y ( ) overnight and treated with 1 µM VEM for 48 h. (b) Similarly, cell viability assay of NLRP1 siRNA transfected VEM-R 1205Lu cells treated with 5 µM VEM for 48 h. (c) NLRP1 siRNA transfected parental or TRA-resistant (TRA-R) SK-MEL-28 cells treated with DMSO or 0.5 µM TRA for 7 days, assessed for colony formation by crystal violet staining (left) and quantitated (right). Representative images are shown and data expressed as the mean ± SEM, n = 4. * p < 0.05 and ** p < 0.01. counterparts and was further upregulated with VEM treatment (Figure 4f and Figure S8). These data support that PKA is a regulator of ATF4/NLRP1 in MAPK inhibitor-resistant melanoma cells. 2.5. NLRP1 Is Required for Cell Growth and Colony Formation of Targeted Therapy-Resistant counterparts and was further upregulated with VEM treatment (Figure 4f and Figure S8). These data support that PKA is a regulator of ATF4/NLRP1 in MAPK inhibitor-resistant melanoma cells. 2.5. NLRP1 Is Required for Cell Growth and Colony Formation of Targeted Therapy-Resistant 2.5. NLRP1 Is Required for Cell Growth and Colony Formation of Targeted Therapy-Resistant Melanoma Cells Melanoma Cells Lastly, we determined the role of NLRP1 in cell growth and colony formation of 2.5. NLRP1 Is Required for Cell Growth and Colony Formation of Targeted Therapy-Resistant Melanoma Cells Melanoma Cells Lastly, we determined the role of NLRP1 in cell growth and colony formation o fested significantly increased NLRP1 promoter activity. However, unlike parental cells, increased NLRP1 promoter activity in resistant cells remained unaffected following RSK 2.4. The cAMP/PKA Pathway Is a Regulator of ATF4/NLRP1 in Resistant Melanoma Cells In (c–f), the band densities of p-PKA IIβ reg and p-ERK1/2 were quantitated and normalized to those of the corresponding loading control β-actin (c,e,f) or total ERK1/2 (d). Representative images are shown and data expressed as the mean ± SEM, n = 3. * p < 0.05 and ** p < 0.01. M, n = 3. * p < 0.05 and ** p < 0.01. In fact, in addition to the ER stress and MAPK/ERK pathways, other pathways, including cAMP/protein kinase A (PKA) and MAPK/c-Jun N-terminal kinase (JNK) pathways, regulate ATF4 expression and activation [22,23]. Therefore, we investigated their effects on NLRP1 promoter activity and found that the PKA inhibitor signiﬁcantly reduced NLRP1 promoter activity in both VEM- and TRA-resistant cells (Figure 4b). Inhibitor of the stressor protein kinase R-like ER kinase (PERK), the upstream regulator of ATF4 in ER stress, partially reduced NLRP1 promoter activity. On the contrary, the JNK inhibitor increased NLRP1 promoter activity in VEM-resistant cells (Figure 4b). To conﬁrm the potential involvement of cAMP-dependent PKA in NLRP1 gene expression, we evaluated PKA activation by determining the PKA IIβ regulatory subunit phosphorylation status in resistant SK-MEL-28 cells. Figure 4c (and Figure S8) shows an increased PKA IIβ regulatory subunit phosphorylation in both VEM- and TRA-resistant cells compared to parental cells, which was further upregulated in the presence of VEM, suggesting that the MAPK/ERK pathway cross-regulates PKA activation to affect NLRP1 expression. p y g p We then used other VEM-resistant cells derived from 1205Lu cells (Figure 4d and Figure S12) and veriﬁed the elevated PKA IIβ regulatory phosphorylation. TMZ-resistant counterparts [12] were used as control. When parental 1205Lu cells were treated with a single dose of TMZ, VEM, or TRA, we found decreased PKA IIβ regulatory phospho- rylation by signaling inhibitors, VEM and TRA, but not TMZ (Figure 4e and Figure S2), consistent with the ﬁndings in Figure 1c. However, in VEM-resistant 1205Lu cells, PKA IIβ regulatory phosphorylation was increased compared to its parental and TMZ-resistant Pharmaceuticals 2021, 14, 23 8 of 15 counterparts and was further upregulated with VEM treatment (Figure 4f and Figure S8). These data support that PKA is a regulator of ATF4/NLRP1 in MAPK inhibitor-resistant melanoma cells. E E IE 9 o 6 2.5. NLRP1 Is Required for Cell Growth and Colony Formation of Targeted Therapy-Resistant 2.5. NLRP1 Is Required for Cell Growth and Colony Formation of Targeted Therapy-Resistant Melanoma Cells Melanoma Cells Lastly, we determined the role of NLRP1 in cell growth and colony formation o (a) Cell viability assay of VEM-resistant (VEM-R) SK-MEL-28 cells transfected with 50 nM control or NLRP1 siRNA overnight and treated with 1 µM VEM for 48 h. (b) Similarly, cell viability assay of NLRP1 siRNA transfected VEM-R 1205Lu cells treated with 5 µM VEM for 48 h. (c) NLRP1 siRNA transfected parental or TRA-resistant (TRA-R) SK-MEL-28 cells treated with DMSO or 0.5 µM TRA for 7 days, assessed for colony formation by crystal violet staining (left) and quantitated (right). Representative images are shown and data expressed as the mean ± SEM, n = 4. * p < 0.05 and ** p < 0.01. Figure 5. Silencing NLRP1 slows down resistant cell growth and colony formation. (a) Cell viabil- ity assay of VEM-resistant (VEM-R) SK-MEL-28 cells transfected with 50 nM control or NLRP1 siRNA overnight and treated with 1 µM VEM for 48 h. (b) Similarly, cell viability assay of NLRP1 siRNA transfected VEM-R 1205Lu cells treated with 5 µM VEM for 48 h. (c) NLRP1 siRNA trans- fected parental or TRA-resistant (TRA-R) SK-MEL-28 cells treated with DMSO or 0.5 µM TRA for 7 days, assessed for colony formation by crystal violet staining (left) and quantitated (right). Rep- resentative images are shown and data expressed as the mean ± SEM, n = 4. * p < 0.05 and ** p < 0.01. Figure 5. Silencing NLRP1 slows down resistant cell growth and colony formation. (a) Cell viability assay of VEM-resistant (VEM-R) SK-MEL-28 cells transfected with 50 nM control or NLRP1 siRNA overnight and treated with 1 µM VEM for 48 h. (b) Similarly, cell viability assay of NLRP1 siRNA transfected VEM-R 1205Lu cells treated with 5 µM VEM for 48 h. (c) NLRP1 siRNA transfected parental or TRA-resistant (TRA-R) SK-MEL-28 cells treated with DMSO or 0.5 µM TRA for 7 days, assessed for colony formation by crystal violet staining (left) and quantitated (right). Representative images are shown and data expressed as the mean ± SEM, n = 4. * p < 0.05 and ** p < 0.01. y y ( ) siRNA overnight and treated with 1 µM VEM for 48 h. (b) Similarly, cell viability assay of NLRP1 siRNA transfected VEM-R 1205Lu cells treated with 5 µM VEM for 48 h. 3 Di i 3. Discussion 3. Discussion NLRP1 participates in multiple activities in melanoma. We previously reported that NLRP1 plays a role in tumorigenesis, progression, and inflammation-associated chemo- therapy resistance [10,12]. Here, we found that NLRP1-mediated IL-1β signaling is down- stream of the MAPK/ERK signaling through the ATF4 regulation, further uncovering its NLRP1 participates in multiple activities in melanoma. We previously reported that NLRP1 plays a role in tumorigenesis, progression, and inﬂammation-associated chemother- apy resistance [10,12]. Here, we found that NLRP1-mediated IL-1β signaling is down- stream of the MAPK/ERK signaling through the ATF4 regulation, further uncovering its involvement in targeted therapy resistance. involvement in targeted therapy resistance. Melanoma is a type of cancer that utilizes IL-1β to shape the tumor microenviron- f i h M i l i i f BRAF ib i i i g py Melanoma is a type of cancer that utilizes IL-1β to shape the tumor microenvironment for its own growth. Mutational activation of BRAF contributes to constitutive activation of Pharmaceuticals 2021, 14, 23 9 of 15 9 of 15 NF-κB, the key transcriptional factor that controls the production of many cytokines, includ- ing IL-1β, and is usually activated by chemotherapy and in cancer drug resistance [24–26]. Other pathways also regulate and augment IL-1β signaling via crosstalk with NF-κB signal- ing. The MAPK/ERK signaling pathway positively regulates the NF-κB pathway through multiple mechanisms, including direct phosphorylation of IκBα by RSK2 or indirect ac- tivation of IKKα/β via maternal embryonic leucine zipper kinase [27,28]. As such, it is not surprising that we observed decreased IL1B mRNA expression and IL-1β production in VEM and TRA-treated 1205Lu cells (Figure S13). However, inhibiting the MAPK/ERK with VEM and TRA also resulted in a decrease in IL-1β secretion (Figure 1), in concordance with previous observations [7,8]. Due to the fact that NLRP1 inﬂammasomes regulate not only IL-1β secretion but also NF-κB activation [10] and IL-1β production [5,10], our data suggest that the MAPK/ERK signaling modulates IL-1β signaling via the NF-κB pathway and NLPR1 inﬂammasomes. ER stress is an adaptive mechanism by which cells can survive in an unfavorable envi- ronment, though it often triggers cell death upon excessive or persistent disturbance [29]. Unfortunately, this ER defense strategy can be fully used by tumor cells to survive under hostile microenvironments, such as nutrient shortage, oxidative stress, and drug pres- sure [30]. Sustained activation of ER stress enables tumor cells with greater tumorigenic, metastatic, and drug-resistant capacity [30,31]. 3 Di i 3. Discussion We have assessed the ER stress dependence of melanoma cell growth in vitro and found that pretreatment of 1205Lu and SK-MEL-28 cells with 4-phenylbutyric acid, an ER stress inhibitor [32], sensitizes cells to TMZ and VEM treatment, respectively (Figure S14). Therefore, manipulating ER stress, including suppressing ATF4, has been regarded as an important strategy for therapeutic intervention in cancer [13,22,33,34]. Indeed, the regulation of ATF4 expression by the MAPK/ERK pathway has been recently reported [13,35]. ATF4 is a master transcriptional factor of the ER stress response, and its expression is typically upregulated in solid tumors compared to normal tissues [33]. We examined ATF4 mRNA expression in human melanoma tis- sues using two publicly available microarray datasets from independent gene proﬁling studies [36,37] and found that metastatic melanoma tissues display higher ATF4 mRNA level than primary melanoma (Figure S15). In agreement with this, we observed elevated levels of ATF4 mRNA and nuclear ATF4 protein in MAPK inhibitor-resistant SK-MEL-28 cells compared to parental cells (Figure 3), suggesting that ATF4 is a critical contributor in MAPK inhibitor resistance. Recently, PERK and/or IRE1α branches of ER stress were found to enhance NLRP1 gene expression through the cAMP response element binding pro- tein (CREB) in HeLa and chronic myelogenous leukemia cells [18,38]. We provide further evidence that NLRP1 lies downstream of the ER stress signaling cascade, possibly through the PERK/ATF4 in melanoma cells. ATF4 transcriptionally regulates many pro-survival genes, including HMOX1, BCL2, and MCL1, and key autophagy genes such as ATG5 and ATG7 [33,35,39]. We added NLRP1 to this pro-survival ATF4 target gene list. p g g NLRP1 is considered to be a tumor suppressor, but aberrant NLRP1 inﬂammasome activation due to gain-of-function mutations has been associated with cancers and au- toimmune diseases [11,40,41], which could reasonably explain why some melanoma cell lines, if not all, are capable of secreting IL-1β. Our current work has linked NLRP1 in- ﬂammasome activation to the MAPK/ERK signaling pathway and indicated that NLRP1 inﬂammasome activation is implicated in the downstream signaling cascades of the acti- vating BRAF mutation. In parental melanoma cells, the RSK2/ATF4 axis acts as the hub between the MAPK/ERK signaling and NLRP1; however, MAPK inhibitor-resistant cells seem to lose this signaling hub and switch to the cAMP/PKA pathway to redirect NLRP1 expression (Figure 6). In fact, there is a crosstalk between the MAPK/ERK signaling and the cAMP/PKA pathway, which are mutually exclusive [42]. 3 Di i 3. Discussion In RAS-mutated melanoma, PKA regulates the MAPK/ERK signaling by switching BRAF to RAF-1 signaling [43]. On the other hand, active RSK inhibits PKA activity while inactive RSK interacts with PKA and sensitizes it to cAMP stimulation [39]. It has been reported that dibutyryl-cAMP, a cAMP analogue, can induce NLRP1 expression in human myeloid leukemia cells, possibly Pharmaceuticals 2021, 14, 23 10 of 15 rified th 10 of 15 rified th through PKA-mediated CREB activation [44]. In the current work, we veriﬁed that the regulation of the NLRP1 gene promoter activity in MAPK inhibitor-resistant melanoma cells is via PKA. It is possible that in the reactivated, BRAF-mutated melanoma cells, there exists a signaling network including the ER stress, MAPK/ERK, and cAMP/PKA signaling pathways, which interact with each other and play an integral role in the self-survival of tu- mor cells. In this network, NLRP1 functions downstream through a shared transcriptional regulation by ATF4. Given the role of these signaling pathways in melanoma tumorigenesis and progression, downstream NLRP1 may be an important executor. This scenario could explain the tumor-promoting role of NLRP1, not only in the targeted therapy resistance observed in this study, but also in the chemotherapy resistance we observed previously [12]. These two different drug treatments activate distinct signaling pathways that converge on the ATF4/NLRP1 axis to regulate melanoma growth. This ATF4/NLRP1 axis may also be a common molecular mechanism behind cancer resistance to other therapeutic approaches. cells is via PKA. It is possible that in the reactivated, BRAF-mutated melanoma cells exists a signaling network including the ER stress, MAPK/ERK, and cAMP/PKA sig pathways, which interact with each other and play an integral role in the self-surv tumor cells. In this network, NLRP1 functions downstream through a shared tran tional regulation by ATF4. Given the role of these signaling pathways in melano morigenesis and progression, downstream NLRP1 may be an important executo scenario could explain the tumor-promoting role of NLRP1, not only in the targete apy resistance observed in this study, but also in the chemotherapy resistance w served previously [12]. These two different drug treatments activate distinct sig pathways that converge on the ATF4/NLRP1 axis to regulate melanoma growth ATF4/NLRP1 axis may also be a common molecular mechanism behind cancer resi to other therapeutic approaches. Figure 6. Hypothetical roles of ATF4/NLRP1 in acquired targeted therapy resistance in mela (a) ATF4/NLRP1 in parental melanoma cells. 3 Di i 3. Discussion Red arrows indicate the inhibitory effects of V and TRA. Bars indicate inhibition. (b) ATF4/NLRP1 in resistant melanoma cells. Red arrows cate the upregulation of expression levels. A dashed line indicates a signal loss. In both a an the thickness of red arrows indicates relative expression levels; the thickness of black arrow cates the relative strength of signaling pathways; and “p” in orange oval indicates phosphor tion. Figure 6. Hypothetical roles of ATF4/NLRP1 in acquired targeted therapy resistance in melanoma. (a) ATF4/NLRP1 in parental melanoma cells. Red arrows indicate the inhibitory effects of VEM and TRA. Bars indicate inhibition. (b) ATF4/NLRP1 in resistant melanoma cells. Red arrows indicate the upregulation of expression levels. A dashed line indicates a signal loss. In both a and b, the thickness of red arrows indicates relative expression levels; the thickness of black arrows indicates the relative strength of signaling pathways; and “p” in orange oval indicates phosphorylation. Figure 6. Hypothetical roles of ATF4/NLRP1 in acquired targeted therapy resistance in mela (a) ATF4/NLRP1 in parental melanoma cells. Red arrows indicate the inhibitory effects of V and TRA. Bars indicate inhibition. (b) ATF4/NLRP1 in resistant melanoma cells. Red arrows cate the upregulation of expression levels. A dashed line indicates a signal loss. In both a an the thickness of red arrows indicates relative expression levels; the thickness of black arrow cates the relative strength of signaling pathways; and “p” in orange oval indicates phosphor tion Figure 6. Hypothetical roles of ATF4/NLRP1 in acquired targeted therapy resistance in melanoma. (a) ATF4/NLRP1 in parental melanoma cells. Red arrows indicate the inhibitory effects of VEM and TRA. Bars indicate inhibition. (b) ATF4/NLRP1 in resistant melanoma cells. Red arrows indicate the upregulation of expression levels. A dashed line indicates a signal loss. In both a and b, the thickness of red arrows indicates relative expression levels; the thickness of black arrows indicates the relative strength of signaling pathways; and “p” in orange oval indicates phosphorylation. NLRP1 is expressed at low levels in melanoma and nonmelanoma skin c [10,45], possibly due to its DNA hypermethylation. However, increased NLRP1 e sion and activated NLRP1 inflammasomes favor drug resistance. Since the inflamm microenvironment provides melanoma tolerance to targeted therapy [9], tar NLRP1/IL-1β may be an important strategy to improve MAPK inhibitor efficacy. 3 Di i 3. Discussion W many inflammasome inhibitors are available currently, most of them target NLR 1β, IL-1 receptor, inflammasome adaptor ASC, and inflammasome effector cas [46,47]. It has been reported that aspirin and green tea component epigallocatechin late suppress NLRP1 expression in humans [48,49]; however, NLRP1 inhibitors NLRP1 is expressed at low levels in melanoma and nonmelanoma skin cancers [10,45], possibly due to its DNA hypermethylation. However, increased NLRP1 expression and activated NLRP1 inﬂammasomes favor drug resistance. Since the inﬂammatory microen- vironment provides melanoma tolerance to targeted therapy [9], targeting NLRP1/IL-1β may be an important strategy to improve MAPK inhibitor efﬁcacy. Whereas many inﬂam- masome inhibitors are available currently, most of them target NLRP3, IL-1β, IL-1 receptor, inﬂammasome adaptor ASC, and inﬂammasome effector caspase-1 [46,47]. It has been reported that aspirin and green tea component epigallocatechin-3-gallate suppress NLRP1 expression in humans [48,49]; however, NLRP1 inhibitors so far have not been at the forefront of development. It is critical to developing such unique drugs for inﬂammation, cancer, and other diseases. pp p [ , ]; , have not been at the forefront of development. It is critical to developing such u drugs for inflammation, cancer, and other diseases. It is worth noting that the majority of our data were generated using SK-MEL- 1205Lu cells. Due to the intertumoral and intercellular heterogeneity in human mela our findings should be verified in more melanoma cell lines. Moreover, though w served increased PKA activation in BRAF/MEK inhibitor-resistant melanoma ce mechanism behind PKA activation and its impact on the resistance acquisition nee It is worth noting that the majority of our data were generated using SK-MEL-28 and 1205Lu cells. Due to the intertumoral and intercellular heterogeneity in human melanoma, our ﬁndings should be veriﬁed in more melanoma cell lines. Moreover, though we ob- served increased PKA activation in BRAF/MEK inhibitor-resistant melanoma cells, the mechanism behind PKA activation and its impact on the resistance acquisition need to be further investigated. In addition to ATF4, NLRP1 may be subject to regulation by other transcription factors. For example, ATF2 and ATF4 share the same binding cAMP response element within the NLRP1 promoter. Our preliminary study shows that knocking down Pharmaceuticals 2021, 14, 23 11 of 15 11 of 15 ATF2 also reduces NLRP1 gene expression in VEM- and TRA-resistant SK-MEL-28 cells (Figure S16). It has been reported that the PKCε-ATF2 axis is involved in melanomagenesis and BRAF inhibitor resistance [50]. 4. Materials and Methods 4.1. Chemicals VEM, TRA, PLX-4720, CI-1040, RSK1/2/3/4 inhibitor BI-D1870, PKA inhibitor H 89 2HCl, JNK1/2/3 inhibitor SP600125, and PERK inhibitor GSK2606414 were from Selleck Chem (Houston, TX, USA). TMZ and TG, ER stress inducers, were from Sigma (St. Louis, MO, USA). These reagents were reconstituted in 100% dimethyl sulfoxide (DMSO) and stored at −20 ◦C in aliquots. Other chemicals and reagents were indicated elsewhere. 3 Di i 3. Discussion Furthermore, ATF2 can be phosphorylated and acti- vated by the MAPK/ERK pathway in collaboration with another MAPK member, p38 [51]. Therefore, it will be interesting to evaluate the regulatory role of ATF2 in NLRP1-mediated targeted therapy resistance in melanoma. 4.2. Cell Culture Human metastatic melanoma cell lines A375 (CRL-1619) and SK-MEL-28 (HTB-72) were obtained from the American Type Culture Collection (Manassas, VA, USA) and 1205Lu cell line (1205Lu-01-0001) was purchased from Rockland (Limerick, PA, USA). Cells were routinely grown in RPMI medium 1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA, USA). These cell lines have been authenticated using short tandem repeat (STR) ﬁngerprinting by the Barbara Davis Center BioResource Core at the University of Colorado Anschutz Medical Campus and were regularly tested for mycoplasma contamination. 4.5. Enzyme-Linked Immunosorbent Assay (ELISA) A human IL-1β ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA). Culture supernatants were collected for quantitating IL-1β secretion, with cell lysates for assaying intracellular pro-IL-1β levels [12]. 4.3. Cell Growth Inhibition To evaluate cell’s sensitivity to VEM and TRA, melanoma cells were seeded into a 96-well plate at a density of 1–2 × 103 cells per well and allowed to adhere for ~2 h before being exposed to increasing concentrations of drugs in triplicate for 72 h. Cell growth inhibition was determined using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA). 4.4. Generation of Acquired Resistance Cells were plated into a 100 mm dish at a density of 1–2 × 105 cells per dish and treated with increasing doses of VEM or TRA per passage for two months [52]. The starting doses of VEM and TRA were 0.05 and 0.005 µM, respectively. Acquired resistance was deﬁned as at least a 10-fold increase of IC50 over that of parental cells. Resistant cells were regularly maintained in a medium with 1 (SK-MEL-28) or 3 (1205Lu) µM VEM or 0.2 µM TRA (SK-MEL-28). TMZ-resistant 1205Lu cells have been described previously [12]. 4.11. Clonogenic Assay Single-cell suspensions were prepared and 1000 cells seeded into 6-well plates per well. Following treatment with TRA for 7 days, the colonies were ﬁxed in 4% paraformaldehyde for 10 min and stained with 0.2% (w/v) crystal violet for 30 min. Digital images of the colonies were obtained using a scanning device and the colonies were counted using Image J software [53]. 4.10. Luciferase Reporter Assay LightSwitch Promoter Reporter vector containing a 905-bp human NLRP1 promoter region cloned upstream of the RenSP luciferase gene was from Active Motif (Carlsbad, CA, USA). Cells were transfected with the construct and an empty promoter vector using FuGENE HD transfection reagent (Active Motif) for 24 h according to the manufacturer’s instructions. Luciferase reporter signals were monitored using the Lightswitch Luciferase assay reagent (Active Motif) as per manufacturer’s protocol. 4.12. Statistical Analysis GraphPad Prism 7 was used for testing the differences between the groups by one- way ANOVA with Bonferroni’s or Dunnett’s post-tests. A value p < 0.05 was considered signiﬁcant. 4.7. Quantitative RT-PCR RNA isolation and quantitative PCR have previously been described [12]. The primers for IL1B, ATF4, NLRP1, NLRP3, MITF, AXL, and GAPDH are listed in Supplemental Table S1. 4.6. siRNA Transfection Cells seeded in appropriate culture plates at 60% conﬂuency were transfected with 50 nM of NLRP1 siRNA (a mixture of two preselected siRNAs; Qiagen, Valencia, CA, USA) or ATF4 siRNA (a pool of three target-speciﬁc 19–25 nt siRNA; Santa Cruz Biotechnology, Santa Cruz, CA, USA), as well as their respective negative control using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in OPTI-MEM1 reduced serum medium (Gibco) for 4–6 h. An equal volume of culture medium with 20% fetal bovine serum was then added. 12 of 15 Pharmaceuticals 2021, 14, 23 12 of 15 4.9. Chromatin Immunoprecipitation (ChIP) Assay ChIP assay of ATF4 binding to NLRP1 gene promoter was performed using the Pierce Magnetic ChIP kit (Pierce Biotechnology, Rockford, IL, USA). Brieﬂy, cells at 60% conﬂuency were treated with DMSO or 1 µM TG for 18 h and cross-linked with 1% formaldehyde for 6 min, followed by quenching with 1× glycine for 5 min at room temperature. After washing, cells were lysed and nuclei digested with the MNase at 37 ◦C for 15 min, followed by sonication for 10 cycles at 4 ◦C with a 20 s pulse at 40% amplitude with a 20 s pause between each cycle. Digested chromatin was obtained by centrifugation and then split into two aliquots incubated with mouse anti-ATF4 or control IgG antibodies for 2 h. Protein A/G magnetic beads were then added and incubated at 4 ◦C for another 2 h. Finally, protein and RNA were removed by digestion of the eluted protein–chromatin complex with proteinase K, and DNA was puriﬁed following the manufacturer’s protocol. ChIP DNA was detected by qPCR using the Power SYBR Green PCR Master Mix with speciﬁc primers for NLRP1 and ATF3 genomic promoter regions (Supplemental Table S1). 4.8. Western Blot As described previously [12], Western blot analysis was carried out. The primary antibodies included mouse anti-NLRP1 (Enzo Life Sciences, Farmingdale, NY, USA), mouse anti-ATF4 (Santa Cruz Biotechnology), rabbit anti-phospho-p44/42 MAPK (p-ERK1/2), rabbit anti-p44/42 MAPK (ERK1/2), rabbit anti-CyPA (Cell Signaling Technology, Danvers, MA, USA), mouse anti-phospho-RSK2, mouse anti-phospho-PKA IIβ reg, goat anti-Lamin B, and rabbit anti-β-actin (Santa Cruz Biotechnology). 5. Conclusions Overall, this work added a new understanding of NLRP1 in melanoma biology. This is the ﬁrst report to link the MAPK/ERK signaling to NLRP1 in melanoma through the ATF4 regulation. ATF4-mediated NLRP1 expression and IL-1β efﬂux may be the mechanistic basis for melanoma tumorigenesis and drug resistance via shaping the inﬂammatory tumor Pharmaceuticals 2021, 14, 23 13 of 15 13 of 15 microenvironment. Our ﬁndings support that NLRP1 may be a promising molecular target for melanoma treatment. microenvironment. Our ﬁndings support that NLRP1 may be a promising molecular target for melanoma treatment. Supplementary Materials: The following are available online at https://www.mdpi.com/1424-8 247/14/1/23/s1, Figure S1: The mRNA expression levels of IL1B in human melanoma cell lines. Figure S2: Original blots of Figures 1c, 2b and 4e. Figure S3: Putative ATF4 binding site within the NLRP1 promoter. Figure S4: Original blots of Figure 1d. Figure S5: Original blots of Figure 2e. Figure S6: Synergistic inhibition of NLRP1 protein expression by RSKi and VEM in A375 and 1205Lu cells. Figure S7: Original blots of Figure 3b. Figure S8: Original blots of Figures 3c and 4c,f. Figure S9: Original blots of Figure 3e. Figure S10: MITF and AXL gene expression in MAPK inhibitor-resistant SK-MEL-28 cells. Figure S11. IL1B gene expression is inversely related to the expression ratio of MITF/AXL in melanoma cells as shown in Figure S1. Figure S12: Original blots of Figure 4d. Figure S13: Decreased IL1B mRNA expression and IL-1β production in 1205Lu cells treated with 1 µM vemurafenib (VEM) or 0.5 µM trametinib (TRA). Figure S14: Pretreatment of 1205Lu and SK-MEL-28 cells with 4-phenylbutyric acid (PBA), an ER stress inhibitor, which sensitizes cells to temozolomide (TMZ) and vemurafenib (VEM), respectively. Figure S15: Metastatic melanoma tissues display higher ATF4 mRNA levels than primary melanoma by examining ATF4 mRNA expression in human melanoma tissues using two publicly available microarray datasets. Figure S16: qRT-PCR analysis of NLRP1 expression in parental and resistant SK-MEL-28 cells transfected with 50 nM control siRNA or ATF2 siRNA overnight. Table S1: Primers used for quantitative qRT-PCR and ChIP. Author Contributions: Funding acquisition, M.F.; conceptualization, M.F.; supervision, M.F.; exper- imentation, Z.Z., P.K.V., J.M.S. and T.T.; formal analysis, Z.Z.; writing—original draft preparation, Z.Z.; writing—review and editing, M.F., P.K.V., J.M.S. and T.T. All authors have read and agreed to the published version of the manuscript. 5. Conclusions Funding: This work has been supported, in whole or in part, by Veterans Affairs Merit Review Award 5I01BX001228 (to M.F.), NIH/NCI R01 CA197919 (to M.F.), and Cancer League of Colorado (to M.F.). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in this article or associ- ated supplementary material. Acknowledgments: We thank the University of Colorado Cancer Center (UCCC) Support Grant (P30CA046934) and the Skin Diseases Research Cores Grant (P30AR057212) for their help. We thank Joanne Domenico and Rasika Mukkamala for their linguistic editing and comments. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Wan, P.T.; Garnett, M.J.; Roe, S.M.; Lee, S.; Niculescu-Duvaz, D.; Good, V.M.; Jones, C.M.; Marshall, C.J.; Springer, C.J.; Barford, D.; et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004, 116, 855–867. 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https://openalex.org/W4389519917	https://aclanthology.org/2023.findings-emnlp.234.pdf	English	null	Exploring Graph Pre-training for Aspect-based Sentiment Analysis	null	2,023	cc-by	7,153	Abstract Existing studies tend to extract the sentiment elements in a generative manner in order to avoid complex modeling. Despite their effec- tiveness, they ignore importance of the relation- ships between sentiment elements that could be crucial, making the large pre-trained gener- ative models sub-optimal for modeling senti- ment knowledge. Therefore, we introduce two pre-training paradigms to improve the gener- ation model by exploring graph pre-training that targeting to strengthen the model in cap- turing the elements’ relationships. Specifically, We first employ an Element-level Graph Pre- training paradigm, which is designed to im- prove the structure awareness of the generative model. Then, we design a Task-level Graph Pre-training paradigm to make the generative model generalizable and robust against vari- ous irregular sentiment quadruples. Extensive experiments show the superiority of our pro- posed method, and validate the correctness of our motivation. Our code can be found in https://github.com/HoraceXIaoyiBao/ EGP4ABSA-EMNLP2023. Figure 1: Two proposed pre-train paradigms. either sequence-level (Qiu et al., 2011; Peng et al., 2020; Cai et al., 2021) or token-level classification problems (Tang et al., 2016) in joint learning or pipeline manner. However, these methods not only require sophisticated and complex modeling of sen- timent elements but also suffer severely from er- ror propagation since the overall prediction perfor- mance hinges on the accuracy of every step (Peng et al., 2020). ∗Corresponding author Exploring Graph Pre-training for Aspect-based Sentiment Analysis Xiaoyi Bao1, Zhongqing Wang1∗, Guodong Zhou1 1Natural Language Processing Lab, Soochow University, Suzhou, China p2213545413@outlook.com {wangzq,gdzhou}@suda.edu.cn Mask Task-level Graph Pre-train Element-level Graph Pre-train Aspect Opinion Category Polarity Figure 1: Two proposed pre-train paradigms. Findings of the Association for Computational Linguistics: EMNLP 2023, pages 3623–3634 December 6-10, 2023 ©2023 Association for Computational Linguistics 1 Introduction surface (aspect) Design (category) smooth (opinion) Positive (polarity) Quad apps (aspect) Software (category) hard (opinion) Negative (polarity) Quad ROOT Opinion Tree Aspect Opinion Aspect Opinion Linearized Tree Decode Generate Generative Model Subtask 𝐹𝑖𝑛𝑒𝑡𝑢𝑛𝑒 Aspect Opinion Category Polarity Generative Model Task-level Graph Pre-train Figure 2: Overview of joint pre-training, the subtasks will be introduced in the following section. We simplify the process of Task-level Graph Pre-training for brief reading, the detailed process will be introduced in the following section. cedure to learn to handle the task in stages. Specifi- cally, we first decompose the quadruple extraction task into multiple subtasks. Each subtask corre- sponds to mapping the steps for manually building an opinion tree from scratch. Afterwards, we fea- ture a prompt-based learning strategy to separately acquire the knowledge of subtasks and finally em- ploy the learned knowledge to tackle the main task, i.e., generating the entire opinion tree. The decom- posed subtasks build fundamental knowledge of irregular sentiment quadruples for generation. strengthen the generative model in modeling aspect- level sentiment structure. We believe the challenges locate in two aspects. First is structural modeling: the huge gap between the pre-training and finetun- ing phases makes it difficult to model its succinct yet distinctive structure : certain components ( e.g. aspect term ) in sentiment structure obviously more important than others. Another challenge is the gen- eralization and robustness of the generative model: the generative model should be generalizable and robust against irregular sentiment quadruples. It is crucial since the structure is built depending on the quadruples and the challenging scenarios in real practice are usually brought by the irregular sentiment quadruples. As shown in Figure 2, we then jointly pre-train the model with the two paradigms above and fine- tune the model with the Finetune task. The ad- vantages of our pre-training method over previ- ous learning methods are threefold: 1) both the Element-level Graph Pre-training and Task-level Graph Pre-training are designed depending on the intrinsic characteristics of the opinion tree instead of treating it as a plain graph.2) the Element-level Graph Pre-training abandons the strategy of cap- turing the complex structure but focuses directly on the core elements. 3) the Task-level Graph Pre- training explicitly forces the model to learn the irregular quadruples with an easy-to-hard routine, making it easier for the model to learn the funda- mental knowledge required. 1 Introduction More recently, studies tend to tackle the ABSA problem with a unified generative approach (Zhang et al., 2021b,a; Yan et al., 2021; Bao et al., 2022). They organize the target sequence in dif- ferent approaches, namely listing (Zhang et al., 2021b): “(apps, hard, Software, Negative)”, in- dexing(Yan et al., 2021): “(1,1,3,3)”, paraphrasing (Zhang et al., 2021a): “(Software is good because apps are hard)” or opinion tree(Bao et al., 2022): “((Root,(Quad,( Aspect ( Software, apps ),( Opinion ( Negative, hard )))))”. However, they ignore the importance of the relationships among elements (e.g. sentiment polarity should be identified based on opinion words, like great identifies a positive polarity and disappointing identifies a negative po- larity). Aspect-based sentiment analysis (ABSA) has drawn increasing attention in the community, which includes four fine-grained elements: aspect term, opinion term, aspect category, and opinion polarity. The first two terms exist as a raw text span in the review sentence while the remaining two are the classification result of aspect and opinion re- spectively. Each four mapped sentiment elements form an aspect-level sentiment quadruple. For in- stance, for the given review "The apps are hard to use.", the corresponding quadruple is (apps, hard, Software, Negative). The joint extraction of quadruples is the most complex and challenging subtask among all the ABSA tasks, previous work usually formulate it as In this situation, a natural question is how to 3623 surface (aspect) Design (category) smooth (opinion) Positive (polarity) Quad apps (aspect) Software (category) hard (opinion) Negative (polarity) Quad ROOT Opinion Tree Aspect Opinion Aspect Opinion Linearized Tree Decode Review Task-level Graph Pre-train Element-level Graph Pre-train Subtask Aspect Generate Generative Model The apps are hard to use. Review [Tree] Prompt Mask Tree + + Tree Input Output Subtask 𝐸𝑃𝐺1 The apps are hard to use. Review [Aspect] Prompt + Input Output Subtask Pair Input The apps are hard to use. Review Aspect [Opinion] Prompt + Output Subtask Triple Input The apps are hard to use. Review Aspect 𝑃𝑜𝑙𝑎𝑟𝑖𝑡𝑦 [Opinion] Prompt + Output Subtask 𝐹𝑖𝑛𝑒𝑡𝑢𝑛𝑒 Input The apps are hard to use. Review Aspect 𝐶𝑎𝑡𝑒𝑔𝑜𝑟𝑦 𝑃𝑜𝑙𝑎𝑟𝑖𝑡𝑦[Opinion] Prompt + Output Subtask 𝐹𝑖𝑛𝑒𝑡𝑢𝑛𝑒 Pre-train Aspect Opinion Category Polarity Figure 2: Overview of joint pre-training, the subtasks will be introduced in the following section. We simplify the process of Task-level Graph Pre-training for brief reading, the detailed process will be introduced in the following section. 1 Introduction The detailed evalua- tion shows that our model significantly advances the state-of-the-art performance on several bench- mark datasets. In this study, we proposed two novel graph pre- training paradigms to address above challenges. As shown in Figure 1, we first introduce an op- timal self-encoding method called Element-level Graph Pre-training. We abandon the traditional indiscriminate masking strategy (equally random masking every node or edge ) and depending on the characteristics of the opinion tree, adopt sen- timent element level masking. Given the opinion tree of the review "The apps are hard to use.", only sentiment nodes (namely apps, hard, Software, Negative ) or the sub-trees they composed in the graph will be masked. In this case, this method can serve as an effective addition to structural modeling in opinion tree generation. We then propose a Task-level Graph Pre-training paradigm, which mimics the human learning pro- 3624 Encoder Decoder Output The surface is smooth, but the apps are hard to use. … (Root, (Quad, ( Aspect (Software, apps ), ( Opinion ( Input Aspect Opinion Category Polarity Reform Opinion Tree Figure 3: Opinion tree generation model. elements, proposed a sentiment tree structure called opinion tree, and employed generative model to extract the linearized tree. However, the genera- tive model is pre-trained to solve textual sequence tasks(e.g. masked language model) but finetuned for structure generation, between which exists a huge gap, making generative models sub-optimal for modeling structural knowledge. Different from previous studies, we introduce two pre-training paradigms for opinion tree gener- ation without treating it as a plain graph. To our knowledge, we are the first to consider designing methods depending on the intrinsic characteristics of the opinion tree. Figure 3: Opinion tree generation model. 3 Opinion Tree Generation Model There are four aspect-level sentiment elements in ABSA, the various combination of these elements form the numerous sub-tasks of ABSA. The re- searches on ABSA generally follow a route from handling single sub-task to complex compositions of them. The starting point usually locates in the prediction of a single sentiment element, which is the target of fundamental sub-tasks, such as extract- ing the aspect term (Qiu et al., 2011; Tang et al., 2016; Wang et al., 2021), classifing the aspect cate- gory mentioned in the sentence (Bu et al., 2021; Hu et al., 2019), and detecting the sentiment polarity for a given aspect (Tang et al., 2016; Chen et al., 2022a; Liu et al., 2021; Seoh et al., 2021; Zhang et al., 2022). In this section, we introduce the basic opinion tree generation model we employed to generate in the pre-train and finetune phases, along with the objec- tive functions and training. 3.1 Opinion Tree Construction For further strengthen the relationship between el- ements, we build a structure called opinion tree, which aims to jointly model all sentiment elements in a tree for a given review sentence. The opinion tree can be considered as a semantic representa- tion in order to better represent the structure of sentiment elements. Inside the opinion tree, each sentiment element would be connected with an- other node as either the child or parent relation to represent the crucial relationship. Since the sentiment elements are naturally cor- related, many studies further focus on exploring the co-extraction of sentiment elements, including aspect and opinion term extraction (Xu et al., 2020; Li et al., 2022); aspect term extraction and its po- larity detection (Zhang and Qian, 2020); aspect category and polarity detection (Cai et al., 2020). Furthermore, recent studies also employed end-to- end models to extract all the sentiment elements in triplet or quadruple format (Peng et al., 2020; Wan et al., 2020; Cai et al., 2021; Zhang et al., 2021a; Chen et al., 2022b; Mukherjee et al., 2021). As shown in Figure 3, we construct the opinion tree using a rooted directed acyclic graph, including nodes of aspect, opinion, category, and polarity, along with the semantic relations between them. After that, we linearize the opinion tree to the target sequence via depth-first traversal. 3.2 Generation Model where p(xi T \|x1 T , x2 T , ...xi−1 T , XO; θ) is calculated by the decoder. 4 Pre-training Paradigms In this study, we introduce two pre-training paradigms for opinion tree generation. As shown in Figure 2, the two paradigms and finetune task share the same input format with a joint input of prompt, encoded text and tree, each method con- sists of a set of subtasks focus on respective train- ing targets. The combination of subtasks forms the joint pre-training in our work, we will introduce the paradigms first in this section. Figure 4: Example of element-level graph masking. After the input token sequence is encoded, the decoder predicts the output sequence token-by- token with the sequential input tokens’ hidden vectors. At the i-th step of generation, the self- attention decoder predicts the i-th token yi in the linearized form, and decoder state hd i as: 3.2 Generation Model We employ the pre-trained language model T5 (Raf- fel et al., 2020) to generate the linearized opin- ion tree. As shown in Figure 3, it is an encoder- decoder architecture model, the input would be the raw review and the output is linearized opinion tree. Given the token sequence x = x1, ..., x\|x\| as input, the sequence-to-sequence model outputs the lin- earized representation y = y1, ..., y\|y\|. To this end, the sequence-to-sequence model first computes the hidden vector representation: More recently, studies tend to design a uni- fied framework to extract quadruples at one stop with pre-trained encoder-decoder language models, achieving great improvements in ABSA (Zhang et al., 2021a). The target sequence of them is formed by either class index (Yan et al., 2021) or the desired sentiment element (Zhang et al., 2021b). OTG (Bao et al., 2022) addressed the im- portance of semantic correlations among sentiment H = (x1, ..., x\|x\|) (1) H = (x1, ..., x\|x\|) (1) 3625 Subtask Input Subtask Prompt Review Tree EGP1 [Tree] The apps are hard to use. (Root,(Quad, ...<Mask>,. . . ) (Root,(Quad, ...(Negative,hard) EGP2 [Sentence] The apps <Mask> use. <Mask> The apps are hard to use. EGP3 [Sentence] The apps <Mask> use. (Root,(Quad, ...(Negative, hard) The apps are hard to use. EGP4 [Tree] The apps <Mask> use. (Root,(Quad, ...<Mask>,. . . ) (Root,(Quad, ...(Negative,hard) EGP5 [Sentence] The apps <Mask> use. (Root,(Quad, ...<Mask>,. . . ) The apps are hard to use. Table 1: Subtasks of Element-level Graph Pre-training. Subtask Input Subtask Prompt Review Tree EGP1 [Tree] The apps are hard to use. (Root,(Quad, ...<Mask>,. . . ) (Root,(Quad, ...(Negative,hard) EGP2 [Sentence] The apps <Mask> use. <Mask> The apps are hard to use. EGP3 [Sentence] The apps <Mask> use. (Root,(Quad, ...(Negative, hard) The apps are hard to use. EGP4 [Tree] The apps <Mask> use. (Root,(Quad, ...<Mask>,. . . ) (Root,(Quad, ...(Negative,hard) EGP5 [Sentence] The apps <Mask> use. (Root,(Quad, ...<Mask>,. . . ) The apps are hard to use. Table 1: Subtasks of Element-level Graph Pre-training. Table 1: Subtasks of Element-level Graph Pre-training. (Root, (Quad, ( Aspect (Software, apps ), ( Opinion (Negative, hard )))) Linearize Mask apps Software hard Negative Quad ROOT Aspect Opinion (Root, (Quad, ( Aspect (Software, apps ), ( Opinion (Negative, hard )))) (Software, apps ) ✓legitimate structure ✓sentiment elements Mask Figure 4: Example of element-level graph masking. 4.2 Task-level Graph Pre-training Table 2: Dynamic masking rate. Table 2: Dynamic masking rate. Inspired by the human-learning process we pro- pose a Task-level Graph Pre-training paradigm, whose subtasks follow the routine of human learn- ing procedure to learn to build the opinion tree from scratch. Specifically, we first decompose the quadruple extraction task into multiple sub- tasks. Each subtask corresponds to mapping the steps for manually building an opinion tree from scratch. The paradigm consists of six subtasks, four (Aspect, Opinion, Category, Polarity) of which extract sentiment structure as the fundamen- tal knowledge for building an opinion tree, the rest (Pair, Triple) target the intermediate state of the procedure with co-extraction. The subtasks and the corresponding steps of building can be found in Appendix A. In this case, we force the model to focus directly on irregular cases with a gradual pro- cess to build fundamental knowledge for OTG. The inputs of Task-level Graph Pre-training are similar to the previous paradigm, which would be a con- cat of a prompt and a sentence. Then the subtasks in Task-level Graph Pre-training paradigm can be given as shown in Figure 5. subtasks. The inputs would be a concat of a prompt, a sentence, and an opinion tree. The sentence and tree will be masked with different masking rates while the prompt illustrates the output tar- get, either the sentence or tree. For a given re- view s = (x1, x2, ...xn−1, xn) and linearized tree t = (t1, t2, ...tn−1, tn), We design the 5 subtasks in the Element-level Graph Pre-training paradigm, which can be found in Table 1. Among which, EPG1 and EPG4 are designed to help the model generate the complete tree t by adding text infor- mation while EPG2, EPG3 and EPG5 help the model to generate the full review s by adding the structural information. To further emphasize the interaction between the pre-training and finetune phases, we designed a dynamic masking rate for Element-level Graph Pre-training paradigms: a small masking rate is used in the initial phase, and then the masking rate increases with training rounds, so that at the end of pre-training, all partially masked pre-training tasks be very close to the finetune tasks (which can be considered as 100% masking rate), the specific masking rate is shown in Table 2. 4.1 Element-level Graph Pre-training The opinion tree is directly composed of subtrees that represent respective quadruples, this naturally decides the noteworthy information must locate within the aspect-level sentiment element instead of the other parts of the opinion tree, which could be other structure nodes. For instance, for a lin- earized opinion tree "(Root,(Quad,(Aspect (Soft- ware, apps),(Opinion (Negative, hard)", the in- discriminate masking may mask a sub-sequence "(Opinion (" that: 1) logically can not be reform into a valid structure due to the non-closing brack- ets. 2) contains nodes (e.g."Opinion" ) not included in the crucial sentiment elements. yi, hd i = ([H; hd 1, ..., hd i−1], yi−1) (2) (2) The conditional probability of the whole output sequence p(y\|x) is progressively combined by the probability of each step p(yi\|y<i, x): p(y\|x) = \|y\| Y i=1 p(yi\|y<i, x) (3) (3) where y<i = y1...yi−1, and p(yi\|y<i, x) are the probabilities over target vocabulary V . On the other hand, our Element-level Graph Pre-training paradigm masks aspect-level element nodes (including aspect term, opinion term, aspect category, and opinion polarity) in the opinion tree, as shown in Figure 4, the masked sequence "(Soft- ware, apps )" represent legitimate struct and covers core sentiment element only. If continuous nodes are masked, the corresponding sub-graph will be masked as a whole. The method can not only make sure the masked node are crucial sentiment ele- ments but also guarantee the corresponding sub- sequence is logically legitimate. The objective functions is to maximize the out- put linearized opinion tree XT probability given the review sentence XO. Therefore, we optimize the negative log-likelihood loss function: L = −1 \|τ\| X (XO,XT )∈τ log p(XT \|XO; θ) (4) (4) where θ is the model parameters, and (XO, XT ) is a (sentence, tree) pair in training set τ, then log p(XT \|XO; θ) = (5) = n X i=1 log p(xi T \|x1 T , x2 T , ...xi−1 T , XO; θ) (5) With the element-level graph mask strategy in- troduced above, we propose a set of pre-training = X log p(xi T \|x1 T , x2 T , ...xi−1 T , XO; θ) 3626 Input Aspect Opinion Category Polarity Pair Triple Prompt Sentence The apps are hard to use. [Aspect] The apps are hard to use. [Opinion] [Category] [Aspect] [Polarity] [Opinion] [Aspect] [Opinion] [Aspect] [Opinion] [Polarity] The apps are hard to use. The apps are hard to use. 4.1 Element-level Graph Pre-training The apps are hard to use. The apps are hard to use. (Root, (Aspect, apps)) (Root,(Opinion, hard)) (Root, Aspect (Software, apps)) (Root, Opinion (Negative, hard)) (Root, Quad (Aspect … hard)) (Root, Quad(Aspect … 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒)) Finetune The apps are hard to use. [Aspect] [Opinion] [Polarity][Category] (Root,(Quad, ...(Negative, hard) Pre-train Loop Output Figure 5: Subtasks of Task-level Graph Pre-training paradigm. Note the finetune task has been added into the pre-training phase. Figure 5: Subtasks of Task-level Graph Pre-training paradigm. Note the finetune task has been added pre-training phase. Figure 5: Subtasks of Task-level Graph Pre-training paradigm. Note the finetune task has been added into the pre-training phase. Epoch range Tree mask rate Text mask rate 0% ∼25% 0.25 0.15 25% ∼50% 0.3 0.15 50% ∼75% 0.35 0.15 75% ∼100% 0.4 0.15 Table 2: Dynamic masking rate. masked text from an opinion tree is unreasonable since opinion tree contains limited information as we discussed before. 4.2 Task-level Graph Pre-training Note our mask- ing rate obviously lower than previous work (Bai et al., 2022), that is because recovering a nearly all- 1https://www.yelp.com/dataset 2http://jmcauley.ucsd.edu/data/amazon/ 5 Experiments In this section, we introduce the datasets used for evaluation and the baseline methods employed for comparison. We then report the experimental re- sults conducted from different perspectives, and analyze the effectiveness of the proposed model with different factors. Particularly, we build two Large Language Model (LLM) baselines: ChatGPT5 is a sibling model to InstructGPT (Ouyang et al., 2022), which is trained to follow instruction in a prompt and pro- vide a detailed response. We ask it to generate all the sentiment elements from the input review sen- tences. LLaMA6 (Touvron et al., 2023) is a collec- tion of foundation language models, these models are trained on trillions of tokens, and have shown that it is possible to train state-of-the-art models us- ing publicly available datasets exclusively. We use LLaMA-7B, and fine-tune it on the ABSA dataset. 5.2 Main Results We compare the proposed method with several classification-based aspect-based sentiment anal- ysis models, including, DP (Qiu et al., 2011), JET (Xu et al., 2020), TAS-BERT (Wan et al., 2020) and Extract-Classify (Cai et al., 2021). In addi- tion, generative models are also compared, such as BARTABSA (Yan et al., 2021), GAS (Zhang et al., 2021b), Paraphrase (Zhang et al., 2021a),TODA (Hu et al., 2022), Seq2Path (Mao et al., 2022) and OTG (Bao et al., 2022).4. 3T5base, https://huggingface.co/transformers/ model_doc/t5.html 4We directly adopt the result from Bao et al. (2022) 5https://openai.com/blog/chatgpt. 6https://huggingface.co/docs/transformers/ main/model_doc/llama. 4.3 Joint Pre-training We use a joint pre-training method to com- bine the advantages of the Element-level Graph Pre-training paradigm and Task-level Graph Pre- training paradigms. In addition, we include the 3627 0% 10% 20% 30% 40% 50% 60% Basic One ToMany MonoImplicit BiImplicit Restaurant Laptop Figure 6: Statistic of regular and irregular situations of opinion trees. We employ T53 and fine-tune its parameters for our opinion tree generation model. We tune the parameters of our models by grid searching on the validation dataset. We select the best models by early stopping using the Accuracy results on the validation dataset. The model parameters are optimized by Adam (Kingma and Ba, 2015), the learning rate of pre-training and finetuning is 3e-5 and 1e-4 respectively. The batch size is 16. Our experiments are carried out with an Nvidia RTX 3090 GPU. The experimental results are obtained by averaging ten runs with random initialization. Figure 6: Statistic of regular and irregular situations of opinion trees. In evaluation, a quadruple is viewed as correct if and only if the four elements, as well as their combination, are exactly the same as those in the gold quadruple. On this basis, we calculate the Precision and Recall, and use F1 score as the final evaluation metric for aspect sentiment quadruple extraction (Cai et al., 2021; Zhang et al., 2021a). finetune task Finetune in the pre-train phase for narrowing the gap between two phases and avoid- ing overfitting. During pre-training, the model will be cyclically trained in the order of a loop started with the subtasks of the Element-level Graph Pre- training, followed by Task-level Graph Pre-training, the gradient will be updated after accumulating the loss in each epoch. After that, we save the model weights and finetune the model with finetune task Finetune. 5.1 Setting In this study, we use ACOS dataset (Cai et al., 2021) for our experiments. Following the setting from (Cai et al., 2021), we divide the original dataset into a training set, a validation set, and a testing set. In addition, we choose 20,000 sentences from Yelp1, and 20,000 sentences from the laptop domain in Amazon2 to pre-train the opinion tree generation model, the sentences are annotated by the OTG model without pre-training. As shown in Table 3, we find that generative models outperform previous classification-based methods and the structural generative method sur- Following the setting of Bao et al. (2023), we divide the quadruples into 4 types, apart from the basic situation, there are 3 irregular situations: One- to-Many, Mono-Implicit and Bi-Implicit. The statistic can be found in Figure 6. 3628 3628 Method Restaurant Laptop P. R. F1. P. R. F1. DP 0.3467 0.1508 0.2104 0.1304 0.0057 0.0800 JET 0.5981 0.2894 0.3901 0.4452 0.1625 0.2381 TAS-BERT 0.2629 0.4629 0.3353 0.4715 0.1922 0.2731 Extract-Classify 0.3854 0.5296 0.4461 0.4556 0.2948 0.3580 BARTABSA 0.5662 0.5535 0.5598 0.4165 0.4046 0.4105 GAS 0.6069 0.5852 0.5959 0.4160 0.4275 0.4217 Paraphrase 0.5898 0.5911 0.5904 0.4177 0.4504 0.4334 TODA 0.5904 0.6029 0.5966 0.4359 0.4367 0.4363 Seq2Path 0.6029 0.5961 0.5995 0.4448 0.4375 0.4411 ChatGPT 0.5014 0.3625 0.4207 0.4492 0.3123 0.3541 LLaMA 0.5963 0.6097 0.6029 0.4461 0.4392 0.4426 OTG 0.6138 0.6190 0.6164 0.4408 0.4381 0.4394 Ours 0.6486 0.6297 0.6390 0.4523 0.4523 0.4512 Table 3: Comparison with baselines. Table 3: Comparison with baselines. Method Restaurant Laptop Ours 0.6390 0.4512 - EGP 0.6339 0.4490 - TGP 0.6334 0.4463 - EGP& TGP 0.6164 0.4393 Table 4: Impact of pre-training paradigm. Method Restaurant Laptop OTG 0.6164 0.4394 + Indiscriminate 0.6287 0.4423 + Finetune 0.6211 0.4411 + EGP1, Finetune 0.6243 0.4413 + EGP1, Finetune EGP2, EGP3 0.6276 0.4421 + All EGPs 0.6334 0.4463 Ours 0.6390 0.4512 Table 5: Impact of subtasks in Element-level Graph Pre-training paradigm. Method Restaurant Laptop Ours 0.6390 0.4512 - EGP 0.6339 0.4490 - TGP 0.6334 0.4463 - EGP& TGP 0.6164 0.4393 passes non-structural methods, this indicates that semantic structure does contribute to quadruple extraction. Meanwhile, our proposed model out- performs all the previous studies significantly (p < 0.05), which has an advantage of 2.36% and 0.92% in Restaurant and Laptop domain respectively. 6 Analysis and Discussion Table 5: Impact of subtasks in Element-level Graph Pre-training paradigm. In this section, we first give some analysis and discussion to show the influence of Element-level Graph Pre-training (EGP) and Task-level Graph Pre-training (TGP) paradigms. After that, we will investigate our search over masking rate, the influ- ence of pre-training subtasks. avgerage drop of 0.52% while EGP’s cause 0.21%, this may due to the generalization and robustness being more effective than the structural association. 5.1 Setting The result shows that the proposed joint pre-training is effective in modeling tree structural constraints for generative model, while the large gap between pre-training and finetuning significantly encumbers previous systems. Furthermore, the results also in- dicate the effectiveness of our Element-level Graph Pre-training and Task Decomposition paradigms, which are used to unify the pre-train and finetune task with special task designs depending on the intrinsic characteristics of the opinion tree instead of treating it as a plain graph. Table 4: Impact of pre-training paradigm. 6.1 Influence of Different Factors It is worth noting that, the par- ticipation of finetune task Finetune demonstrates an obviously positive effect in both paradigms, which improves two domains with an average of 0.31%, this phenomenon gives us a conclusion that adding the finetune task in the pre-train phase is an effective solution for narrowing the gap between them. We then investigate the impact of subtasks in TGP paradigm. We remove the subtasks in the paradigms gradually. Table 7 shows the result for Task Decomposition paradigm: the contributions of subtasks stay in a similar scope, among which the Aspect surpasses others with a tiny gap, this may due to the lower implicit rate of aspect terms7.i Table 7: Impact of subtasks in Task-level Graph Pre- training paradigm. Table 7: Impact of subtasks in Task-level Graph Pre- training paradigm. this situation, there will be one intuitive question: Whether the element-level masking design does achieve a performance better than the indiscrimi- nate paradigm as we expect? In addition, all the subtasks are beneficial to ex- tract the opinion tree. It is worth noting that, the par- ticipation of finetune task Finetune demonstrates an obviously positive effect in both paradigms, which improves two domains with an average of 0.31%, this phenomenon gives us a conclusion that adding the finetune task in the pre-train phase is an effective solution for narrowing the gap between them. We investigate this question by employing abla- tion experiments. We first design an indiscriminate paradigm under similar settings, then we give the performance of using different paradigms in Ta- ble 5. As we can see, our element-level paradigm outperforms the indiscriminate paradigm, this re- sult shows the superiority of our element-level masking design, and also validated our motivation: for target graphs that contain limited knowledge like opinion tree, indiscriminate masking strategies would be sub-optimal and fine-grained masking should be adopted. 7 Conclusion In this study, we propose two novel pre-train paradigms for opinion tree generation, which are designed depending on the intrinsic characteristics of the opinion tree. Specifically, the Element-level Graph Pre-training paradigm abandons the strategy of capturing the complex structure but focuses di- rectly on the core elements. While the Task-level Graph Pre-training explicitly focuses on improving the generalization and robustness against irregu- lar quadruples with an easy-to-hard routine. Fur- thermore, we explore a dynamic masking rate and a cyclical train method for jointly combining the pre-training paradigms in order to bridge the gap between the pre-training and finetuning phases in modeling structural knowledge. We then investigate the impact of subtasks in EGP paradigm. We add the subtasks in paradigm gradually. As we can see in Table 5, the sub- task pair of EPG5 and EPG4 (+All EGPs) con- tributes the most to the performance (0.58% and 0.42% in each domain respectively), which aims to integrate the complementary information from both formations to generate text and tree respectively, indicating the significance of the complementary association. 7The average implicit rate of aspect term and opinion term is 22.63% and 24.19% respectively 6.1 Influence of Different Factors We first investigate the difference between the two paradigms, from Table 4 we can find, all the paradigms are beneficial to extract the opinion tree. Among which TGP paradigm’s contribution outper- forms EGP paradigm, the removal of TGP cause an Under the setting of our element-level masking de- sign for graph pre-train, previous graph-masking strategies can be classified into the indiscriminate paradigm, which means indiscriminately mask- ing random nodes and words in tree or text. In 3629 Method OTG Ours Basic 0.6517 0.6610 + 0.93% OneToMany 0.4503 0.4720 + 2.17% MonoImplicit 0.4035 0.4261 + 2.26% BiImplicit 0.4184 0.4341 + 1.57% Table 6: The average performance of different situations in Restaurant and Laptop domain. the basic situation. Thus we mimic the human learning procedure for building an opinion tree from scratch with Task-level Graph Pre-training to strengthen its fundamental knowledge. We investigate the paradigm’s effect by com- paring the model’s performance on each irregu- lar quadruple situation. As shown in Table 6 , our model’s improvement in all of the irregular classes surpasses the basic situation when com- pared with OTG. This result indicates that our pre- train method significantly improves the model’s performance with a burst in generalization and robustness against irregular sentiment quadruples, which accomplish the foundation for building an opinion tree and should be taken into consideration apart from improving the structural awareness. Table 6: The average performance of different situations in Restaurant and Laptop domain. Method Restaurant Laptop OTG 0.6164 0.4394 + Aspect 0.6251 0.4439 + Aspect, Opinion 0.6275 0.4447 + Aspect, Opinion, Pair, Triple 0.6294 0.4473 + Aspect, Opinion, category polarity, Pair, Triple 0.6294 0.4473 + Aspect, Opinion, category polarity, Pair, Triple finetune 0.6339 0.4490 Table 7: Impact of subtasks in Task-level Graph Pre- training paradigm. We then investigate the impact of subtasks in TGP paradigm. We remove the subtasks in the paradigms gradually. Table 7 shows the result for Task Decomposition paradigm: the contributions of subtasks stay in a similar scope, among which the Aspect surpasses others with a tiny gap, this may due to the lower implicit rate of aspect terms7. In addition, all the subtasks are beneficial to ex- tract the opinion tree. References Xuefeng Bai, Yulong Chen, and Yue Zhang. 2022. Graph pre-training for AMR parsing and generation. In Proceedings of the 60th Annual Meeting of the Association for Computational Linguistics (Volume 1: Long Papers), pages 6001–6015, Dublin, Ireland. Association for Computational Linguistics. Mengting Hu, Shiwan Zhao, Li Zhang, Keke Cai, Zhong Su, Renhong Cheng, and Xiaowei Shen. 2019. CAN: Constrained attention networks for multi-aspect sen- timent analysis. 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Association for Computational Linguistics. 6.3 Effect of Task-level Graph Pre-training As shown in Table 6, the OTG model obviously be short in its generalization and robustness against ir- regular sentiment quadruples when compared with 3630 Experimental results show that our proposed model can achieve state-of-the-art performance in ABSA. In addition, the results also validate that, for target graphs that contain certain knowledge like opinion tree, the improving strategy should be made based on the intrinsic characteristics of the structure instead of treating it as a plain graph. of the 59th Annual Meeting of the Association for Computational Linguistics and the 11th International Joint Conference on Natural Language Processing (Volume 1: Long Papers), pages 340–350, Online. Association for Computational Linguistics. Chenhua Chen, Zhiyang Teng, Zhongqing Wang, and Yue Zhang. 2022a. Discrete opinion tree induction for aspect-based sentiment analysis. 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Association for Computational Linguistics. Duyu Tang, Bing Qin, Xiaocheng Feng, and Ting Liu. 2016. Effective lstms for target-dependent sentiment classification. In COLING 2016, pages 3298–3307. Hugo Touvron, Thibaut Lavril, Gautier Izacard, Xavier Martinet, Marie-Anne Lachaux, Timothée Lacroix, Baptiste Rozière, Naman Goyal, Eric Hambro, Faisal Azhar, Aurelien Rodriguez, Armand Joulin, Edouard Grave, and Guillaume Lample. 2023. Llama: Open and efficient foundation language models. A Building Procedure In Task-level Graph Pre-training paradigm, the sub- tasks are set to follow the routine of building the opinion tree from scratch. For building an opinion tree manually, humans often learn to find funda- mental elements, such as aspects or opinions, fol- lowed by finding the corresponding classification result such as category and polarity to build a sin- gle quadruple unit, then composing multiple units to fulfill a more challenging goal, i.e., writing the entire opinion tree. Hai Wan, Yufei Yang, Jianfeng Du, Yanan Liu, Kunxun Qi, and Jeff Z. Pan. 2020. Target-aspect-sentiment joint detection for aspect-based sentiment analysis. In AAAI 2020, pages 9122–9129. Qianlong Wang, Zhiyuan Wen, Qin Zhao, Min Yang, and Ruifeng Xu. 2021. Progressive self-training with discriminator for aspect term extraction. In Proceed- ings of the 2021 Conference on Empirical Methods in Natural Language Processing, pages 257–268, 3632 Based on the process introduced, we design the subtasks in Task-level Graph Pre-training paradigm. Based on the process introduced, we design the subtasks in Task-level Graph Pre-training paradigm. Each subtask corresponds to mapping the steps for manually building an opinion tree from scratch. The paradigm consists of six subtasks: Aspect, Opinion, Category, Polarity,Pair and Triple. Their prompts and target graph can be found in Fig- ure 7. Among which, Aspect and Opinion focus on searching the basic elements of each quadruple: • Aspect: Extract all the aspect terms in the review in the form of a tree, Figure 7 (a). • Opinion: Extract all the Opinion terms in the review in the form of a tree, Figure 7 (b). Category and Polarity further explore the clas- sification results with the corresponding basic ele- ments: • Category: On the base of Aspect, extract the category classification result of the aspect terms in the review in the form of a tree, Fig- ure 7 (c). • Polarity: On the base of Opinion, extract the polarity classification result of the opin- ion terms in the review in the form of a tree, Figure 7 (d). Pair and Triple fulfill the mapping between quadruples. • Pair: On the base of Aspect and Opinion, map the corresponding aspect term and opin- ion term within a quadruple, Figure 7 (e). • Triple: On the base of Aspect and Polarity, map the corresponding aspect term and opin- ion term and polarity within a quadruple, Fig- ure 7 (f). A Building Procedure 3633 surface aspect apps aspect ROOT Aspect Aspect smooth opinion hard opinion ROOT Opinion Opinion Design category Software category ROOT Aspect Aspect surface aspect surface aspect smooth opinion Positive polarity ROOT Opinion hard opinion Negative polarity Opinion [Aspect] [Opinion] [Category][Aspect] [Polarity][Opinion] surface aspect smooth opinion Quad apps aspect hard opinion Quad ROOT Aspect Opinion Aspect Opinion [Aspect][Opinion] [Aspect][Opinion][Polarity] surface aspect Design category smooth opinion Positive polarity Quad apps aspect Software category hard opinion Negative polarity Quad ROOT Aspect Opinion Aspect Opinion [Tree] surface aspect smooth opinion Positive polarity Quad apps aspect hard opinion Negative polarity Quad ROOT Aspect Opinion Aspect Opinion (a) Aspect Extraction (b) Opinion Extraction (c) Category Classification (d) Polarity Classification (e) Pair Mapping (f) Triple Mapping (g) Finetune Figure 7: Building procedure of subtasks in Task-level Graph Pre-training. 3634 Aspect Opinion Opinion (a) Aspect Extraction hard opinion Quad (e) Pair Mapping Quad (f) Triple Mapping Quad Quad Aspect Opinion Aspect Opinion (g) Finetune Figure 7: Building procedure of subtasks in Task-level Graph Pre-training.
https://openalex.org/W2911247790	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0212437&type=printable	English	null	Frequency of reported pain in adult males with muscular dystrophy	PloS one	2,019	cc-by	9,190	RESEARCH ARTICLE OPEN ACCESS Citation: Jacques MF, Stockley RC, Bostock EI, Smith J, DeGoede CG, Morse CI (2019) Frequency of reported pain in adult males with muscular dystrophy. PLoS ONE 14(2): e0212437. https://doi. org/10.1371/journal.pone.0212437 Abstract a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Matthew F. JacquesID1, Rachel C. StockleyID2, Emma I. BostockID1, Jonathon Smith3, Christian G. DeGoede4, Christopher I. Morse1 Matthew F. JacquesID1, Rachel C. StockleyID2, Emma I. BostockID1, Jonathon Smith3, Christian G. DeGoede4, Christopher I. Morse1 1 Musculoskeletal Science & Sports Medicine Research Centre, School of Healthcare Science, Faculty of Science and Engineering, Manchester Metropolitan University, Manchester, United Kingdom, 2 School of Nursing, University of Central Lancashire, Preston, United Kingdom, 3 The Neuromuscular Centre, Winsford, Cheshire, United Kingdom, 4 Department of Paediatric Neurology, Royal Preston Hospital, Preston, United Kingdom * matthew.jacques@stu.mmu.ac.uk Editor: Denis Martin, Teesside University, UNITED KINGDOM All types of MD reported more VAS pain than CTRL, with 97% of all MD participants report- ing pain; however, no differences were reported between types of MD. The generalised body map approach identified more frequent pain in the shoulders of FSHD (93%) than other groups (13–43%), hips of DMD (87%) and LGMD (75%) than other groups (0–29%), and legs of all MD (64–78%) than CTRL (25%). The localised body map approach identified common areas of frequent pain across types of MD, posterior distal leg and distal back, as well as condition specific regions of frequent pain, for example posterior trapezius in FSHD, and anterior hip pain in DMD and LGMD. Received: August 3, 2018 Accepted: February 1, 2019 Published: February 14, 2019 Copyright: © 2019 Jacques 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. Methods Pain was assessed using 1) a whole body visual analogue scale (VAS) of pain, 2) a general- ised body map and 3) a localised body map. * matthew.jacques@stu.mmu.ac.uk Results Editor: Denis Martin, Teesside University, UNITED KINGDOM Introduction The purpose of this study was to present and compare pain between adult males with Duchenne (DMD), Becker’s (BMD), Limb-Girdle (LGMD) Facioscapulohumeral (FSHD) forms of Muscular Dystrophy (MD), and healthy controls (CTRL), using three different meth- ods of assessment. Introduction Muscular Dystrophy (MD) is an umbrella term for a set of myopathic conditions which are classi- fied by their genetic defect and characterised by their location, rate of progression and age of onset, of muscle weakness [1]. A large amount of research has focussed on describing the distribu- tion of weakness within the MDs associated with defects in the dystrophin-glycoprotein complex [2], namely Duchenne MD (DMD), Becker’s MD (BMD), Limb Girdle MD (LGMD) and Facios- capulohumeral MD (FSHD) [3–5]. Pain has been reported in each of the four MD’s described [6, 7], and shown to influence quality of life (QoL) [8, 9]. When pain has been reported in MD, it has typically been presented using whole body scales [10], or broad anatomical regions [11–13], which lack specificity and don’t reflect pain investigations in clinical assessment. The impact of pain on QoL within neuromuscular diseases is well established [8, 9]; specifi- cally, pain has been described as “serious, disabling and difficult to control” and consistently disturbing sleep, in adults with FSHD [14]. For clinicians, pain assessment results in detailed questioning of a patient, such as adopted previously [14], and is essential for the diagnostic process. Within the context of research, broad and generic methods are typically used, with pain presented using quantifiable and reliable methods, which are key for making population comparisons and determining the effectiveness of non-clinical interventions [15], such as exer- cise or manual therapy [16]. Given the differing pattern of impairments observed in MDs [17], detailed descriptions of where pain is most frequently observed within adults with MD is essential for understanding what is described as “the most disabling symptom” [14]. Within FSHD, pain has been quantified using pain diaries, analogue scales and body maps, with the predominance of MD pain research focusing on this condition [11, 14, 18]. In other MDs, pain has been presented using a Visual Analogue Scale (VAS) [19–22], a reliable and valid, single measure, providing a whole body score of pain [23]. A whole body measure of pain however, offers little information in relation to body regions [24]. By comparison, body maps can be used to localise pain [7, 25]; but within MD have only been applied using broad anatomical regions, such as the eight anatomical regions presented in FSHD [7, 11]. Conclusions Data Availability Statement: All data are contained with the manuscript and Supporting Information files. Using a single pain value (VAS), increased pain was reported by adults with MD compared to CTRL, with no clear differences between different MD groups, suggesting pain is symp- tomatic of MD. The use of the generalised body map approach, and to an even greater extent the localised body map approach, identified specific areas of frequent pain relevant to each individual condition. These results indicate that whist the commonly used general- ised approach can be used to identify broad anatomical regions, the localised approach Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 1 / 16 Pain in muscular dystrophy provides a more comprehensive understanding of pain, reflective of clinical assessment, and should be utilised in future research. provides a more comprehensive understanding of pain, reflective of clinical assessment, and should be utilised in future research. Introduction Body map presentation of pain may not be sufficient for describing the multifocal nature of pain experienced by adults with MD [14], having previously failed to distinguish anterior-posterior pain (e.g. DMD, LGMD and FSHD [10, 26, 27]), or proximal-distal pain (e.g. DMD, BMD and FSHD [6, 12]). In comparison, Bergsma, Janssen (27) used a more localised body map approach to compare pain in adults with DMD, LGMD and FSHD, however only assessed the upper limbs [27]. A whole-body localised pain map (e.g. 60 regions) requires no further work from the patient, but allows greater distinction in the presentation and description of pain within MDs [28], more reflective of clinical assessment. This research aimed to present and compare pain across four types of MD and a healthy control group using three methods: 1) whole body pain score using a visual analogue scale of pain, 2) frequency of reported pain using a generalised 8-point body map, and 3) frequency of reported pain using a localised 60-point body map. Anthropometrics All participants were weighed in a digital seated scales system (6875, Detecto, Webb City, Mo, USA). Slings, shoes, splints etc. were weighed separately and subtracted from the gross weight, when necessary. All participants stature was calculated as point to point of arm span (index finger, elbow, shoulder and across midline) to replicate the method used on non-ambulatory participants [32, 33]. A correction of 3.5% was applied to the raw data, consistent with regres- sion data from Caucasian males (all participants were of Caucasian ethnicity) in order to account for the known discrepancy between height and arm span measures [34]. Functional scales Upper and lower limb function was assessed using Brooke [35] and Vignos [36] scales, respec- tively [37]. The Brooke scale ranges from 1–6, with 1 meaning the participant is able to “start with arms at the sides and can abduct the arms in a full circle until the touch above the head”, and 6 “Cannot raise hands to the mouth and has no useful function of hands”. The Vignos scale ranges from 1–10, with 1 being able to “Walk and climb stairs without assistance”, and 10 “Confined to a bed”. Functional scales were performed by a chartered physiotherapist on the MD participants only, and are commonly used as functional assessment scales in MD [33, 38, 39]. Procedures All participants were tested in a single session; the MD groups were recruited from and tested at a neuromuscular clinic and the Control group (CTRL) were tested at the local university. 2 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 Pain in muscular dystrophy Only male participants were recruited to reflect the x-linked nature of DMD and BMD [29], as well as previous evidence of increased pain perception/reporting in females compared to males in CTRL [30, 31] and FSHD [11, 12] populations. Of the 75 participants initially recruited, all completed the required experimental procedures. Anthropometric measures were performed first, followed by Visual Analog Scale of pain and Body Map, which were com- pleted independently by participants, however the principal investigator was present to aid with any questions, or in some cases for participants with severely limited upper-limb func- tion, mark the forms upon participant instruction. Participants were also asked to report any currently prescribed pain medication. Ethical approval was obtained through the Department of Exercise and Sport Science Ethics Committee, Manchester Metropolitan University, and all participants signed informed consent forms prior to participation. Only male participants were recruited to reflect the x-linked nature of DMD and BMD [29], as well as previous evidence of increased pain perception/reporting in females compared to males in CTRL [30, 31] and FSHD [11, 12] populations. Of the 75 participants initially recruited, all completed the required experimental procedures. Anthropometric measures were performed first, followed by Visual Analog Scale of pain and Body Map, which were com- pleted independently by participants, however the principal investigator was present to aid with any questions, or in some cases for participants with severely limited upper-limb func- tion, mark the forms upon participant instruction. Participants were also asked to report any currently prescribed pain medication. Ethical approval was obtained through the Department of Exercise and Sport Science Ethics Committee, Manchester Metropolitan University, and all participants signed informed consent forms prior to participation. Visual analogue pain scale A Visual Analog Scale (VAS) of pain was used to quantify the level of whole body pain felt by participants over the last 7 days. VAS is a common method of pain assessment [40] and has been used in many conditions [19, 20, 41]. Participants were given a 10cm straight line, at one end “No Pain”, and the other “Worst Possible Pain”, and instructed to mark where on the scale best represented the pain they had felt over the last 7 days. The marks were measured and presented as distance (cm) from the “No Pain” end. Body maps A single non-segmented, blank schematic drawing of a body (Fig 1A; hereafter referred to as a body map), showing anterior and posterior aspects of the body, was given to participants who were asked to mark any location or area where they had experienced pain in the last 3 months [26]. This approach of freely identifying regions of pain using body maps has previously been described as a valid and reliable method of pain assessment [42]. The body map was then man- ually analysed using two segmentation methods. The first method segmented the body map using a broad eight anatomical region diagram (Fig 1B; hereafter referred to as “generalised”) 3 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 Pain in muscular dystrophy Fig 1. Example body map schematics. A = The example body map given to participants; B = Example of the generalised body map segmented for analysis; C = Example of the localised body maps segmented for analysis. h //d i /10 1371/j l 0212437 001 Fig 1. Example body map schematics. A = The example body map given to participants; B = Example of the generalised body map segmented for analysis; C = Example of the localised body maps segmented for analysis. https://doi.org/10.1371/journal.pone.0212437.g001 https://doi.org/10.1371/journal.pone.0212437.g001 consistent with that used by Moris et al. [12]. The second segmentation method used a 60 region diagram (Fig 1C; hereafter referred to as localised), to provide a more comprehensive assessment of pain [43]. The Generalised and Localised methods of segmentation and analysis are explained in more detail below. Generalised method. The first method of body map segmenting uses a generalised approach grouping of eight areas, namely: Head, Shoulders, Arms, Abdomen, Lower Back, Hips, Legs and Feet (Fig 1B). This segmentation method can be seen in Fig 1B and is replica- tive of that used previously [12]. Single marks in a segment of the body schematic are recorded, with percentage of the total sample reporting pain in respective anatomical regions presented. If more than one mark fell within the same body map segment, a single occurrence was recorded. Localised method. The second method of body map analysis is a localised method, devel- oped by the authors from previous methods [44, 45], to include sixty specific regions of the body e.g. Anterior Left Hand, Anterior Left Distal Arm, Anterior Left Elbow, Anterior Left Proximal Arm, Anterior Left Deltoid, Anterior Left Trapezius (Fig 1C). Body maps Participants’ pain in a relevant segment was recorded and presented as percentage of the total sample. Percentage of participants indicating pain using the localised method is presented using the 3D-Power Maps add-in on Excel (Professional Plus 2016, Microsoft, USA), where each segment is converted to X and Y co-ordinates relevant to a .Jpeg image of the body schematic diagrams. Percentages are presented topographically, with dark red indicating high frequency of reported pain, and blue indicating low frequency of reported pain, and white indicating no reported pain. Similar topographic methods of presentation have been used previously [27, 46, 47]. Pain in muscular dystrophy participants (pooled BMD, LGMD and FSHD) and participants using pain medication and those not using pain medication (pooled DMD, BMD, LGMD and FSHD) were performed using grouped data. Due to the progressive nature of MD, Spearman’s Rank co-efficient was used to determine associations of VAS Pain with age, Brookes scale and Vignos scale in indi- vidual types of MD. Chi-Squared was used to identify significant differences in frequency of pain medication and reported pain using the generalised body maps method. However, due to the more comprehensive approach to body segmentation and topographical analysis, no statis- tical analysis was performed upon the body maps analysed using the localised method, and have instead been described. Data are presented as mean (SD), or median (range) where relevant. Anthropometrics As seen in Table 1, CTRL were younger than FSHD (25%, P = .020) and older than DMD (32%, P = .012). DMD participants were younger than those with LGMD (43%, P < .001), BMD (42%, P < .001) and FSHD (49%, P < .001). CTRL were lighter than LGMD (19%, P = .018), while DMD were lighter than BMD (15%, P = .031), LGMD (25%, P = .001) and FSHD (15%, P = .028). There were no other differences in participant characteristics between any groups (P>0.05, Table 1). DMD participants scored 50–58% higher than BMD, LGMD, and FSHD on the Brooks scale (P < .05), with no other differences identified. FSHD participants scored 59–61% lower than DMD, BMD and LGMD on the Vignos scale (P < .05). Pain medi- cation was 30–50% more frequent in FSHD than all other groups (P < .05). Data presented sn mean ±SD, except Brooke and Vignos scales, which are presented as mean (range). DMD = Duchenne Muscular Dystrophy; BMD = Beckers Muscular Dystrophy; LGMD = Limb-Girdle Muscular Dystrophy; FSHD = Facioscapulohumeral Muscular Dystrophy; cm = centimetres; Kg = Kilograms B denotes significant difference from BMD Data presented sn mean ±SD, except Brooke and Vignos scales, which are presented as mean (range). DMD = Duchenne Muscular Dystrophy; BMD = Beckers Muscular Dystrophy; LGMD = Limb-Girdle Muscular Dystrophy; FSHD = Facioscapulohumeral Muscular Dystrophy; cm = centimetres; Kg = Kilograms B d t i ifi t diff f BMD Brooke and Vignos scales, which are presented as mean (range). DMD = Duchenne Muscular Dystrophy; BMD = Beckers -Girdle Muscular Dystrophy; FSHD = Facioscapulohumeral Muscular Dystrophy; cm = centimetres; Kg = Kilograms BMD https://doi.org/10.1371/journal.pone.0212437.t001 g LG denotes significant difference from LGMD F denotes significant difference from FSHD. Statistical analysis All analysis was performed using IBM Statistics v21 software. The critical level of statistical sig- nificance was set at 5%. Tests for parametricity were performed upon all variables. Age, Body Mass, VAS Pain, Brooke scale and Vignos scale were nonparametric and analysed using Krus- kall Wallis tests with post-hoc Mann-Whitney U pairwise used where appropriate. Height was parametric and compared between groups using a one-way ANOVA, with Tukey’s used for post-hoc comparison. Differences between VAS Pain of ambulant and non-ambulant PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 4 / 16 Pain VAS Pain. All MD groups scored higher than CTRL on the VAS Pain scale (P < .05, Fig 2). 97% of adults with MD reported to have experienced pain within a seven day period (Fig 2), comparably 18% of CTRL reported pain. No differences were reported between MD groups, or between ambulatory status using grouped data, for VAS Pain (P>0.05). Participants currently taking pain medication were found to have higher VAS pain than those not taking Table 1. Participants characteristics. Table 1. Participants characteristics. Table 1. Participants characteristics. DMD BMD LGMD FSHD CTRL n 15 18 12 14 16 Age (Years) 24.2 ±6.1 B,LG,F,C 42.4 ±13.5 41.6 ±11.7 47.1 ±11.1C 35.4 ±12.7 Stature (cm) 172.0 ±4.3 177.4 ±6.0 179.6 ±7.2 178.6 ±8.1 177.5 ±9.3 Mass (Kg) 73.1 ±14.6 B,LG,F 86.5 ±20.3 97.0 ±18.1C 86.0 ±11.2 81.1 ±18.2 Ambulant 0/15 10/18 3/12 10/14 16/16 Brooks 6.0 (5–6) B,LG,F 3.0 (1–4) 3.0 (2–6) 2.5 (1–4) - Vignos 9.0 (9)F 8.5 (2–9)F 9.0 (3–9)F 3.5 (1–9) - Pain Medication 3/15 F 2/18 F 1/12 F 7/14 C 0/16 Data presented sn mean ±SD, except Brooke and Vignos scales, which are presented as mean (range). DMD = Duchenne Muscular Dystrophy; BMD = Beckers Muscular Dystrophy; LGMD = Limb-Girdle Muscular Dystrophy; FSHD = Facioscapulohumeral Muscular Dystrophy; cm = centimetres; Kg = Kilograms B denotes significant difference from BMD LG denotes significant difference from LGMD F denotes significant difference from FSHD. C denotes significant difference from CTRL. https://doi.org/10.1371/journal.pone.0212437.t001 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 Pain in muscular dystrophy Fig 2. VAS Pain Box-Plots. = Outlier; DMD = Duchenne Muscular Dystrophy; BMD = Beckers Muscular Dystrophy; LGMD = Limb-Girdle Muscular Dystrophy; FSHD = Facioscapulohumeral Muscular Dystrophy; CTRL = Control; Kg = Kilograms; B denotes significant difference from BMD; LG denotes significant difference from LGMD; F denotes significant difference from FSHD. C denotes significant difference from CTRL. Fig 2. VAS Pain Box-Plots. = Outlier; DMD = Duchenne Muscular Dystrophy; BMD = Beckers Muscular Dystrophy; LGMD = Limb-Girdle Muscular Dystrophy; FSHD = Facioscapulohumeral Muscular Dystrophy; CTRL = Control; Kg = Kilograms; B denotes significant difference from BMD; LG denotes significant difference from LGMD; F denotes significant difference from FSHD. C denotes significant difference from Fig 2. VAS Pain Box-Plots. Pain = Outlier; DMD = Duchenne Muscular Dystrophy; BMD = Beckers Muscular Dystrophy; LGMD = Limb-Girdle Muscular Dystrophy; FSHD = Facioscapulohumeral Muscular Dystrophy; CTRL = Control; Kg = Kilograms; B denotes significant difference from BMD; LG denotes significant difference from LGMD; F denotes significant difference from FSHD. C denotes significant difference from CTRL. https://doi.org/10.1371/journal.pone.0212437.g002 pain medication (P < .05). Age was associated with VAS Pain in FSHD (r = .674, P = .008), however no other associations were reported between age, Brooke scal or Vignos scale and VAS Pain in any other MD (P>.05). pain medication (P < .05). Age was associated with VAS Pain in FSHD (r = .674, P = .008), however no other associations were reported between age, Brooke scal or Vignos scale and VAS Pain in any other MD (P>.05). PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 Pain in muscular dystrophy Table 2. Frequency of reported pain using a generalised method. DMD BMD LGMD FSHD CTRL Head (%) 7% 6% 0% 7% 0% Shoulders (%) 33% F 39% F 42% F 93% C 13% Arms (%) 0% LG, F 22% C 25% C 29% C 0% Abdomen (%) 0% 0% 0% 0% 0% Lower Back (%) 47% C 61% C 33% 43% C 13% Hips (%) 87% B, F, C 22% LG, C 75% F, C 29% C 0% Legs (%) 67% C 78% C 75% C 64% C 25% Feet (%) 0% 22%C 17% 14% 0% Table 2. Frequency of reported pain using a generalised method. DMD = Duchenne Muscular Dystrophy; BMD = Beckers Muscular Dystrophy; LGMD = Limb-Girdle Muscular Dystrophy; FSHD = Facioscapulohumeral Muscular Dystrophy; CTRL = Control B denotes significant difference from BMD LG denotes significant difference from LGMD F denotes significant difference from FSHD. C denotes significant difference from CTRL. https://doi.org/10.1371/journal.pone.0212437.t002 https://doi.org/10.1371/journal.pone.0212437.t002 CTRL group (BMD, P = 0.045; FSHD, P = 0.022). No differences were reported between groups for the frequency of reported pain in the hips region (P>0.05, Table 2). CTRL group (BMD, P = 0.045; FSHD, P = 0.022). No differences were reported between groups for the frequency of reported pain in the hips region (P>0.05, Table 2). g p q y p p p g Legs region. All MD groups reported 35–53% more frequent pain than the CTRL group (DMD, P = 0.048; BMD, P = 0.045; LGMD, P = 0.009; FSHD, P = 0.022) No differences were reported between MD groups for the frequency of reported pain in the legs region (P>0.05, Table 2). Legs region. All MD groups reported 35–53% more frequent pain than the CTRL group (DMD, P = 0.048; BMD, P = 0.045; LGMD, P = 0.009; FSHD, P = 0.022) No differences were reported between MD groups for the frequency of reported pain in the legs region (P>0.05, Table 2). Feet region. The BMD group reported 22% more frequent pain in the feet region than the CTRL group (P = 0.045). No other differences were reported between groups for the frequency of reported pain in the feet region (P>0.05, Table 2). Frequency of pain using generalised body maps Head region. No differences were reported between groups for the frequency of reported pain in the head region (P>0.05, Table 2). Shoulder region. The FSHD group reported 51–80% more frequent pain in the shoulder region than all other groups (DMD, P = 0.001; BMD, P = 0.002; LGMD, P = 0.005; CTRL, P<0.001). No other differences between groups in frequency of reported pain were identified for the shoulder region (P>0.05, Table 2). Hips region. The DMD group reported 58–87% more frequent pain in the hips region than BMD, FSHD and CTRL groups (BMD, P<0.001; FSHD, P = 0.002; CTRL, P<0.001). The LGMD group reported 53–75% more frequent pain in the hips region than BMD, FSHD, and CTRL groups (BMD, P = 0.004; FSHD, P = 0.018; CTRL, P = 0.034). Furthermore, BMD and FSHD groups reported 22% and 29%, respectively, more frequent pain in the hips than the 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 DMD = Duchenne Muscular Dystrophy; BMD = Beckers Muscular Dystrophy; LGMD = Limb-Girdle Muscular Dystrophy; FSHD = Facioscapulohumeral Muscular Dystrophy; CTRL = Control PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 Frequency of pain using localised body maps Duchenne muscular dystrophy. DMD showed the highest frequency of reported pain across the medial (33%) and lateral (87%) regions of the hip, as well as the posterior distal legs (67%). In addition, the posterior distal region of the back (47%), anterior proximal legs (47%) and posterior aspect of the trapezius (20%) areas are also noteworthy areas of pain frequency in adults with DMD (Fig 3). Beckers muscular dystrophy. The highest frequency of reported pain in the BMD group appear at posterior distal region of the legs (67%), as well as the distal (61%) and medial (50%) aspects of the back, respectively. The next highest frequency areas of pain reported appear at the posterior region of the neck (22%), the posterior proximal region of the legs (28%) and the anterior aspect of the knees (28%). Beyond these areas of pain frequency in the BMD group, less frequently reported pain (6–22%) seems relatively widespread across the rest body (Fig 3). Limb-Girdle muscular dystrophy. The highest frequency of reported pain in the LGMD group appears across the lateral (67%) and medial (25%) aspects of the anterior hip region, and the anterior proximal aspects of the lower leg, specifically the thigh (33%) and knee (42%) regions. Additional areas of frequently reported pain in the LGMD group are at the distal region of the back (33%). Other areas of frequently reported pain are across the superior and inferior limbs (8–17%), and the posterior regions of the proximal girdles (17–25%, Fig 3). Facioscapulohumeral muscular dystrophy. The FSHD group shows a high frequency of reported pain across the proximal posterior aspect of the back, specifically across the neck PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 7 / 16 Pain in muscular dystrophy (79%), trapezius (71%) and shoulder (43%) areas of participants, as well as the anterior aspect Fig 3. Body maps. Topographic presentation of reported pain frequency across four types of Muscular Dystrophy using a localised method. A = Anterior; B = Posterior; DMD = Duchenne Muscular Dystrophy; BMD = Beckers Muscular Dystrophy; LGMD = Limb-Girdle Muscular Dystrophy; FSHD = Facioscapulohumeral Muscular Dystrophy; CTRL = Control. https://doi.org/10.1371/journal.pone.0212437.g003 Fig 3. Body maps. Topographic presentation of reported pain frequency across four types of Muscular Dystrophy using a localised method. A = Anterior; Fig 3. Body maps. Topographic presentation of reported pain frequency across four types of Muscular Dystrophy using a localised method. Pain in muscular dystrophy Control. Areas of reported pain in the CTRL group are anterior right deltoid (13%), ante- rior right knee (13%), anterior left knee (6%), anterior left ankle (6%) and distal back (13%). Whole body pain Pain is reported as symptomatic of FSHD [14], and reported as a frequent problem in the other discussed MD [7, 27], the present study shows that 97% of adults with MD reported pain, with no differences in pain reported between types of MD. By comparison 18% of CTRL reported pain, consistent with previous reports of pain in the general population of 15–20% [48, 49]. The VAS pain score of the FSHD group in the present study, while lower, appears comparable with previous reports of adults with FSHD with and without (pre-established) chronic pain [11, 20]. By comparison, VAS pain scores in adults with DMD is higher than that reported previously in adolescents with DMD [6]. Although no association was found, an increase in pain with age in DMD may be the long-term effect of wheelchair-use or progres- sion of the condition through adolescence to adulthood. Pain scores are however comparable to those previously reported using the single VAS in adults with MD [10, 20], suggesting that the present adults with MD are consistent with expected, despite recruitment from a neuro- muscular centre (see “limitations” below). The similarity in VAS pain scores between the MDs are presented with a very wide range of variance. This likely reflects the varied clinical presen- tation both between and within the MD types. It should be noted that no associations were identified with functional scales, suggesting pain at the whole body level may not be sensitive to the specific functional impairments and progression that describe the conditions. Further- more, the variability presented in DMD of VAS pain is comparably smaller than the three other types of MD in the present study, as well as previous reports of pain DMD when using broader functional abilities of young men (aged 11–21) [50] or combining types of MD (DMD and BMD) [6, 7]. The use of a homogenous sample of adults (aged 18+, all long-term wheel- chair users and consistent Brookes and Vignos scores) with DMD may explain the reduced variability in this condition compared to previous research. Discussion This research presents rating and frequency of reported pain in adults within four types of MD and a CTRL comparison. MD groups showed increased pain compared to CTRL, however no differences were identified between types of MD. Using a localised pain body map, specific regions of high frequency pain were identified: lateral and medial aspects of the hips and poste- rior distal region of legs in DMD; distal back and posterior distal region of the legs in BMD; lateral and medial aspects of the hips in LGMD; posterior aspects of the trapezius in FSHD. PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 Frequency of pain using localised body maps A = Anterior; B = Posterior; DMD = Duchenne Muscular Dystrophy; BMD = Beckers Muscular Dystrophy; LGMD = Limb-Girdle Muscular Dystrophy; FSHD = Facioscapulohumeral Muscular Dystrophy; CTRL = Control. https://doi.org/10.1371/journal.pone.0212437.g003 https://doi.org/10.1371/journal.pone.0212437.g003 (79%), trapezius (71%) and shoulder (43%) areas of participants, as well as the anterior aspect of the trapezius’ (43%). In addition, the two other main areas of frequent pain in the condition appear at the distal region of the back (43%) and the distal region of the lower limbs, specifi- cally the calves (43%). Other notable areas include pain at the lateral aspects of the hips (29%) and along upper extremities (7–21%, Fig 3). (79%), trapezius (71%) and shoulder (43%) areas of participants, as well as the anterior aspect of the trapezius’ (43%). In addition, the two other main areas of frequent pain in the condition appear at the distal region of the back (43%) and the distal region of the lower limbs, specifi- cally the calves (43%). Other notable areas include pain at the lateral aspects of the hips (29%) and along upper extremities (7–21%, Fig 3). 8 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 Limb-Girdle muscular dystrophy Adults with LGMD report a high frequency of pain around the limb-girdle regions, particu- larly the pelvic girdles, which appears consistent with the classic areas of muscle weakness [17] and previous identification of the shoulders as a specific area of pain [27]. In addition, the non-ambulatory nature of the sample in the present study likely exacerbates pain around the pelvic girdles and lower limbs. Pain identified within the upper limbs is likely caused by muscle weakness from the shoulder girdles, which could be exacerbated by unstabilised, yet some maintenance of arm function (Brooke score of 3) in this population. Pain in muscular dystrophy anterior (43%) and posterior (71%) aspects. Therefore, while the generalised approach offers an effective overview of pain, it fails to distinguish key aspects of pain presentation. In the subsequent sections, we provide an overview of pain described using the localised pain map for each MD condition. Where possible, we have referenced previous aetiological In the subsequent sections, we provide an overview of pain described using the localised pain map for each MD condition. Where possible, we have referenced previous aetiological factors associated with pain. As we have mentioned, pain is multi-factorial in nature and unlikely to be due to one single factor, therefore the following is an overview of each condition based on the highest incidence of reported pain and is not meant as an exhaustive description of the aetiology. Duchenne muscular dystrophy The present study shows a high frequency of pain across the hip and lower back, possibly due to the imposed body position from long-term power-wheelchair use [51]. The long-term use of power-wheelchairs likely exacerbates contractures associated with DMD by further limiting muscle lengthening, leading to pain around the hips and calves. Furthermore, pain in the pos- terior distal leg could be attributed to myofascial pain syndrome, whereby taut regions within the muscle compartment, possibly caused by increased calf size and contractures, could mani- fest itself as pain [52, 53]. In addition, other factors such as the specific sitting position, scolio- sis, and foot deformity have all previously been identified as important parts of long-term management in DMD and could contribute to pain [54, 55]. Beckers muscular dystrophy Adults with BMD reported pain over numerous areas of the body, which is consistent with the whole body nature of this condition, as presented previously [17]. Frequent areas of pain appear posteriorly, especially though the spine and calf areas. Increased pain in the calves may be similar to the myofascial pain syndrome noted in the DMD group, whereby increased pain is associated with oedema, consistent with pseudohypertrophy in the gastrocnemius in adults with BMD [32]. Within the spine, impaired muscular stabilisation, likely due to reduced mus- cle strength, has been previously associated with lower back pain and could be a contributing factor in this population [56]. In addition, the frequency of neck pain could be associated with increased sitting time [57, 58]. Body maps method The current study presents frequency of reported pain using a generalised method of grouping eight anatomical areas of the body, and a novel method of topographic presentation of pain using sixty anatomical regions. Previous research using body maps have typically grouped ana- tomical regions [12, 21, 26], consistent with the generalised body map method used in the present study. This approach however, generalises pain across regions, when in fact, as evi- denced in the current study and noted previously, pain can be multifocal [14], with large dif- ferences identified between localised areas of pain within the same anatomical region. For example, using the generalised approach, leg pain was reported in 67% of DMD, this was dis- tinguished, using the localised approach, as almost entirely posterior, specifically posterior-dis- tal leg pain (67%), rather than posterior-proximal leg pain (7%). Similarly, the generalised approach reported pain in the shoulders (93%) of FSHD, failing to distinguish between PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 9 / 16 Limitations Recall methods have been criticised previously for a lack of sensitivity, however are frequently used in cross-sectional, longitudinal and intervention studies [11, 64, 65]. Recall methods may not be sensitive enough to identify minor pain, but identify the most clinically significant regions, which would impact quality of life [9, 12, 20, 26]. The VAS scale specifically, is limited in the information it gives, but is essential as part of broader clinical assessment, and is a reli- able method of acute and chronic pain assessment [66–68]. While future studies should look at methods of pain diaries to gain further insight into the onset and implications of pain [14]. All MD participants from the present study were recruited from a neuromuscular clinic. The recruitment of participants from a health centre has two possible contrasting, implications for reported pain in the present study. Firstly, the influence of a long-term management plan focussing on condition and pain management may mean pain in a non-managed sample could be higher, however VAS pain scores were comparable with previous [6, 11]. Secondly, participants are part of long-term condition management and may be better at, and more comfortable, reporting pain, as difficulties in reporting pain have previously been identified [50]. The results and their interpretation, are therefore presented (as always) within the con- straints of the participant demographics. This study recruited male participants only, due to DMD and BMD both being x-linked conditions, and previous research in CTRL [30, 31] and FSHD [11, 12] populations identifying increased prevalence of pain perception/reporting in females compared to males. Therefore, to allow comparisons between types of MD, only males were recruited. Within the participant demographic data we have also reported pain medication use, which was, as expected, shown to be associated with higher reports of VAS pain. It would be unethical to withdraw medica- tion to identify the extent of the influence of medication on the present data. Future research is required to identify the possible influence of medication on pain in adults with MD, and sex differences in adults with FSHD and LGMD on reported pain. The sample sizes in this study are relatively small compared to some previous pain in MD research [11, 12], however are consistent with other previous research of pain using multiple types of MD [7, 50]. Facioscapulohumeral muscular dystrophy Pain in FSHD was reported around the scapula region, largely consistent with the classical areas of weakness [17, 27], and is similar to “shoulder” pain previously reported in FSHD, using a seven-region pain map [12]. Specific to the localised approach, in the present study we observed particularly high frequency of pain around the posterior aspect of the neck and trape- zius, and a high frequency of pain in the calf areas. Scapular winging is seen as a common fea- ture of FSHD [59, 60], and has been associated with pain in non-FSHD groups [61–63]. 10 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 Pain in muscular dystrophy Similar to the LGMD body maps, the extent of pain goes beyond the classic areas of predomi- nant weakness [17], but reflects the whole body nature of these conditions. The locality of fre- quently reported areas of pain and muscle weakness suggests work should be done to maintain and improve muscle strength in these areas, particularly for postural control around the neck, spine and scapula. Strength training and Albuterol interventions have previously shown no impact on pain in adults with FSHD [22], however these interventions were based on strength training of elbow flexors and ankle dorsi-flexors, two muscle groups not identified in the pres- ent study as areas of frequently reported pain. PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 S1 File. Basic Information. S1 File. Basic Information. (XLSX) Limitations The larger sample size adopted previously in FSHD ([11, 12], n = 398 and 104, respectively) were conducted through postal questionnaire in males and females, rather than a face-to-face format conducted in the present study. It should be noted that our data from FSHD is largely consistent with that reported previously, based on broader VAS and 8–10 region pain body maps. The smaller sample sizes in the present study however may explain the lack of associations identified between functional scales and VAS Pain, as previous larger studies have shown relationships between functional scales and VAS Pain [10]. The localised pain maps adopted in the present study, although conducted in smaller participant groups from face-to-face recall, better reflects clinical practice, and has identified more specific regions, not previously described in larger participant groups. We therefore acknowledge that the current study is not providing an exhaustive description of pain in adults with MD, but do PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 11 / 16 Pain in muscular dystrophy however, propose that the localised body map method from this study should be used as a tool for subsequent larger studies. Clinical implications The consistent VAS rating of pain across the four types of MD in the present study suggest that rather than being symptomatic of just FSHD [14], pain is more likely symptomatic of MD as a whole. Although no differences were observed between types of MD, the wide variations in reported VAS pain across all types of MD suggests a greater need for investigation into indi- vidual types. Despite comparable VAS pain scores between types of MD, the limitations of this whole-body method are evident as each type of MD presented with specific areas of frequent pain relevant to its own condition, as observed using the localised body map approach. The presentation of pain through pain maps, appears (at least superficially) to be largely consistent with the areas of muscle weakness proposed previously [17]. Specific locations of frequently reported pain around the hip have been identified in the largely non-ambulant populations (DMD and LGMD). The authors have suggested that the muscle shortening positions imposed by wheelchairs could be a cause of this high frequency [51]. The aetiology of pain outlined in the present study (such as prolonged sitting) is speculative. Future research is required to fur- ther understand pain within types of MD. While there may be evidence from the presented body maps that frequent areas of pain could be associated with areas of weakness and the use of wheelchairs, identification of triggers for episodes of pain are required. The presentation of pain using the localised body map approach appears to be more reflective of clinical practice than generic methods typically used in research, providing greater insight into pain. By com- parison, the whole body measure of VAS pain couldn’t identify any differences between types, while the generalised body map approach is unable to identify differences found between ante- rior-posterior or proximal-distal pain. Therefore, the localised body map approach is recom- mended for future pain assessment research in MD, as a method reflective of clinical practice. Conclusions In conclusion, pain appears as a common characteristic in MD with no differences identified in pain rating between the four types of MD in the present study. Using a localised body map approach however specific areas of frequent pain became evident, which appear to be consis- tent with previous work of areas of predominant muscle weakness in these conditions; how- ever, the authors have noted the possible influence of long-term wheelchair use on location of pain. The novel aspect of this research has been the identification of localised areas of pain, compared to typically presented generalised areas of pain, and propose this method for future research. Project administration: Matthew F. Jacques, Christopher I. Morse. Project administration: Matthew F. Jacques, Christopher I. Morse. Resources: Matthew F. Jacques, Jonathon Smith, Christopher I. Morse. Software: Matthew F. Jacques, Christopher I. Morse. Supervision: Rachel C. Stockley, Emma I. Bostock, Jonathon Smith, Christian G. DeGoede, Christopher I. Morse. Validation: Matthew F. Jacques, Christopher I. Morse. Validation: Matthew F. Jacques, Christopher I. Morse. Visualization: Matthew F. Jacques, Christian G. DeGoede, Christopher I. Morse. Writing – original draft: Matthew F. Jacques, Christopher I. Morse. Writing – review & editing: Matthew F. Jacques, Rachel C. Stockley, Emma I. Bostock, Jona- thon Smith, Christian G. DeGoede, Christopher I. Morse. Author Contributions Conceptualization: Matthew F. Jacques, Emma I. Bostock, Jonathon Smith, Christian G. DeGoede, Christopher I. Morse. Data curation: Matthew F. Jacques, Christopher I. Morse. Data curation: Matthew F. Jacques, Christopher I. Morse. Formal analysis: Matthew F. Jacques, Christopher I. Morse. Formal analysis: Matthew F. Jacques, Christopher I. Morse. 12 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0212437 February 14, 2019 Pain in muscular dystrophy Funding acquisition: Christopher I. Morse. 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https://openalex.org/W2553917251	https://he02.tci-thaijo.org/index.php/bkkmedj/article/download/218116/151126	English	null	Accuracy of Stress Echocardiography in Detecting Ischemic Heart Disease, Experience at the Bangkok Heart Hospital 2012	THE BANGKOK MEDICAL JOURNAL	2,013	cc-by	2,400	Original Article Original Article Original Article The Bangkok Medical Journal Vol. 6; September 2013 ISSN 2287-0237 (online)/ 2287-9674 (print) Accuracy of Stress Echocardiography in Detecting Ischemic Heart Disease, Experience at the Bangkok Heart Hospital 2012 Angkasuwapala K, MD email : ped@post.com OBJECTIVES: Stress echocardiography is a good test for detecting ischemic heart disease. The sensitivity, speciÀ city and accuracy should be veriÀ ed. This test was compared to standard coronary angiographic results. This is the À rst study by Bangkok Heart Hospital’s echocardiography lab to verify the effectiveness of stress echocardiography. MATERIAL AND METHODS: This study was a retrospective study that reviewed data from records. The 149 selected cases underwent stress echocardiography and a coronary angiogram (within 2 months after the stress echocardiography) between 1st January and 31st December 2012. The sensitivity, speciÀ city and accuracy were assessed. Angkasuwapala K, MD email : ped@post.com RESULTS: The overall sensitivity, speciÀ city, and accuracy were 81.98%, 42.10%, and 71.81% respectively. There was a variation of sensitivity, speciÀ city and accuracy in each day of the week. There were many factors such as number of cases, reporting physicians, technically poor studies (including poor echogenicity of subject), technician experience, abnormal wall motion at rest, atrial À brillation and bundle branch block. The sensitivity ranged from 57.14 % to 92.3%. The speciÀ city ranged from 16.67% to 70%. The accuracy ranged from 50.0 % to 86.67 %. Kitiporn Angkasuwapala, MD1 Uthaiwan Chanyeam, BS, RT2 Kitiporn Angkasuwapala, MD1 Uthaiwan Chanyeam, BS, RT2 Keywords: accuracy, stress echocardiography, sensitivity, speci¿ city, ischemic heart disease CONCLUSION: Stress echocardiography is a good test for evaluating ischemic heart disease. Our echo lab had lower sensitivity, speciÀ city and accuracy than other previous studies. The limitation of this study was that it was a retrospective study. But it showed our routine work. We hope this will help our echo lab to improve the quality of its stress echocardiography testing. 1 Heart Clinic, Bangkok Heart Hospital, Bangkok Hospital Group, Bangkok, Thailand. 2 Non Invasive Department, Bangkok Heart Hospital, Bangkok Hospital Group, Bangkok, Thailand. S S tress testing is the most frequent investigation to diagnose ischemic heart disease. The most common technique is electrocardiography during a treadmill stress period. Electrocardiography has limitations because there are many artifacts from movement. There are many techniques to improve the detection of ischemic heart disease, such as magnetic resonance imaging (MRI), thallium scintigraphy,1 radionuclide ventricu- lography and echocardiography. The dobutamine and exercise stress echocardiography test were shown to detect coronary artery disease.2-4 Address Correspondence to author: Heart Clinic, Bangkok Heart Hospital, 2 Soi Soonvijai 7, New Petchburi Road, Bangkapi, Huaykwang, Bangkok 10310, Thailand. Accuracy of Stress Echocardiography in Detecting Ischemic Heart Disease, Experience at the Bangkok Heart Hospital 2012 E-mail: ped@post.com Address Correspondence to author: Heart Clinic, Bangkok Heart Hospital, 2 Soi Soonvijai 7, New Petchburi Road, Bangkapi, Huaykwang, Bangkok 10310, Thailand. E-mail: ped@post.com *Address Correspondence to author: Heart Clinic, Bangkok Heart Hospital, 2 Soi Soonvijai 7, New Petchburi Road, Bangkapi, Huaykwang, Bangkok 10310, Thailand. E-mail: ped@post.com In the Bangkok Heart Hospital, we use treadmill or dobutamine in this test depending on the patient’s status. The dobutamine is used in patients who cannot exercise. We often use echocardiography as a technique to diagnose ischemia. Received March 27, 2013. Revision received June 10, 2013. Accepted after revision June 16, 2013. Bangkok Med J 2013;6:17-20 E-journal: http//www.bangkokmedjournal.com Received March 27, 2013. Revision received June 10, 2013. Accepted after revision June 16, 2013. Bangkok Med J 2013;6:17-20 E-journal: http//www.bangkokmedjournal.com This study retrospectively shows the clinical use of stress echocardiography for diagnosis of coronary artery disease to detect ischemia in patients with known or suspected ischemic heart 17 17 The Bangkok Medical Journal Vol. 6; September 2013 ISSN 2287-0237 (online)/ 2287-9674 (print) Angkasuwapala K and Chanyeam U disease. This study will show the sensitivity, speciÀ city, and accuracy of stress echocardiography in our non-invasive department. It will help us to improve the quality of stress echocardiography in our echo lab. The treadmill was used with Bruce protocol. Patients walked at least 85% of predicted heart rate. If they could not reach 85% of predicted heart rate (the test was labeled as an inadequate test), they were excluded. Echocardiographic images were scanned before and after the treadmill test. Electrocardiogram and blood pressure were monitored during exercise. 18 The Bangkok Medical Journal Vol. 6; September 2013 ISSN 2287-0237 (online)/ 2287-9674 (print) The Bangkok Medical Journal Vol. 6; September 2013 Results The calculated values shown in Table 2 detail each day of the week for a week. The sensitivity of this echo lab was 81.98%. The speciÀ city was 42.1%. The accuracy was 71.81%. There were no cases of true negative on Day 3, so the speciÀ city and negative predictive value was 0 (presented as NA) (Table 2). Coronary angiogram results: The positive angio- gram was deÀ ned as 50% diameter stenosis. Coronary artery disease was present in 111 patients. Thirty-eight cases were single vessel disease. There were 73 cases with multivessel disease. The left main lesion was deÀ ned as multivessel disease. Materials and Methods Case studies: The patients underwent a stress echo- cardiography test in the non-invasive department of the Bangkok Heart Hospital between 1st January and 31st December 2012. There were 2,035 patients who underwent stress echocardiography. Their ages ranged from 17 to 96 years. There were 1,284 (63.1 %) men and 751 (36.9 %) women. There were 149 cases that had undergone stress echocardiography testing before undergoing a coronary angiogram (CAG) within 2 months. These patients were the subject of this study. All cases were reviewed retro- spectively from data recorded. Eighty-three cases received dobutamine stress echo- cardiography. Dobutamine was step infused at the doses of 10, 20, 30, 40 g/kg/min every 2 minutes and every 1 minute to capture echocardiography images. The test was stopped when a new wall motion presented or reached 85% of predicted heart rate. If a 40 g/kg/min dose could not achieve the target heart rate, atropine was injected and an image was captured at 85% of predicted heart rate or higher.5 Echocardiography: 2D echocardiography was recorded with a parasternal short axis, apical 4 chamber 2 chamber, and 3 chamber views. The echocardiographic machines used were GE vivid 7 and vivid E9. The transducer frequency range was 1.5-2.3 MHz. Echocardiographic wall motion was graded as normal, hypokinesia, akinesia or dyskinesia. A 16-segment model (Figure 1) was used for grading wall motion. A coronary angiogram was performed within 2 months after the stress echocardiography testing. SigniÀ cant nar- rowing was determined as 50 % diameter stenosis of the major coronary artery or its major branch. Stress echocardiography protocols: Sixty-six cases performed the treadmill stress echocardiography testing. Figure 1: 16-segment model Figure 1: 16-segment model Figure 1: 16-segment model The Bangkok Medical Journal Vol. 6; September 2013 18 g ISSN 2287-0237 (online)/ 2287-9674 (print) Accuracy of Stress Echocardiography in Detecting Ischemic Heart Disease, Experience at the Bangkok Hear Discussion Exercise (treadmill) stress echocardiography was used in 66 cases. Dobutamine stress echocardiography was used in 83 cases. Positive stress echocardiography was found in 113 cases, 91 cases were true positive and 22 cases were false positive. There were 36 cases with negative stress echocardiography and 16 cases were true negative and 20 cases were false negative (Table 1). Stress echocardiography has an advantage over stress electrocardiography (ECG). This test evaluated baseline echocardiography before the stress test. Left ventricle (LV) function, regional wall motion, heart valves, chambers and other abnormalities were detected before the stress test. The indication of stress echocardiography is an evaluation of chest pain, diagnosis of coronary artery disease, detection of viable myocardium, preoperative evaluation, evaluation of status of coronary revascularization. Sensitivity varied from 54% to 96% and speciÀ city varied from 50% to 100% 6-8 in previous studies. Sensitivity was calculated by (true positive) (true positive + false negative) SpeciÀ city was calculated by (true negative) (true negative + false positive) Accuracy was deÀ ned by (true positive + true negative) (total cases) Positive predictive value was (true positive) (true positive + false positive) Negative predictive value was (true negative) (true negative + false negative) Sensitivity was calculated by (true positive) (true positive + false negative) SpeciÀ city was calculated by (true negative) (true negative + false positive) Accuracy was deÀ ned by (true positive + true negative) (total cases) Positive predictive value was (true positive) (true positive + false positive) Negative predictive value was (true negative) (true negative + false negative) In this study, the overall sensitivity was 81.98%, and this was lower than previous studies.6-9 The reason might be that this study was retrospective. But this might reÁ ect real life, and routine work. The speciÀ city was very low because there were more false positive cases than true negative cases. Perhaps there was a bias on the part of the physician wanting to recommend that the patient should have an angiogram which resulted in more false positive cases. Some cases did not have an angiogram when there was a negative stress echocardiography, especially on Day 3 ( no true negative test). Because of the low true negative and high false positive, the accuracy was too low. g y Bold = highest, Underlined = lowest, PPV = positive predictive value, NPV = negative predictive value 19 The Bangkok Medical Journal Vol. 6; September 2013 ISSN 2287-0237 (online)/ 2287-9674 (print) Discussion Table 1: Comparision of Stress Echocardiography and Coronary Angiogram Stress echocardiography Coronary Angiogram Positive Negative Positive 91 (true positive) 22 (false positive) Negative 20 (false negative) 16 (true negative) Table 1: Comparision of Stress Echocardiography and Coronary Angiogram Table 2: The calculated values show sensitivity, speci¿ city, accuracy for each day of the week (Unordered) NA = not available , because there was no true negative case on Wednesday. Bold = highest, Underlined = lowest, PPV = positive predictive value, NPV = negative predictive value n Sensitivity Speci¿ city Accuracy PPV NPV All 149 81.98% 42.10% 71.81% 80.53% 44.44% Day 1 30 92.0% 60.0% 86.67% 92.0% 60.0% Day 2 32 72.73% 70.0% 71.87% 84.2% 53.85% Day 3 13 70.0% NA 53.85% 70.0% NA Day 4 15 72.73% 50.0% 66.67% 80.0% 40.0% Day 5 10 57.14% 33.33% 50.0% 66.67% 25.0% Day 6 32 92.30% 16.67% 78.12% 82.76% 33.33% Day 7 17 90.0% 28.57% 64.70% 64.28% 66.67% Table 2: The calculated values show sensitivity, speci¿ city, accuracy for each day of the week (Unordered) 19 The Bangkok Medical Journal Vol. 6; September 2013 ISSN 2287-0237 (online)/ 2287-9674 (print) Angkasuwapala K and Chanyeam U There was variation in the sensitivity, speciÀ city and accuracy on each day of the week examined. The most sensitive day was Day 6. The most speciÀ c day was Day 2. The most accurate day was Day 1. There were many factors that inÁ uenced sensitivity, speciÀ city and accuracy. For example, the number of cases, the reporting physician, technically poor studies (including poor echo- genicity of subject), technician experience, abnormal wall motion at rest, atrial À brillation and bundle branch block were important factors that had an effect on each day. atrial À brillation, and bundle branch block. The quantitative evaluation was suggested but this is time consuming, and requires good technical studies (good images, good equipment, good views, good operators) and needs experienced reporters. Conclusion Stress echocardiography is a good test to evaluate ischemic heart disease but it needs good technical studies and experienced reporters. This study was a retrospective study that showed the routine work of our echo lab. This study showed low sensitivity, speciÀ city and accuracy in our lab compared to previous studies (from another site). This study had limitations but it has shown an opportunity to improve the accuracy, sensitivity, and speciÀ city of the test in the lab. There are many quantitative evaluations, hardware, and software to help. The accuracy, sensitivity, and speciÀ city of stress echocardiography will continue to improve. The Bangkok Medical Journal Vol. 6; September 2013 ISSN 2287-0237 (online)/ 2287-9674 (print) Limitation This study was a retrospective study. There was some bias from the physician, these results are not randomized. There were many cases that underwent a stress echocar- diography test but only selected cases had anangiogram. There was a subjective interpretation that depends on the reporter’s experience. The lack of quantitative evaluation reduced the accuracy, sensitivity, andspeciÀ city of the test. Especially in the case of abnormal wall motion at rest, References 1. Kotler TS, Diamond GA. Exercise thallium-201 scintig- raphy in the diagnosis and prognosis of coronary artery disease. Ann Intern Med 1990;113:684-702. 1. Kotler TS, Diamond GA. Exercise thallium-201 scintig- raphy in the diagnosis and prognosis of coronary artery disease. Ann Intern Med 1990;113:684-702. 6. Cohen JL, Greene TO, Ottenweller J, et al. Dobutamine digital echocardiography for detecting coronary artery disease. Am J Cardiol 1991;67:1311-8. 7. Marcovitz PA, Armstrong WF. Accuracy of dobutamine stress echocardiography in detecting coronary artery disease. Am J Cardiol 1992;69:1269-73. 2. Grouse LJ, Harbrecht JJ, Vacek JL, et al. Exercise echo- cardiography as a screening test for coronary artery disease and correlation with coronary arteriography. Am J Cardiol 1991;67:1213-8. 8. Armstrong WF, O’Donnell J, Dillon JC, et al. Comple- mentary value of two-dimensional exercise echocar- diography to routine treadmill exercise testing. Ann Intern Med 1986;105:829-35. 3. Sawada SG, Segar DS, Ryan T, et al. Echocardiographic detection of coronary artery disease during dobutamine infusion. Circulation 1991;83:1605-14. 3. Sawada SG, Segar DS, Ryan T, et al. Echocardiographic detection of coronary artery disease during dobutamine infusion. Circulation 1991;83:1605-14. 9. Marcovitz PA, Matbias W, Dick R, et al. Accuracy of dobutamine stress echocardiography for the diagnosis of coronary artery disease: correlation with quantitative coronary arteriography (abstr). J Am Soc Echo 1991;4:279. 4. Segar DS, Brown SE, Sawada SG, et al. Dobutamine stress echocardiography: Correlation with coronary lesion severity as determined by quantitative angiography. J Am Coll Cardiol 1992;19:1197-202. 4. Segar DS, Brown SE, Sawada SG, et al. Dobutamine stress echocardiography: Correlation with coronary lesion severity as determined by quantitative angiography. J Am Coll Cardiol 1992;19:1197-202. 5. McNeill AJ, Fioretti PM, el-Said SM, et al. Enhanced sensitivity for detection of coronary disease by addition of atropine to dobutamine stress echocardiography. Am J Cardiol 1992;70:41-6. 5. McNeill AJ, Fioretti PM, el-Said SM, et al. Enhanced sensitivity for detection of coronary disease by addition of atropine to dobutamine stress echocardiography. Am J Cardiol 1992;70:41-6. 20 The Bangkok Medical Journal Vol. 6; September 2013 ISSN 2287-0237 (online)/ 2287-9674 (print) 20
https://openalex.org/W2110317479	https://europepmc.org/articles/pmc3281779?pdf=render	English	null	Histological alterations in the liver of rats induced by different gold nanoparticle sizes, doses and exposure duration	Journal of nanobiotechnology	2,012	cc-by	5,440	RESEARCH Open Access Abstract Keywords: gold nanoparticles, size, hepatic tissue, histology, hydropic degeneration, nanotoxicity, rats * Correspondence: abdelhalimmak@yahoo.com 1Department of Physics and Astronomy, College of Science, King Saud University, Saudi Arabia Full list of author information is available at the end of the article Histological alterations in the liver of rats induced by different gold nanoparticle sizes, doses and exposure duration Mohamed Anwar K Abdelhalim1* and Bashir M Jarrar2 © 2012 Abdelhalim and Jarrar; 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. Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Abstract Background: Nanoparticles (NPs) can potentially cause adverse effects on organ, tissue, cellular, subcellular and protein levels due to their unusual physicochemical properties. Advances in nanotechnology have identified promising candidates for many biological and biomedical applications. Since the properties of NPs differ from that of their bulk materials, they are being increasingly exploited for medical uses and other industrial applications. The aim of the present study was to investigate the particle-size effect of gold nanoparticles (GNPs) on the hepatic tissue in an attempt to cover and understand the toxicity and the potential threat of their therapeutic and diagnostic use. Methods: To investigate particle-size effect of GNPs on the hepatic tissue, a total of 70 healthy male Wistar-Kyoto rats were exposed to GNPs received 50 or 100 ul of GNPs infusion of size (10, 20 and 50 nm for 3 or 7 days). Results: In comparison with respective control rats, exposure to GNPs doses has produced alterations in the hepatocytes portal triads and the sinusoids The alterations in the hepatocytes were mainly summarized as Methods: To investigate particle-size effect of GNPs on the hepatic tissue, a total of 70 healthy male Wistar-Kyoto rats were exposed to GNPs received 50 or 100 ul of GNPs infusion of size (10, 20 and 50 nm for 3 or 7 days). Results: In comparison with respective control rats, exposure to GNPs doses has produced alterations in the hepatocytes, portal triads and the sinusoids. The alterations in the hepatocytes were mainly summarized as hydropic degeneration, cloudy swelling, fatty degeneration, portal and lobular infiltrate by chronic inflammatory cells and congestive dilated central veins. Conclusions: The induced histological alterations might be an indication of injured hepatocytes due to GNPs toxicity that became unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these NPs. These alterations were size-dependent with smaller ones induced the most effects and related with time exposure of GNPs. The appearance of hepatocytes cytoplasmic degeneration and nuclear destruction may suggest that GNPs interact with proteins and enzymes of the hepatic tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may induce stress in the hepatocytes to undergo atrophy and necrosis. More histomorphologcal, histochemical and ultrastrucural investigations are needed in relation of the application of GNPs with their potential threat as a therapeutic and diagnostic tool. Introduction reactive than their bulk counter parts due to their small size and larger surface area to volume ratio [1,2]. In vivo studies in rats exposed to aerosols of GNPs revealed that the NPs were rapidly taken into the system with the highest accumulation in the lungs, aorta, eso- phagus and olfactory bulb [1]. Moreover, particles of nano-dimension are believed to be more biologically Gold in its bulk form has long been considered an inert, noble metal with some therapeutic and even med- icinal value hence GNPs are thought also to be relatively non-cytotoxic [3]. Yet there are differing reports of the extent of the toxic nature of these particles owing to the different modifications of the GNPs, surface functional attachments and shape and diameter size of the nano- spheres [4,5]. Introduction Moreover, the metallic nature of the metal * Correspondence: abdelhalimmak@yahoo.com 1Department of Physics and Astronomy, College of Science, King Saud University, Saudi Arabia Full list of author information is available at the end of the article Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Page 2 of 9 Page 2 of 9 Table 1 The intraperitoneal administration of 10, 20 and 50 nm GNPs in rats for periods of 3 or 7 days Groups Infusion dose, size and exposure duration of GNPs Control received no gold nanoparticles (GNPs; n = 10) G1A received infusion of 50 μl GNPs of size 10 nm for 3 days (n = 5) G1B received infusion of 50 μl GNPs of size 10 nm for 7 days (n = 5) G2A received infusion of 50 μl GNPs of size 20 nm for 3 days (n = 5) G2B received infusion of 50 μl GNPs of size 20 nm for 7 days (n = 5) G3A received infusion of 50 μl GNPs of size 50 nm for 3 days (n = 5) G3B received infusion of 50 μl GNPs of size 50 nm for 7 days (n = 5) G4A received infusion of 100 μl GNPs of size 10 nm for 3 days (n = 5) G4B received infusion of 100 μl GNPs of size 10 nm for 7 days (n = 5) G5A received infusion of 100 μl GNPs of size 20 nm for 3 days (n = 5) G5B received infusion of 100 μl GNPs of size 20 nm for 7 days (n = 5) G6A received infusion of 100 μl GNPs of size 50 nm for 3 days (n = 5) G6B received infusion of 100 μl GNPs of size 50 nm for 7 days (n = 5) The intraperitoneal administration of 10, 20 and 50 nm GNPs in rats for periods of 3 or 7 days Infusion dose, size and exposure duration of GNPs derived NPs and the presence of transition metals encourages the production of reactive oxygen species (ROS) leading to oxidative stress [6,7]. intraperitoneal administration of GNPs and, if so, whether are related to the size of these NPs and the time of exposure. Introduction The present study was carried out to investigate the particle-size effect of GNPs on the hepatic tissue in an attempt to cover and understand the toxicity and poten- tial threat of their therapeutic and diagnostic use in rela- tion with the time of exposure. Although some scientists consider NPs as nontoxic, other studies reporting the toxic effects induced by NPs [8-10]. Although some NPs may appear to be nontoxic, other cellular mechanisms such as cell signaling and other normal cellular functions may be disrupted and are currently undergoing further investigation [11,12]. The toxicity of NPs is being addressed by a number of standardized approaches with in vitro, in vivo as well as detailed genomic or biodistribution studies [12]. Materials and methods A total of 70 healthy male Wistar-Kyoto rats obtained from the Laboratory Animal Center (College of Phar- macy, King Saud University, Saudi Arabia). The rats nearly of the same age (12 weeks old) and weighing 220-240 gm of King Saud University colony were used. Animals were randomly divided into groups, 12 GNPs- treated rats groups and one control group (NG). It has been shown that NPs may produce in vitro toxi- city in some cell-based assays, but not in others. This may be a result of interference with the chemical probes, differences in the innate response of particular cell types, or other factors [13]. In addition, GNPs are used as carriers for the delivery of drugs and genes [14]. The histological and the histochemical characteriza- tion in the hepatic tissues due to GNPs have not yet been identified and documented. In the present study, an attempt has been made to characterize the possible histological alterations in the hepatic tissues following The 10, 20 and 50 nm GNPs were administered intra- peritonealy in rats for periods of 3 or 7 days as shown in Table 1. Materials and methods The rats were maintained on standard laboratory rodent diet pellets and were housed in humidity and temperature-controlled ventilated cages on a 12 h day/ 40 x 1 40 x 3 Figure 1 40 x 3 40 x 1 - 40 x 3 40 x 7 100 x 3 Figure 2 40 x 3 40 x 5 Figure 3 40 3 40 4 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Page 3 of 9 Page 3 of 9 - 40 x 3 40 x 7 100 x 3 Figure 2 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Page 3 of 9 Page 3 of 9 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 - 40 x 3 40 x 7 100 x 3 Figure 2 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Page 3 of 9 - 40 x 3 40 x 7 - 40 x 3 40 x 7 100 x 3 Figure 2 40 x 3 40 x 5 Figure 3 40 x 3 40 x 4 Figure 4 40 x 3 40 x 3 40 x 7 100 x 3 Figure 2 40 x 3 40 x 5 Figure 3 40 x 3 Figure 3 40 x 5 40 x 3 40 x 4 Figure 4 40 x 4 40 x 3 Page 4 of 9 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 40 x 2 Figure 5 night cycle. Two animals from each group were killed by dislocation of the neck at intervals of 3 and 7 days of treatment with GNPs. All experiments were conducted in accordance with the guidelines approved by King Saud University Local Animal Care and Use Committee. Fresh portions of the lateral lobes of the liver from each rat were cut rapidly, fixed in neutral buffered for- malin (10%), then dehydrated, with grades of ethanol (70, 80, 90, 95 and 100%). Dehydration was then fol- lowed by clearing the samples in 2 changes of xylene. Samples were then impregnated with 2 changes of mol- ten paraffin wax, then embedded and blocked out. Materials and methods Par- affin sections (4-5 um) were stained with hematoxylin (40 x 1) 40 x 10 40 x 8 (100 x 2) Figure 6 (40 x 1) (40 x 1) (40 x 1) 40 x 10 40 x 8 40 x 10 40 x 8 (100 x 2) Figure 6 Figure 6 Page 5 of 9 Page 5 of 9 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Results and discussions and eosin the conventional histological and stain according to Pearse [15]. Stained sections of control and treated rats were examined for alterations in the archi- tecture, portal triads, hepatocytes, sinusoids and for the presence of degeneration, necrosis, fatty change and portal fibrosis. Size and morphology of different gold nanoparticles The 10 and 20 nm GNPs show spherical shape while the 50 nm GNPs show hexagonal shape. The mean size for GNPs was calculated from the images taken by the transmission electron microscope (TEM). The 10 nm presence of degeneration, necrosis, fatty change and portal fibrosis. for GNPs was calculated from the images taken by the transmission electron microscope (TEM). The 10 nm (40 x 1) 40 x 3 100 x 3 (40 x 9) (40 x 1) 40 x 3 100 x 3 (40 x 9) Figure 7 (40 x 1) (40 x 1) (40 x 1) 100 x 3 40 x 3 40 x 3 (40 x 9) (40 x 9) (40 x 9) Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Page 6 of 9 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 100 x 1 100 x 4 100 x 2 100 x 3 Figure 8 100 x 4 100 x 1 100 x 4 100 x 3 100 x 2 Fig re 8 observed in Figures 1,2,3,4,5,6,7,8,9,10,11 and 12 and can be summarized as shown in Table 2. GNPs was of mean size of 9.45 ± 1.33 nm, 20 nm GNPs was of mean size of 20.18 ± 1.80 and the 50 nm GNPs was of mean size of 50.73 ± 3.58. The high electron densities of GNPs as well as the homogeneity of the par- ticles shape and size make them highly conspicuous under the TEM. In addition, relatively simple methods can be applied to obtain the populations of GNPs of dif- ferent average sizes which allow simultaneous detection of several targets. GNPs was of mean size of 9.45 ± 1.33 nm, 20 nm GNPs was of mean size of 20.18 ± 1.80 and the 50 nm GNPs was of mean size of 50.73 ± 3.58. The high electron densities of GNPs as well as the homogeneity of the par- ticles shape and size make them highly conspicuous under the TEM. Results and discussions In addition, relatively simple methods can be applied to obtain the populations of GNPs of dif- ferent average sizes which allow simultaneous detection of several targets. The cloudy swelling might be exhibited as a result of disturbances of membranes function that lead to mas- sive influx of water and Na+ due to GNPS effects. Cellu- lar swelling might be accompanied by leakage of lysosomal hydrolytic enzymes that lead to cytoplasmic degeneration and macromolecular crowding [16]. Hydropic degeneration is a result of ion and fluid homestasis that lead to an increase of intracellular water [17]. The vacuolated swelling of the cytoplasm of the hepatocytes of the GNPs treated rats might indicate acute and subacute liver injury induced by these NPs. Some studies indicate that nuclear polymorphism is seen in hepatic dysplasia and carcinomatous lesion [18]. Binucleation represents a consequence of cell injury and is a sort of chromosomes hyperplasia which is usually seen in regenerating cells [19]. Histological Alterations In comparison with the control group, the following his- tological alterations were detected in the liver tissue of GNPs-treated rats. These histological alterations were 40 x 4 Figure 9 Occasional inflammatory cells infiltration was seen in the portal triads and the perioral zones of GNPs treated rats. The infiltrate cells were mainly lymphocytes and plasma cells. This infiltration was more prominent after 7 days of administration and in rats received 100 μl than those received 50 μl. The appearance of inflamma- tory cells in the hepatic tissue may suggest that GNPs could interact with proteins and enzymes of the hepatic interstitial tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Page 7 of 9 Page 7 of 9 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 100 x 1 40 x 2 Figure 10 http://www.jnanobiotechnology.com/content/10/1/5 40 x 2 (ROS) generation which in turn may imitate an inflam- matory response [20]. represents a defense mechanism of detoxification. Kupf- fer cell hyperplasia is contributed to hepatic oxidative stress [21]. The sinusoidal Kupffer cells became prominent and increased in number due to GNPs exposure. This change was more prominent at 10 nm GNPs with dose of 100 μl than 20 nm and 50 nm GNPS and more after 7 days of administration than rats exposed to GNPs for 3 days. Kupffer cells activation might indicate that GNPs activate the phagocytic activity of the sinusoidal cells by increasing the number of Kupffte cells to help in removing the accumulated GNPs where lysosomes are involved in the intracellular breakdown into small metabolic products. The produced Kupffer cells hyper- plasia might be correlated with the amount of injurious to the hepatic tissue induced by GNPs intoxication and Fatty change was observed in some swelling hepato- cytes of rats exposed to 100 μl of 10 nm GNPs and to les- ser extent in the ones exposed to larger particles. This hepatic liposis was more frequent in rat exposed to the GNPs for 7 days than those received the treatment for 3 days. Hepatocytes fatty change might be due to lipid per- oxidation that leads to rough endoplasmic damage and detachment of the cytoplasmic lipoprotein and indicate abnormal fat metabolism [22]. Authors’ contributions d h Histological alterations by GNPs exposure as shown in the results of the present work could be an indication of AMAK and JBM have analyzed data, interpreted and written the final draft of this manuscript. The animal model used in this study was obtained from the Acknowledgements Th th The authors are very grateful to National Plan of Science and Technology (NPST). This research was financially supported by the National Science and Technology The authors are very grateful to National Plan of Science and Technology (NPST). This research was financially supported by the National Science and Technology Innovation Plan (NSTIP), Research No. 08-ADV206-02 and Research No. 09- NAN670-02, College of Science, King Saud University, Saudi Arabia. The authors are very grateful to National Plan of Science and Technology (NPST). This research was financially supported by the National Science and Technology Innovation Plan (NSTIP), Research No. 08-ADV206-02 and Research No. 09- NAN670-02, College of Science, King Saud University, Saudi Arabia. Innovation Plan (NSTIP), Research No. 08-ADV206-02 and Research No. 09- NAN670-02, College of Science, King Saud University, Saudi Arabia. Histological Alterations The seen hepatocytes abnormal retention of lipids in the present investigation induced by GNPs might indicate toxic injury to liver in the form of hepatocytes liposis by these particles. 40 x 7 100 x 8 40 x 8 Figure 11 40 x 7 100 x 8 40 x 8 Figure 11 100 x 8 40 x 7 40 x 7 Figure 11 Page 8 of 9 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 Abdelhalim and Jarrar Journal of Nanobiotechnology 2012, 10:5 http://www.jnanobiotechnology.com/content/10/1/5 100 x 2 Figure 12 injured hepatocytes due to GNPs toxicity that become unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these particles. One might conclude that these alterations are size-dependent with smaller ones induced more damage with relation with the time exposure of GNPs. The appearance of hepatocytes cytoplasmic degenera- tion and nuclear destruction may suggest that GNPs interact with proteins and enzymes of the hepatic tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may induce stress in the hepatocytes to undergo atrophy and necrosis. More histomorphologcal, histochemical and ultrastru- cural investigations are needed to correlate the biomedi- cal application of GNPs with the potential threat of their therapeutic and diagnostic use. Sporadic spotty well-defined necrosis was noticed in some hepatocytes of GNPs treated rats. This alteration was detected in the liver of rats exposed to 10 nm size particles and to lesser extent with 20 nm particles but was not seen with those exposed to 50 nm size particles. Apoptic altera- tion might be followed organelles swelling specially mito- chondria, endoplasmic reticulum and rupture of lysosomes which might lead to amorphous eosinophilic cytoplasm as an initial sign in the sequence of hepatocytes necrosis before shrinking and dissolution of nulei [23]. The seen hepatocytes necrosis due to GNPs exposure might indicate oxidative stress on these cells by glutathione depletion. Author details 1 1Department of Physics and Astronomy, College of Science, King Saud University, Saudi Arabia. 2College of Applied Medical Sciences, Al-Jouf University, P.O. Box 2014, Skaka-Al-Jouf, Saudi Arabia. Table 2 Histological alterations induced in GNPs-treated rats gure 1 GNPs-normal rat demonstrating benign looking hepatic lobule and hepatocytes surrounding a central vein Figure 2 GNPs-treated rat received 50 μl of 10 nm particles for 3 days demonstrating: Congested central vein with portal infiltrated by chronic inflammatory cells (40 × 3); Liver section showed widely dilated congested central vein (40 × 7); necrotic hepatocytes (100 × 3) Figure 3 GNPs-treated rat received 100 μl of 10 nm particles for 7 days demonstrating: Liver section widely dilated congested central vein (40 × 3) with hepatocytes disarray and cloudy swelling of liver hepatocytes (40 × 5) Figure 3 GNPs-treated rat received 100 μl of 10 nm particles for 7 days demonstrating: Liver section widely dilated congested central vein (40 × 3) with hepatocytes disarray and cloudy swelling of liver hepatocytes (40 × 5) Figure 4 GNPs-treated rat received 50 μl of 20 nm particles for 3 days demonstrating: lobular infiltrate by chron × 3) congested hepatic sinusoids containing red blood cells (40 x4) Figure 5 GNPs-treated rat received 50 μl of 10 nm particles for 7 days demonstrating dilated congested blood si GNPs-treated rat received 50 μl of 10 nm particles for 7 days demonstrating dilated congested blood sinusoids (40 Figure 6 GNPs-treated rat received 50 μl of 50 nm particles for 3 days demonstrating: Widely dilated congested central vein with dilated congested liver sinusoids with hepatocytes cloudy swelling (40 × 1); Fatty degeneration focally affecting the liver lobules (40 × 10) and (40 × 8); Fatty degeneration focally affecting the liver lobules (100 × 2) Figure 6 GNPs-treated rat received 50 μl of 50 nm particles for 3 days demonstrating: Widely dilated congested central vein with dilated congested liver sinusoids with hepatocytes cloudy swelling (40 × 1); Fatty degeneration focally affecting the liver lobules (40 × 10) and (40 × 8); Fatty degeneration focally affecting the liver lobules (100 × 2) Figure 7 GNPs-treated rat received 100 μl of 10 nm particles for 3 days demonstrating: Liver hepatocytes with lobular lymphocytic infiltrate (40 × 1); prominent diffuse fatty degeneration (40 × 3) & (100 × 3); prominent diffuse fatty degeneration (40 × 3) & (100 × 3); congested dilated blood sinusoids (40 × 9); scattered fatty degeneration of liver hepatocytes with focus of inflammatory cells infiltrate of liver lobules; diffuse fatty degeneration Figure 7 GNPs-treated rat received 100 μl of 10 nm particles for 3 days demonstrating: Liver hepatocytes with lobular lymphocytic infiltrate (40 × 1); prominent diffuse fatty degeneration (40 × 3) & (100 × 3); prominent diffuse fatty degeneration (40 × 3) & (100 × 3); congested dilated blood sinusoids (40 × 9); scattered fatty degeneration of liver hepatocytes with focus of inflammatory cells infiltrate of liver lobules; diffuse fatty degeneration Figure 8 GNPs-treated rat received 100 μl of 10 nm particles for 7 days demonstrating: Dilated congested blood congested blood sinusoids (40 × 4); fatty degeneration of liver hepatocytes (100 × 2); increased sinusoidal Kupff Figure 8 GNPs-treated rat received 100 μl of 10 nm particles for 7 days demonstrating: Dilated congested blood vessels (100 × 1); congested blood sinusoids (40 × 4); fatty degeneration of liver hepatocytes (100 × 2); increased sinusoidal Kupffer cells (100 × 4) Figure 9 GNPs-treated rat received 100 μl of 20 nm particles for 3 days demonstrating: Inflammatory cells infiltrate of liver lobules and dilated congested central vein (40 × 4) Figure 8 GNPs-treated rat received 100 μl of 10 nm particles for 7 days demonstrating: Dilated congested blood vessels (100 × 1); congested blood sinusoids (40 × 4); fatty degeneration of liver hepatocytes (100 × 2); increased sinusoidal Kupffer cells (100 × 4) Figure 9 GNPs-treated rat received 100 μl of 20 nm particles for 3 days demonstrating: Inflammatory cells infiltrate of liver lobules and dilated congested central vein (40 × 4) Figure 9 GNPs-treated rat received 100 μl of 20 nm particles for 3 days demonstrating: Inflammatory cells infiltrate of liver lobules and dilated congested central vein (40 × 4) Figure 9 GNPs-treated rat received 100 μl of 20 nm particles for 3 days demonstrating: Inflammatory cells infiltrate of liver lobules and dilated congested central vein (40 × 4) Figure 10 GNPs-treated rat received 100 μl of 20 nm particles for 7 days demonstrating: Liver hepatocytes with binucleated cells (100 × 1); dilated congested central vein (40 × 2) Figure 10 GNPs-treated rat received 100 μl of 20 nm particles for 7 days demonstrating: Liver hepatocytes with binucleated cells (100 × 1); dilated congested central vein (40 × 2) Figure 11 GNPs-treated rat received 100 μl of 50 nm particles for 3 days demonstrating: Scattered lobular inflammatory cells infiltrate (40 × 7) and (100 × 8); scattered few cloudy swellings on (40 × 8) Figure 11 GNPs-treated rat received 100 μl of 50 nm particles for 3 days demonstrating: Scattered lobular inflammatory cells infiltrate (40 × 7) and (100 × 8); scattered few cloudy swellings on (40 × 8) Figure 12 GNPs-treated rat received 100 μl of 50 nm particles for 7 days demonstrating: No major pathological effect seen. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit yp 17. Schrand AM, Rahman MF, Hussain SM, Schlager JJ, 1 David A, Smith DA, Syed2 AF: Metal-based nanoparticles and their toxicity assessment. Nanomed Nanobiotechnol 2010, 2:544-568. yp 17. 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Pandey G, Srivastava DN, Madhuri S: A standard hepatotoxic model produced by paracetamol in rat. Toxicology International 2008, 15(1):69-70. doi:10.1186/1477-3155-10-5 Cite this article as: Abdelhalim and Jarrar: Histological alterations in the liver of rats induced by different gold nanoparticle sizes, doses and exposure duration. Journal of Nanobiotechnology 2012 10:5. 21. Neyrinck A: Modulation of Kupffer cell activity: physio-pathological consequences on hepatic metabolism. Bull Mem Acad R Med Belg 2004, 159(5-6):358-66. 22. Reddy JK, Rao MS: Lipid metabolism and liver inflammation. II. Fatty liver disease and fatty acid oxidation. Am J Physiol Gastrointest Liver Physiol 2006, 290(5):G852-8. 23. Pandey G, Srivastava DN, Madhuri S: A standard hepatotoxic model produced by paracetamol in rat. Toxicology International 2008, 15(1):69-70. doi:10.1186/1477-3155-10-5 Cite this article as: Abdelhalim and Jarrar: Histological alterations in the liver of rats induced by different gold nanoparticle sizes, doses and exposure duration. Journal of Nanobiotechnology 2012 10:5. Laboratory Animal Center (College of Pharmacy, King Saud University, Saudi Arabia). AMAK has conceived the study and its design and obtained research grants for this study. Moreover, both authors have read and approved the final manuscript. Received: 20 May 2011 Accepted: 25 January 2012 Published: 25 January 2012 doi:10.1186/1477-3155-10-5 Cite this article as: Abdelhalim and Jarrar: Histological alterations in the liver of rats induced by different gold nanoparticle sizes, doses and exposure duration. Journal of Nanobiotechnology 2012 10:5. Competing interests h h d l h The authors declare that they have no competing interests. Received: 20 May 2011 Accepted: 25 January 2012 Published: 25 January 2012 Received: 20 May 2011 Accepted: 25 January 2012 Published: 25 January 2012 References Zusman I, Kozlenko M, Zimber A: Nuclear polymorphism and nuclear size in precarcinomatous and carcinomatous lesions in rat colon and liver. Cytometry 1991, 12(4):302-7. 19. Gerlyng1 P, Åbyholm1 A, Grotmol T, Erikstein B, Huitfeldt HS, Stokke T, Seglen1 PO: Binucleation and polyploidization patterns in developmental and regenerative rat liver growth. Cell Proliferation 2008, 26(6):557-565. 19. Gerlyng1 P, Åbyholm1 A, Grotmol T, Erikstein B, Huitfeldt HS, Stokke T, Seglen1 PO: Binucleation and polyploidization patterns in developmental and regenerative rat liver growth. Cell Proliferation 2008, 26(6):557-565. 20. Johar D, Roth JC, Bay GH, Walker JN, Kroczak TJ, Los M: Inflammatory response, reactive oxygen species, programmed (necrotic-like and apoptotic) cell death and cancer. Rocz Akad Med Bialymst 2004, 49:31-9. 20. 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https://openalex.org/W3118824966	https://vuzbiochemi.elpub.ru/jour/article/download/483/325	Russian	null	Some regularities in the process of anthocyanin extraction from vegetable sources	Izvestiâ vuzov. Prikladnaâ himiâ i biotehnologiâ	2,021	cc-by	5,902	Некоторые закономерности экстракции антоцианов из растительных источников © Я.Ю. Саласина, Д.А. Калиникин, В.И. Дейнека, Л.А. Дейнека Белгородский государственный национальный исследовательский университет, г. Белгород, Российская Федерация Резюме: Работа посвящена определению закономерностей экстракции антоцианов из различных растительных источников в некоторых экстрагентах. Для экстракции использовали свежий рас- тительный материал: корнеплоды фиолетовой моркови, корнеплоды фиолетового картофеля сорта Аметист, свежие плоды аронии Мичурина, плоды паслена садового, плоды кизила обыкно- венного, чернику и краснокочанную капусту, а также высушенные лепестки пиона. Экстракцию осуществляли настаиванием растительного материала в избранном экстрагенте (оставляли на ночь). Концентрацию антоцианов определяли спектрофотометрическим методом. Показано, что 0,1 М водный раствор HCl является эффективным и экологически безопасным экстрагентом, поз- воляющим осуществлять экстракцию антоцианов из многих объектов. При значениях кислотно- сти среды больше 1 возможны значительные потери антоцианов: 5–45% – при рН = 2; 33–88% – при рН = 3; 41–92% – при экстракции дистиллированной водой. Ацилирование не способствует ро- сту степени экстрагирования антоцианов. Добавки органических растворителей (этанола, аце- тонитрила и глицерина) в ряде случаев позволяют существенно ускорить экстракцию антоциа- нов, особенно в случае плодов кизила обыкновенного. Влияние экстрагента на степень экстраги- рования нивелируется для измельченного материала. Приведены данные по сольватохромному эффекту, влияющему на спектральные характеристики растворов некоторых антоцианов. Пока- зано, что сдвиг максимума полосы поглощения велик в случае неацилированных антоцианов и уменьшается для ацилированных соединений, по всей вероятности, вследствие внутримолеку- лярной копигментации ацилированных антоцианов в водных растворах. Отмечено, что при опре- делении концентрации антоцианов в растворителях с различной концентрацией органической добавки необходимо учитывать сольватохромный эффект. Показано, что добавка органического растворителя приводит не только к смещению максимумов полос поглощения, но и к значитель- ным гиперхромным эффектам. При игнорировании этого эффекта погрешности в определении антоцианов могут превысить 70%. Предложен простой и эффективный способ учета указанных эффектов по схеме перекрестных разбавлений. Таким образом, экспериментально обоснован вы- бор экстрагентов для эффективной экстракции антоцианов из некоторых видов растительного сырья и предложен метод оценки сольватохромных эффектов. лючевые слова: антоцианы, экстракция, влияние рН, сольватохромные эффекты, спо Для цитирования: Саласина Я.Ю., Калиникин Д.А., Дейнека В.И., Дейнека Л.А. Некоторые законо- мерности экстракции антоцианов из растительных источников. Известия вузов. Прикладная химия и биотехнология. 2020. Т. 10. N 4. С. 691–699. https://doi.org/10.21285/2227-2925-2020-10-4-691-699 ИЗВЕСТИЯ ВУЗОВ. ПРИКЛАДНАЯ ХИМИЯ И БИОТЕХНОЛОГИЯ 2020 Том 10 N 4 EEDINGS OF UNIVERSITIES. APPLIED CHEMISTRY AND BIOTECHNOLOGY 2020 Vol. 10 No ИЗВЕСТИЯ ВУЗОВ. ПРИКЛАДНАЯ ХИМИЯ И БИОТЕХНОЛОГИЯ 2020 Том 10 N 4 EEDINGS OF UNIVERSITIES. APPLIED CHEMISTRY AND BIOTECHNOLOGY 2020 Vol. 10 No Yaroslava Yu. Salasina, Danila S. Kalinikin, Viktor I. Deineka, Lyudmila A. Deineka Belgorod National Research University, Belgorod, Russian Federation Yaroslava Yu. Salasina, Danila S. Kalinikin, Viktor I. Deineka, Lyudmila A. Deineka Belgorod National Research University, Belgorod, Russian Federation Abstract: This work investigates regularities in the process of extracting anthocyanins from various plant sources in the medium of extractants. For extraction, fresh plant samples were used, including the roots of purple carrots, roots of purple potatoes of the Amethyst variety, fresh fruits of Michurin's aronia, fruits of the garden nightshade, cornelian cherries, blueberries and red cabbage, as well as dried peony petals. The ex- traction was carried out by infusing the plant material under study in a selected extractant (left overnight). The concentration of anthocyanins was determined spectrophotometrically. It was shown that a 0.1 M aque- ous solution of HCl is an effective and environmentally friendly extractant allowing for the extraction of an- thocyanins from various sources. Provided that the medium acidity is greater than 1, significant losses of 691 anthocyanins are possible: 5–45% at pH = 2; 33–88% at pH = 3; 41–92% when extracted with distilled water. Acylation does not contribute to an increase in the degree of anthocyanin extraction. The addition of organic solvents (ethanol, acetonitrile and glycerin) can significantly accelerate the anthocyanin extraction in some cases, which is particularly true for cornelian cherries. When using crushed material, the influence of the ex- tractant on the degree of extraction is minimal. Information is presented on the solvatochromic effect, which affects the spectral characteristics of some anthocyanin solutions. It was shown that the shift of the absorp- tion band maximum is the greatest for non-acylated anthocyanins, while decreasing for acylated compounds. The latter is most likely to be associated with the intramolecular copigmentation of acylated anthocyanins in aqueous solutions. When determining the concentration of anthocyanins in solvents with different concentra- tions of organic additives, the solvatochromic effect should be taken into account. The addition of an organic solvent leads not only to a shift in the absorption band maxima, but also to significant hyperchromic effects. If this effect is ignored, errors in determination of anthocyanins can exceed 70%. This paper proposes a simple and effective approach to considering these effects using a cross-dilution scheme. Therefore, the choice of extractants for efficient anthocyanin extraction from various plant raw materials was experimentally substan- tiated, and a method for evaluating solvatochromic effects was proposed. Yaroslava Yu. Salasina, Danila S. Kalinikin, Viktor I. Deineka, Lyudmila A. Deineka Belgorod National Research University, Belgorod, Russian Federation Keywords: anthocyanins, extraction, pH influence, solvatochromic effects, accounting method Keywords: anthocyanins, extraction, pH influence, solvatochromic effects, accoun For citation: Salasina YYu, Kalinikin DA, Deineka VI, Deineka LA. Some regularities in the process of an- thocyanin extraction from vegetable sources. Izvestiya Vuzov. Prikladnaya Khimiya i Biotekhnologiya = Pro- ceedings of Universities. Applied Chemistry and Biotechnology. 2020;10(4):691–699. (In Russian) https://doi.org/10.21285/2227-2925-2020-10-4-691-699 ЭКСПЕРИМЕНТАЛЬНАЯ ЧАСТЬ следствием подкисления экстракта органически- ми кислотами, присутствующими в образцах, уровень биосинтеза которых может сильно зави- сеть даже от сорта в пределах одного и того же вида растения. В работе использовали свежие корнеплоды фиолетовой моркови (Daucus carota ssp. sativus var. atrorubens Alef.), свежие корнеплоды карто- феля сорта Аметист с фиолетовой мякотью, свежие плоды аронии Мичурина (Arónia mit- schurinii Skvortsov & Maitul), плоды паслена садо- вого (Solanum retroflexum Dunal.), выращенные в г. Белгород, а также плоды кизила обыкновен- ного (Cornus mas L.), черники (Vaccinium myrtil- lus L.) и краснокочанную капусту (Brassica oleracea var. capitata F. rubra), приобретенные на рынке г. Белгорода, и высушенные лепестки пиона. д р В экспериментах использовали водные рас- творы HCl с концентрациями 0,1 М, 0.01 М, 0.001 М и водные растворы без добавок кислоты. После- довательные порции трех различных источников антоцианов, получаемые его настаиванием в течение 12 часов, объединяли перед определе- нием концентрации антоцианов. В фиолетовой моркови по нашим данным содержатся основные два антоциана – цианидин-3-гексозилпентозил- гексозид, ацилированный феруловой кислотой и без ацилирования. В корнеплодах фиолетового картофеля обнаружены 3-рутинозид-5-глюкози- ды трех метилированных антоцианидинов (пету- нидина, пеонидина и мальвидина), ацилирован- ные пара-кумаровой и феруловой кислотами. Антоцианы плодов аронии Мичурина образованы неацилированными гликозидами цианидина. При выборе объектов исследования руководствова- лись информацией о большей устойчивости ацилированных антоцианов по сравнению с неа- цилированными [17]. Экстракцию продолжали до достижения практически бесцветного экстракта на последней стадии. Перед измерением опти- ческой плотности во всех полученных экстрактах рН (при разбавлении) доводили до 1. Получен- ные результаты для суммы экстрактов всех ста- дий представлены в табл. 1. Для приготовления экстрагентов использова- ли дистиллированную воду, этанол, ацетонит- рил, глицерин, соляную кислоту. Экстракцию выполняли в статических усло- виях настаиванием смеси растительного мате- риала и экстрагента в течение 12 ч. Экстракты отделяли от остатка фильтрованием через бу- мажный фильтр на воронке Бюхнера под вакуу- мом. Электронные спектры поглощения записыва- ли в кварцевых кюветах на спектрофотометре Shimadzu UV 2550 при рН = 1 без поправок на абсорбцию «полимерных» антоцианов. При рас- четах использовали пересчет на цианидин-3- глюкозид хлорид с ε = 26900 л·моль-1·см-1 [16]. ВВЕДЕНИЕ динений, из-за которых возможны проблемы при хранении и использовании антоцианов. В каче- стве основных органических растворителей ис- пользуют этанол и метанол, но известно исполь- зование и других растворителей, в том числе ацетона [10], некоторых одноатомных спиртов [11, 12], глицерина [13, 14]. Антоцианы как подкласс обширного класса флавоноидов – это вещества, хорошо раствори- мые в воде, существующие в нескольких рН- зависимых формах, часть которых окрашена [1, 2]. Высокая антиоксидантная активность ан- тоцианов [2, 3] и красящая способность [4] поз- воляют рассматривать эти соединения как при- родные антиоксиданты и красители для пищевой и медицинской промышленности. Природные антоцианы являются гликозидами антоцианиди- нов, синтезируемых в плодах, цветках, листьях и других частях многих растений [5], поэтому для их извлечения из растительных источников на первом этапе необходима экстракция. Суще- ствуют различные технологии экстракции анто- цианов из растительного сырья, представлен- ные, например, в работах [6–9]. Антоцианы не- растворимы в неполярных органических раство- рителях, поэтому базовые компоненты экстра- гентов – вода, смешивающиеся с водой органи- ческие растворители и подкислитель. Подкисли- тель добавляется для перевода различных форм антоцианов в самую устойчивую флавили- евую форму (I) (рис. 1). В качестве подкислителя можно использовать минеральные кислоты, ча- ще всего используют соляную кислоту, добавки которой (до рН ≤ 1) обеспечивают условия для существования антоцианов только во флавилие- вой форме. При этом подавляется и реакция гидратации антоцианов до не отличающихся устойчивостью бесцветных псевдооснований (II), которые легко превращаются в слабоокрашен- ные цис-халконную (III) и далее – в транс- халконную (IV) формы (см. рис. 1). Рис. 1. Схема одного из направлений превращения антоцианов с повышением рН Fig. 1. One of the directions of anthocyanins conversion with increasing pH Рис. 1. Схема одного из направлений превращения антоцианов с повышением рН Fig. 1. One of the directions of anthocyanins conversion with increasing pH При сравнении экстракционной эффективности органических добавок возникает проблема сопо- ставления концентрации антоцианов из-за сольва- тохромного эффекта, приводящего не только к смещению максимумов полос поглощения [15], но и к изменению коэффициентов экстинкции, кото- рые для многих систем не известны. Поэтому нашей целью являлась разработка подхода, поз- воляющего сопоставлять коэффициенты экстинк- ции антоцианов в растворителях любых составов и методы экстракции антоцианов, подкисленных водными и водно-органическими смесями. Одна из важных функций добавок органиче- ских растворителей – исключение экстракции полимерных и олигомерных гидрофильных сое- ФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯ / PHYSICOCHEMICAL BIOLOGY 692 ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ Некоторые закономерности … Salasina Y.Yu., Kalinikin D.A., Deineka V.I., Deineka L.A. Some regularities … Дейнека Л.А. Некоторые закономерности … V.I., Deineka L.A. Some regularities … Рис. 2. Гиперхромный эффект спектров антоцианов как функция концентрации ацетонитрила. Экстракты: 1 – плодов черники; 2 – листьев краснокочанной капусты; 3 – плодов паслена садового Fig. 2. Hyperchromic effect of anthocyanin spectra as function of acetonitrile concentration. Extracts from: 1 – bilberry fruits; 2 – red cabbage leaves; 3 f i f d i h h d Добавка органического растворителя обычно осуществляется тогда, когда желают увеличить растворимость веществ, но в случае антоцианов, находящихся в кислой среде в виде положи- тельно заряженных ионов флавилия в этом нет необходимости. Даже антоцианы дважды ацили- рованные замещенными коричными кислотами (как наиболее липофильные среди антоцианов) легко растворяются в подкисленных водных рас- творах. При этом антоцианы очень плохо рас- творяются в воде без добавок кислоты из сухого состояния. За многолетний опыт работы с анто- цианами был обнаружен единственный объект, для экстракции антоцианов из которого необхо- димо в экстрагент добавлять органический рас- творитель – это плоды кизила (Cornus mas L.). Рис. 2. Гиперхромный эффект спектров антоцианов как функция концентрации ацетонитрила. Экстракты: 1 – плодов черники; 2 – листьев краснокочанной капусты; 3 – плодов паслена садового Рис. 2. Гиперхромный эффект спектров антоцианов как функция концентрации ацетонитрила. Экстракты: 1 – плодов черники; р р 2 – листьев краснокочанной капусты; 3 – плодов паслена садового ( ) Экспериментально было установлено, что при экстракции 0,1 М водным раствором для обесцвечивания растительного материала (пло- дов кизила) требуется до шести и более стадий, хотя на первой стадии экстрагируется около 75% от суммы антоцианов, (табл. 2). При добавлении 40% (по объему) ацетонитрила удается экстра- гировать практически все антоцианы за одну стадию. При повышении концентрации ацетони- трила эффективность экстракции снижается. И не удивительно, поскольку при очень высокой концентрации ацетонитрила в смеси с кислотой растворимость дигликозидов антоцианидинов сильно снижается и их не удается быстро элюи- ровать с концентрирующих патронов С18 при очистке антоцианов методом твердофазной экс- тракции. Впрочем, экологически неблагоприят- ный ацетонитрил удобен при пробоподготовке перед определением антоцианов методами об- ращенно-фазовой ВЭЖХ или гидрофильной хроматографии [18]. Fig. 2. Hyperchromic effect of anthocyanin spectra as function of acetonitrile concentration. ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ В большинстве опубликованных работ, к со- жалению, при сопоставлении экстрагирующей способности различных растворителей исполь- зуется только однократная экстракция. Но при отсутствии данных по исчерпывающей экстрак- ции невозможно оценить эффективность экстра- гентов. В этом отношении важен результат пер- вой экстракции, но только на фоне результатов экспериментов по исчерпывающей экстракции. Рассмотрим несколько важных параметров экс- трагентов. Как следует из представленных данных, пони- жение концентрации кислоты ниже 0,1 М нежела- тельно вследствие падения выхода антоцианов, причем роль стабилизации антоцианов ацилиро- ванием не всегда проявляется – наибольшие по- тери при экстракции водой без подкисления полу- чены для полностью ацилированных антоцианов корнеплодов фиолетового картофеля. Следует также учесть, что могут срабатывать собственные ферментные системы растения, разрушающие антоцианы. Так, например, по нашему опыту, ан- тоцианы из цветков цикория обыкновенного (Cichorium intybus L.) быстро разрушаются после сбора лепестков, поэтому для предотвращения потерь антоцианов требуется немедленное поме- щение их в 0,1 М водный раствор HCl. Аналогично, красные недозрелые ягоды калины гордовины (Vi- burnum lantana L.) вне раствора кислоты быстро (за 2-3 часа) становятся мягкими и черными, из кото- рых антоцианы уже не экстрагируются. Водные растворы соляной кислоты являются не только наиболее простыми по составу экстра- гентами, но и могут быть отнесены к так называ- емым «зеленым» технологиям, не наносящим вреда окружающей среде. Но при этом возника- ет вопрос о значимости рН водного экстрагента для сохранности экстрагируемых антоцианов вследствие возможных превращений, представ- ленных на схеме 1. Возможное различие в ре- зультатах использования воды может быть Таблица 1. Зависимость концентрации антоцианов в экстракте от концентрации HCl в водных растворах (n = 2) Table 1. Relationship between anthocyanin concentration in extracts and HCl concentration in water solutions (n = 2) Таблица 1. Зависимость концентрации антоцианов в экстракте от концентрации HCl в водных раст Table 1. Relationship between anthocyanin concentration in extracts and HCl concentration in water solu ФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯ / PHYSICOCHEMICAL BIOLOGY 693 Таблица 1. Зависимость концентрации антоцианов в экстракте от концентрации HCl в водных растворах (n = 2) Table 1. Relationship between anthocyanin concentration in extracts and HCl concentration in water solutions (n = 2) Концентрация HCl Морковь Картофель Арония с, г/100 г % с, г/100 г % с, г/100 г % 0,1 0,225±0,002 100 0,114±0,001 100 0,845±0,003 100 0,01 0,183±0,007 81 0,062±0,006 55 0,553±0,003 95 0,001 0,151±0,007 67 0,013±0,001 12 0,273±0,006 47 0 0,124±0,008 59 0,009±0,001 8 0,258±0,017 44 ФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯ / PHYSICOCHEMICAL BIOLOGY 693 асина Я.Ю., Калиникин Д.А., Дейнека В.И., Дейнека Л.А. ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ Some regularities … 𝑐𝑖= 𝐷𝑖 𝜀𝑖×𝑙, (2) 𝑐𝑖= 𝐷𝑖 𝜀𝑖×𝑙, 1) вначале получали экстракт Э1 некоторого растительного материала в 0,1 М водном рас- творе HCl с концентрацией антоцианов в нем с1, для которого известно значение ε1 для пересчета на цианидин-3-глюкозид (26900 л×моль-1×см-1) и определяли оптическую плотность экстракта при λmax(1), D1; 1) вначале получали экстракт Э1 некоторого растительного материала в 0,1 М водном рас- творе HCl с концентрацией антоцианов в нем с1, для которого известно значение ε1 для пересчета на цианидин-3-глюкозид (26900 л×моль-1×см-1) и определяли оптическую плотность экстракта при λmax(1), D1; (2) где l – длина оптического пути, см, то для расче- та неизвестного коэффициента экстинкции ε2 использовали формулу, полученную комбинаци- ей уравнений (1) и (2): 2) затем получали экстракт этого же матери- ала в другом растворителе Э2 с неизвестным значением ε2 при λmax(2) и определи оптическую плотность полученного экстракта (с концентра- цией антоцианов в нем с2), D2; 2) затем получали экстракт этого же матери- ала в другом растворителе Э2 с неизвестным значением ε2 при λmax(2) и определи оптическую плотность полученного экстракта (с концентра- цией антоцианов в нем с2), D2; 𝐷1×𝜀2 𝐷2×𝜀1 = 𝐷12 𝐷21. 𝜀2 = 𝐷12×𝐷2×𝜀1 𝐷1 . (3) 𝐷1×𝜀2 𝐷2×𝜀1 = 𝐷12 𝐷21. 𝜀2 = 𝐷12×𝐷2×𝜀1 𝐷1 . (3) (3) 3) смешивали равные объемы экстракта Э1 и экстрагента Э2, определив оптическую плотность полученной смеси, D12; 3) смешивали равные объемы экстракта Э1 и экстрагента Э2, определив оптическую плотность полученной смеси, D12; На рис. 3 представлены результаты экстрак- ции антоцианов в пяти различных растворителях из высушенных лепестков пиона в двух вариан- тах – неразрушенные и измельченные. Расчет показал, что при не учете сольватохромных эф- фектов погрешности измерения концентрации антоцианов превышают 70%. 4) смешивали равные объемы экстракта Э2 и экстрагента Э1, определив оптическую плотность полученной смеси, D21. Поскольку растворители в двух последних случаях одинаковы, соотношение концентраций антоцианов в Э1 и Э2 равно соотношению опти- ческих плотностей D12/ D21: Первый вариант интересен тем, что позволя- ет оценить эффективность экстрагента по спо- собности разрушать клеточные мембраны (рис. 2, а). При соотношении массы растительно- го материала и объема экстрагента 1 : 50, г/мл, лучший результат получен для 1%-го раствора HCl в этаноле, который позволил за первую экс- тракцию извлечь более 65% антоцианов, что бо- лее чем в 2 раза превысило аналогичный пока- затель для 0,1 М водного раствора HCl. с1 с2 = 𝐷12 𝐷21. (1) с1 с2 = 𝐷12 𝐷21. ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ Extracts from: 1 – bilberry fruits; 2 – red cabbage leaves; 3 – fruits of garden nightshade – для экстракта плодов паслена садового, содержащего ацилированный пара-кумаровой кислотой 3,5-дигликозид петунидина – от 522 до 525 нм; – для экстракта краснокочанной капусты, со- держащей моно- и диацилированные производные 3,5 дигликозида цианидина – от 522 до 525 нм. При этом, как следует из представленных на рис. 1 данных, наблюдается гиперхромный эф- фект – рост оптической плотности при λmax, прак- тически линейно зависящий от концентрации ацетонитрила во всем диапазоне концентраций для экстракта плодов черники. Для ацилирован- ных антоцианов линейность также наблюдается, но при содержании ацетонитрила выше 20% об. Изменение на начальном участке в этих случаях, вероятно, являются следствием внутримолеку- лярной копигментации известной для ацилиро- ванных антоцианов [19, 20]. Для трех экстрактов после одинакового раз- бавления, но с различной долей ацетонитрила при одинаковой концентрации соляной кислоты были получены результаты, представленные на рис. 2. Основная проблема при использовании рас- творителей, отличающихся от 0,1 М водного рас- твора соляной кислоты – сольватохромный эф- фект, который не позволяет использовать из- вестные величины коэффициентов экстинкции для расчета концентрации антоцианов в экстрак- тах. Для учета сольватохромного эффекта нами использована следующая процедура: При этом было установлено батохромное смещение полос поглощения для всех экстрак- тов по мере добавления ацетонитрила: – для экстракта плодов черники, содержаще- го неацилированные моногликозиды пяти основ- ных антоцианидинов (дельфинидина, петуниди- на, мальвидина цианидина и пеонидина) – от 515 до 529 нм; Таблица 2. Экстракция антоцианов из плодов кизила за одну стадию Table 2. Extractivity of dogwood fruit anthocyanins for one stage Условия экстракции Образец 1 2 3 4 5 Навеска плодов кизила, m, г 0,493 0,525 0,498 0,620 0,550 Состав экстрагента, мл 0,1 М HCl 25 20 15 10 5 CH3CN 0 5 10 15 20 Экстрагировано антоцианов, г/100 г 0,073 0,086 0,095 0,076 0,079 % 76,8 90,5 100 80,0 83,2 Таблица 2. Экстракция антоцианов из плодов кизила за одну стадию Table 2. Extractivity of dogwood fruit anthocyanins for one stage Таблица 2. Экстракция антоцианов из плодов кизила за одну стадию Table 2. Extractivity of dogwood fruit anthocyanins for one stage ФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯ / PHYSICOCHEMICAL BIOLOGY 694 на Я.Ю., Калиникин Д.А., Дейнека В.И., Дейнека Л.А. Некоторые закономерности … Salasina Y.Yu., Kalinikin D.A., Deineka V.I., Deineka L.A. ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ (1) (1) Поскольку по закону Бугера – Ламберта – Бера концентрация вещества рассчитывается по формуле Рис. 3. Экстрагируемость антоцианов из неразрушенных (а) и растертых (b) высушенных лепестков цветков пионов от числа последовательных экстракций. Экстрагенты: 1 – 0,1 М водный раствор HCl; 2 – 1%-й раствор концентрированной HCl в этаноле; 3 – экстрагент, содержащий 80% об. глиценина и 20% об. 0,1 М водного раствора HCl; 4 – экстрагент, содержащий 50% об. глицерина и 50% об. 0,1 М водного раствора HCl; 5 – экстрагент, содержащий 50% об.1%-го раствора концентрированной HCl в этаноле и 50% об. 0,1 М водного раствора HCl Fig. 3. Extractivity of anthocyanins from whole (a) and crushed (b) dried peony flower petals at consecutive extraction stages. Extragents: 1 – 0,1 М HCl solution in water; 2 – 1% HCl solution in ethanol; 3 – mixture of 80% vol. of glycerol and 20% vol. of 0,1 М HCl solution in water; 4 – mixture of 50% vol. glycerol and 50% vol. of 0,1 М HCl solution in water; 5 – mixture of 50% vol. of 1% HCl solution in ethanol and 50 % vol. of 0,1 М HCl solution in water Рис. 3. Экстрагируемость антоцианов из неразрушенных (а) и растертых (b) высушенных лепестков цветков пионов от числа последовательных экстракций. Экстрагенты: 1 – 0,1 М водный раствор HCl; 2 – 1%-й раствор концентрированной HCl в этаноле; 3 – экстрагент, содержащий 80% об. глиценина и 20% об. 0,1 М водного раствора HCl; 4 – экстрагент, содержащий 50% об. глицерина и 50% об. 0,1 М водного раствора HCl; 5 – экстрагент, содержащий 50% об.1%-го раствора концентрированной HCl в этаноле и 50% об. 0,1 М водного раствора HCl Fig. 3. Extractivity of anthocyanins from whole (a) and crushed (b) dried peony flower petals at consecutive extraction stages. Extragents: 1 – 0,1 М HCl solution in water; 2 – 1% HCl solution in ethanol; 3 – mixture of 80% vol. of glycerol and 20% vol. of 0,1 М HCl solution in water; 4 – mixture of 50% vol. glycerol and 50% vol. of 0,1 М HCl solution in water; 5 – mixture of 50% vol. of 1% HCl solution in ethanol and 50 % vol. of 0,1 М HCl solution in water Рис. 3. Экстрагируемость антоцианов из неразрушенных (а) и растертых (b) высушенных лепестков цветков пионов от числа последовательных экстракций. ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ 4. Экстрагируемость антоцианов из листьев краснокочанной капусты от кратности последовательных экстракций. Условия как в подписях к рис. 3. Рис. 4. Экстрагируемость антоцианов из листьев краснокочанной капусты от кратности последовательных экстракций. Условия как в подписях к рис. 3. Fig. 4. Extractivity of red cabbage leaves anthocyanins at consecutive extraction stages. Conditions as in Fig.3 Fig. 4. Extractivity of red cabbage leaves anthocyanins at consecutive extraction stages. Conditions as in Fig.3 Fig. 4. Extractivity of red cabbage leaves anthocyanins at consecutive extraction stages. Conditions as in Fig.3 ВЫВОДЫ 0,1 М водный раствор HCl является эффек- тивным и экологически чистым экстрагеном ан- тоцианов из многих растительных объектов. До- бавки органических модификаторов эффективны при экстракции из неизмельченного высушенно- го сырья, но при экстракции антоцианов из из- мельченного сырья их добавка не обязательна. При оценке их эффективности следует учиты- вать сольватохромный эффект по предложенно- му в работе способу. При уменьшении концентрации этанола вдвое степень экстракции несколько снижалась, но оказывалась выше, чем без добавок органи- ческого модификатора. Глицерин в количестве 80% об. позволил экстрагировать за две после- довательные экстракции антоцианов больше, чем без органических добавок, но эффектив- ность добавки глицерина уменьшалась при сни- жении его концентрации. Меньшая степень экс- ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ Экстрагенты: 1 – 0,1 М водный раствор HCl; 2 – 1%-й раствор концентрированной HCl в этаноле; 3 – экстрагент, содержащий 80% об. глиценина и 20% об. 0,1 М водного раствора HCl; 4 – экстрагент, содержащий 50% об. глицерина и 50% об. 0,1 М водного раствора HCl; 5 – экстрагент, содержащий 50% об.1%-го раствора концентрированной HCl в этаноле и 50% об. 0,1 М водного раствора HCl Fig. 3. Extractivity of anthocyanins from whole (a) and crushed (b) dried peony flower petals at consecutive extraction stages. Extragents: 1 – 0,1 М HCl solution in water; 2 – 1% HCl solution in ethanol; 3 – mixture of 80% vol. of glycerol and 20% vol. of 0,1 М HCl solution in water; 4 – mixture of 50% vol. glycerol and 50% vol. of 0,1 М HCl solution in water; 5 – mixture of 50% vol. of 1% HCl solution in ethanol and 50 % vol. of 0,1 М HCl l ti i t Рис. 3. Экстрагируемость антоцианов из неразрушенных (а) и растертых (b) высушенных лепестков цветков пионов от числа последовательных экстракций. Экстрагенты: 1 – 0,1 М водный раствор HCl; 2 – 1%-й раствор концентрированной HCl в этаноле; 3 – экстрагент, содержащий 80% об. глиценина и 20% об. 0,1 М водного раствора HCl; 4 – экстрагент, содержащий 50% об. глицерина и 50% об. 0,1 М водного раствора HCl; 5 – экстрагент, содержащий 50% об.1%-го раствора концентрированной HCl в этаноле и 50% об. 0,1 М водного раствора HCl ФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯ / PHYSICOCHEMICAL BIOLOGY 6 695 Саласина Я.Ю., Калиникин Д.А., Дейнека В.И., Salasina Y.Yu., Kalinikin D.A., Deineka Рис. 4. Экстрагируемость антоцианов из листьев краснокочанной капусты от кратности последовательных экстракций. Условия как в подписях к рис. 3. Fig. 4. Extractivity of red cabbage leaves anthocyanins at consecutive extraction stages. Conditions as in Fig.3 сина Я.Ю., Калиникин Д.А., Дейнека В.И., Дейнека Л.А. Некоторые закономерности … Salasina Y.Yu., Kalinikin D.A., Deineka V.I., Deineka L.A. Some regularities … тракции в случае высокой концентрации глице- рина может быть связана с необходимостью предварительного смачивания лепестков перед экстракцией. Во втором варианте при соотноше- нии масса измельченных лепестков : объем экс- трагента 1 : 100, г/мл – при экстракции из размо- лотых лепестков эффективность экстрагентов в значительной степени выровнялась, поэтому добавки органического растворителя не обяза- тельны. В случае свежих листьев краснокочанной ка- пусты добавление органического модификатора в экстрагент оказалось ненужным, наивысшую эффективность в процессах экстракции проявил 0,1 М водный раствор HCl (см. рис. 3). Рис. БИБЛИОГРАФИЧЕСКИЙ СПИСОК 1. Trouillas P., Sancho-García J.C., De Freitas V., Gierschner J., Otyepka M., Dangles O. Stabiliz- ing and modulating color by copigmentation: In- sights from theory and experiment // Chemical re- views. 2016. Vol. 116. P. 4937−4982. https://doi.org/ 10.1021/acs.chemrev.5b00507 R. (eds.) Phenolic Compounds. 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Determina- tion of total monomeric anthocyanin pigment content of fuit juices, beverages, natural colorants, and wines by the pH differential method: Collaborative study. Journal of AOAC International. 2005;88(5):1269–1278. 20. Escribano-Bailon MT, Santos-Buelga C. An- thocyanin Copigmentation – Evaluation, Mecha- nisms and Implications for the Colour of Red Wines. Current Organic Chemistry. 2012;16(6):715–723. https://doi.org/10.2174/138527212799957977 ( ) 17. Bąkowska-Barczak A. Acylated anthocya- nins as stable, natural food colorants – A review. Polish Journal of Food and Nutrition Sciences. 17. Bąkowska-Barczak A. Acylated anthocya- nins as stable, natural food colorants – A review. Polish Journal of Food and Nutrition Sciences. Саласина Ярослава Юрьевна, Саласина Ярослава Юрьевна, аспирант, Институт фармации, химии и биологии Белгородского государственного национального исследовательского университета, 308015, г. Белгород, ул. Победы, 85, Российская Федерация, e-mail: kulchenko@bsu.edu.ru аспирант, Институт фармации, химии и биологии Белгородского государственного национального исследовательского университета, Калиникин Данила Андреевич, Институт фармации, химии и биологии Белгородского государственного национального исследовательского университета, 308015, г. Белгород, ул. Победы, 85, Российская Федерация, e-mail: 1318753@bsu.edu.ru Danila S. Kalinikin, Institute of Pharmacy, Chemistry and Biology, Belgorod State National Research University, 85, Pobedy St., Belgorod, 308015, Russian Federation, e-mail: 1318753@bsu.edu.ru Danila S. Kalinikin, Institute of Pharmacy, Chemistry and Biology, Belgorod State National Research University, 85, Pobedy St., Belgorod, 308015, Russian Federation, e-mail: 1318753@bsu.edu.ru Viktor I. Deineka, Dr. Sci (Chemistry), Professor, Department of General Chemistry, Institute of Pharmacy, Chemistry and Biology, Belgorod State National Research University, 85, Pobedy St., Belgorod, 308015, Russian Federation, e-mail; deineka@bsu.edu.ru Lyudmila A. Deineka, Cand. Sci. (Chemistry), Associate Professor, Department of General Chemistry, Institute of Pharmacy, Chemistry and Biology, Belgorod State National Research University, 85, Pobedy St., Belgorod, 308015, Russian Federation, e-mail; deyneka@bsu.edu.ru INFORMATION ABOUT THE AUTHORS Yaroslava Yu. Salasina, Postgraduate Student, Institute of Pharmacy, Chemistry and Biology, Belgorod State National Research University, 85, Pobedy St., Belgorod, 308015, Russian Federation, e-mail: kulchenko@bsu.edu.ru Yaroslava Yu. Salasina, Postgraduate Student, Institute of Pharmacy, Chemistry and Biology, Belgorod State National Research University, 85, Pobedy St., Belgorod, 308015, Russian Federation, e-mail: kulchenko@bsu.edu.ru Yaroslava Yu. Salasina, Дейнека Людмила Александровна, Дейнека Людмила Александровна, к.х.н., доцент, доцент кафедры общей химии, Институт фармации, химии и биологии Белгородского государственного национального исследовательского университета, 308015, г. Белгород, ул. Победы, 85, ФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯ / PHYSICOCHEMICAL BIOLOGY 698 ласина Я.Ю., Калиникин Д.А., Дейнека В.И., Дейнека Л.А. Некоторые закономерности … Salasina Y.Yu., Kalinikin D.A., Deineka V.I., Deineka L.A. Some regularities … Российская Федерация, e-mail: deyneka@bsu.edu.ru Российская Федерация, Российская Федерация, e-mail: deyneka@bsu.edu.ru д р ц , e-mail: deyneka@bsu.edu.ru Contribution of the authors The authors contributed equally to this article. Заявленный вклад авторов Все авторы сделали эквивалентный вклад в подготовку публикации. Заявленный вклад авторов Все авторы сделали эквивалентный вклад в подготовку публикации. Conflict interests The authors declare no conflict of interests re- garding the publication of this article. Conflict interests The authors declare no conflict of interests re- garding the publication of this article. Конфликт интересов Авторы заявляют об отсутствии конфликта интересов. Все авторы прочитали и одобрили оконча- тельный вариант рукописи. The final manuscript has been read and approved by all the co-authors. Статья поступила в редакцию 20.05.2020; одобрена после рецензирования 14.11.2020; принята к публикации 30.11.2020. The article was submitted 20.05.2020; approved after reviewing 14.11.2020; accepted for publication 30.11.2020. ФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯ / PHYSICOCHEMICAL BIOLOGY 699
https://openalex.org/W2169798151	http://diposit.ub.edu/dspace/bitstream/2445/127749/1/645393.pdf	English	null	Mfn2 downregulation in excitotoxicity causes mitochondrial dysfunction and delayed neuronal death	EMBO journal	2,014	public-domain	20,916	Mfn2 downregulation in excitotoxicity causes mitochondrial dysfunction and delayed neuronal death Alejandro Martorell-Riera1,2, Marc Segarra-Mondejar1,2, Juan P. Muñoz3,4,5, Vanessa Ginet6, Jordi Olloquequi7, Jeús Pérez-Clausell1, Manuel Palacín3,4, Manuel Reina1,2, Julien Puyal6, Antonio Zorzano3,4,5, Francesc X. Soriano1,2* Alejandro Martorell-Riera1,2, Marc Segarra-Mondejar1,2, Juan P. Muñoz3,4,5, Vanessa Ginet6, Jordi Olloquequi7, Jeús Pérez-Clausell1, Manuel Palacín3,4, Manuel Reina1,2, Julien Puyal6, Antonio Zorzano3,4,5, Francesc X. Soriano1,2* Alejandro Martorell-Riera1,2, Marc Segarra-Mondejar1,2, Juan P. Muñoz3,4,5, Vanessa Ginet6, Jordi Olloquequi7, Jeús Pérez-Clausell1, Manuel Palacín3,4, Manuel Reina1,2, Julien Puyal6, Antonio Zorzano3,4,5, Francesc X. Soriano1,2* 1 Department of Cell Biology, 2 CELLTEC-UB, 3 Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Av. Diagonal, 643, 08028 Barcelona, Spain 1 Department of Cell Biology, 2 CELLTEC-UB, 3 Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Av. Diagonal, 643, 08028 Barcelona, Spain 4 Institute for Research in Biomedicine (IRB Barcelona), C/ Baldiri Reixac 10, 08028 Barcelona, Spain 5 CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III 6 Department of Fundamental Neuroscience, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland 7 Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, 5 Poniente Nº 1670, 3460000 Talca (Chile) 4 Institute for Research in Biomedicine (IRB Barcelona), C/ Baldiri Reixac 10, 08028 Barcelona, Spain 5 CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III 6 Department of Fundamental Neuroscience, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland 7 Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, 5 Poniente Nº 1670, 3460000 Talca (Chile) 7 Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, 5 Poniente Nº 1670, 3460000 Talca (Chile) * Correspondence to: Francesc X. Soriano, Department of Cell Biology, Faculty of Biology, University of Barcelona, Av. Diagonal, 643, 08028 Barcelona, Spain Tel. +34 934029046, Fax +34934034607. f.x.soriano@ub.edu Runnig tittle: Mfn2 downregulation in delayed excitotoxicity 1 Abstract Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented but the mechanism underlying this process is poorly understood. Here we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeostasis and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons, and that excitotoxicity-dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke. Key words: Excitotoxicity/ mitochondrial dynamics/ neuron/ transcriptional regulation. 2 Introduction Glutamate is the main excitatory neurotransmitter in the central nervous system. It plays an essential role in development, synaptic plasticity and neuronal survival but sustained elevated levels of extracellular glutamate kill neurons in a process called excitotoxicity (Arundine & Tymianski, 2003). Excitotoxicity takes place in both chronic neurological diseases, such as Huntington’s and Alzheimer’s disease, and in acute episodes such as traumatic brain injury and ischemic stroke. The NMDA receptor (NMDAR) is the main ionotropic glutamate receptor in the CNS and the excessive flux of Ca2+ that passes through it is a major cause of excitotoxicity. Despite evidence indicating a crucial role of NMDAR activation in brain damage during stroke, clinical trials with NMDAR blockers have failed because of poor tolerance and efficacy (Ikonomidou & Turski, 2002; Muir, 2006). In contrast to excessive NMDAR activity that causes cell death, its physiological activity triggers pro-survival signals that may play a role in promoting recovery and preventing delayed neuronal loss in the penumbra (Ikonomidou & Turski, 2002; Lo, 2008). Thus, future therapies to reduce excitotoxicity must target pro-death events downstream of NMDAR without affecting the pro-survival signals. Several mechanisms are implicated in cell death triggered by Ca2+ influx through NMDAR. Mitochondrial dysfunction caused by excessive Ca2+ uptake acts as a signaling hub for many pro-death events (Almeida et al, 1999; Reynolds & Hastings, 1995; Soriano et al, 2008; Stout et al, 1998; Yu et al, 2002). Mitochondria are dynamic organelles that constantly fuse and divide, changing shape Mitochondria are dynamic organelles that constantly fuse and divide, changing shape and localization. The equilibrium between fission and fusion is important for mitochondrial function, which is not only limited to supplying energy to the cell, but also intervenes in anabolic and catabolic biochemical pathways and the regulation of 3 Ca2+ homeostasis, and is a key regulator of cell death progression. The core mitochondrial fusion and fission machineries are formed by a group of dynamin-related large GTPases (Liesa et al, 2009; Westermann, 2010). Inner mitochondrial membrane fusion is mediated by Opa1. Two Mitofusins (Mfn), Mfn1 and 2, mediate mitochondrial outer membrane fusion. Mfn1 and Mfn2 display high homology (81%) and around 60% identity, but nonetheless they have non- redundant roles (de Brito & Scorrano, 2008b; Liesa et al, 2009). Introduction In addition to its fusion role Mfn2 localization in the ER is necessary to maintain the reticular morphology of the ER and control the ER–mitochondria interaction (de Brito & Scorrano, 2008a). Mfn2 may also play a role in neuronal mitochondrial trafficking, and disruption of this function can lead to axon degeneration (Baloh et al, 2007; Misko et al, 2012). Mitochondrial fission is mediated by Drp1. Drp1 is mainly cytoplasmic and its translocation to mitochondria, recruited by Fis1 and/or Mff, is essential for mitochondrial fission. Drp1 is subjected to several posttranscriptional modifications, including phosphorylation, ubiquitination, SUMOylation and nitrosylation, which can either activate or repress fission activity (Cho et al, 2010; Oettinghaus et al, 2012). Although mitochondrial fission per se does not cause cell death, fragmentation of mitochondria has been shown to play a key role in cell death progression. Mitochondrial fragmentation occurs early in apoptosis and can be delayed by expressing a dominant negative Drp1 (Breckenridge et al, 2003; Frank et al, 2001). Recently, Drp1 has also been implicated in the induction of necrosis (Wang et al, 2012). Mutations in Mfn2 are the most commonly identified cause of Charcot-Marie-Tooth type 2 (CMT2), a dominantly inherited disease characterized by degeneration of peripheral sensory and motor axons (Zuchner et al, 2004). Purkinje cells require Mfn2 but not Mfn1 for dendritic outgrowth, spine formation, and cell survival (Chen et al, 2007). 4 Mitochondrial fragmentation is an early event that occurs before the release of mitochondrial proteins and neurite degeneration in an in vivo animal model of stroke (Barsoum et al, 2006). Despite the importance of mitochondrial dynamics in cell death progression the exact mechanism that underlies the mitochondrial fragmentation in excitotoxicity is incompletely understood. In this study, we assessed how the proteins of the core mitochondrial fusion/fission machinery are regulated in excitotoxicity. We found that Mfn2 levels are reduced in both in-vitro and in-vivo models of excitotoxicity, via MEF2 degradation that, by acting on the Mfn2 promoter, regulates basal levels of Mfn2. Downregulation of Mfn2 causes mitochondrial dysfunction and altered Ca2+ homeostasis, and facilitate Bax recruitment to mitochondria during excitotoxicity. 5 Mfn2 protein expression is reduced in excitotoxicity Mitochondrial dynamics plays a pivotal role in cell death. Changes in mitochondrial morphology have been observed in excitotoxicity but the precise mechanism has not been fully defined. For a better understanding of the mechanism by which mitochondria are fragmented during excitotoxicity we exposed primary cortical cultures to moderate doses (30 M) of the glutamate receptor agonist NMDA over a time course of 1, 2, 4 and 8 hours and analyzed the expression of the proteins of the mitochondrial fission/fusion machinery. During the first 2 hours after NMDA application there were no significant changes in either fusion or fission proteins but after 4 hours of NMDA treatment we observed a 40% reduction in the fusion protein Mfn2 with no changes in the other fusion proteins, Mfn1 and Opa1. Surprisingly, Drp1 showed a tendency to reduce its protein levels (Fig. 1A, B). To rule out the possibility that changes in the expression of mitochondrial fission/fusion protein were due to changes in mitochondrial mass, volume normalization was also performed with the mitochondrial protein porin, achieving similar results (Fig. E1D). Using oxygen and glucose deprivation (OGD) as another in vitro model of excitotoxicity, we also found that the level of Mfn2 was reduced 4 hours after reoxigenation with no changes in the other proteins of the fission/fusion machinery (Fig. E1B and C). To check the pathophysiological relevance of these in vitro findings, we then used an in vivo model of cerebral ischemia in 12 days old rats consisting in applying a permanent middle cerebral artery occlusion combined with 90 minutes of transient occlusion of the ipsilateral common carotid artery (Vaslin et al, 2009). During an ischemic episode, glutamate levels build up as a result of synaptic release and impaired and/or reversed 6 uptake mechanisms (Camacho & Massieu, 2006), which induces excessive activation of NMDA glutamate receptors (NMDARs) and Ca2+-mediated cell death (Arundine & Tymianski, 2003). We analyzed the expression of the mitochondrial fusion/fission machinery at several time points after the ischemic insult. The results were in the same trend to those found in vitro. Two hours after restoration of the common carotid artery blood flow, i.e. 3.5 hours after the ischemia onset, Mfn2 protein levels started to decline, and reached a significant 50% reduction 6 hours after the restoration of the common carotid artery blood flow (Fig. 1C,D and Fig. 1SD). Mfn2 protein expression is reduced in excitotoxicity Drp1 also showed a slight but significant reduction (20%) 2 hours after the restoration of blood flow. Neither of the other mitochondrial fusion proteins (Mfn1 and Opa1) showed changes in levels during the 24 hours after the onset of ischemia. Thus, our in vitro neuronal cultures recapitulate well the excitotoxic in vivo model. Activation of Drp1 induces mitochondrial fragmentation We observed the downregulation of Mfn2 4 hours after initiating the insult, but nonetheless the kinetics of excitotoxicity-mediated mitochondrial fragmentation has been reported to be fast (Barsoum et al, 2006; Rintoul et al, 2003; Young et al, 2010). To clarify this, we analyzed the mitochondrial morphology of those neurons expressing RFP targeted to the mitochondrial matrix (mtRFP) that were still alive (as shown by nuclear DAPI staining; Fig. E2A) after NMDA treatment of different durations. Untreated neurons contained mainly well-connected tubular mitochondria (60%). After only 30–60 minutes of NMDA application most of the neurons contained globular mitochondria that remained intact for the following 2 hours with a small amount of additional fragmentation 4 hours after NMDA treatment (Fig. 2A and B). Although the 7 7 secondary fragmentation correlates with the diminished Mfn2 expression, it is unlikely that Mfn2 is responsible for the first and early phase of mitochondrial fragmentation. We observed a tendency for a decline in the level of Drp1 in excitotoxicity (Fig. 1). Nonetheless, what determines Drp1 activity is its subcellular localization. Drp1 is mainly cytosolic and by posttranslational modifications it is recruited to the mitochondria by Mff and/or Fis1 where it promotes fission (Gandre-Babbe & van der Bliek, 2008; James et al, 2003; Otera et al, 2010; Yoon et al, 2003). In basal conditions GFP-Drp1 transfected neurons showed a weak diffuse signal within the neuron. Application of NMDA for 1 hour produced a strong punctuate signal that co-localized with mitochondria (Fig. 2C). Overexpression of a mutant form of Drp1 (Drp1-K38A), which acts as a dominant negative (Smirnova et al, 1998), or using the Drp1 inhibitor mdivi-1, significantly attenuated the NMDA-mediated mitochondrial fragmentation during the first hour of NMDA treatment (Fig. 2D-E). Excessive calcium uptake by mitochondria in excitotoxicity causes mitochondrial depolarization (Nicholls, 2009; Soriano et al, 2006b), this produces an increase in cytosolic calcium that can activate calcineurin which has been shown to de-phosphorylate Drp1 to promote its recruitment to mitochondria and fission (Cereghetti et al, 2008). We analyzed whether mitochondrial fission preceded or was posterior to mitochondrial depolarization. We found that one minute before fission mitochondria have lost 60% of the mitochondrial membrane potential , which continues to dissipate progressively after fission (Fig. E2B and C). We next analyzed the possible involvement of calcineurin in the NMDA- mediated mitochondrial fission. Inhibition of calcineurin with cyclosporine A (CsA) did not prevent mitochondrial fission (Fig. Activation of Drp1 induces mitochondrial fragmentation E2D) as neither did the inhibition of CaMK, a calcium dependent kinase that activates Drp1 (Han et al, 2008), with KN-62. Drp1 is also activated by nitrosylation (Cho et al, 2009). As it had been reported previously We observed a tendency for a decline in the level of Drp1 in excitotoxicity (Fig. 1). Nonetheless, what determines Drp1 activity is its subcellular localization. Drp1 is mainly cytosolic and by posttranslational modifications it is recruited to the mitochondria by Mff and/or Fis1 where it promotes fission (Gandre-Babbe & van der Bliek, 2008; James et al, 2003; Otera et al, 2010; Yoon et al, 2003). In basal conditions GFP-Drp1 transfected neurons showed a weak diffuse signal within the neuron. Application of NMDA for 1 hour produced a strong punctuate signal that co-localized with mitochondria (Fig. 2C). Overexpression of a mutant form of Drp1 (Drp1-K38A), which acts as a dominant negative (Smirnova et al, 1998), or using the Drp1 inhibitor mdivi-1, significantly attenuated the NMDA-mediated mitochondrial fragmentation during the first hour of NMDA treatment (Fig. 2D-E). Excessive calcium uptake by mitochondria in excitotoxicity causes mitochondrial depolarization (Nicholls, 2009; Soriano et al, 2006b), this produces an increase in cytosolic calcium that can activate calcineurin which has been shown to de-phosphorylate Drp1 to promote its recruitment to mitochondria and fission (Cereghetti et al, 2008). We analyzed whether mitochondrial fission preceded or was posterior to mitochondrial depolarization. We found that one minute before fission mitochondria have lost 60% of the mitochondrial membrane potential , which continues to dissipate progressively after fission (Fig. E2B and C). We next analyzed the possible involvement of calcineurin in the NMDA- mediated mitochondrial fission. Inhibition of calcineurin with cyclosporine A (CsA) did not prevent mitochondrial fission (Fig. E2D) as neither did the inhibition of CaMK, a calcium dependent kinase that activates Drp1 (Han et al, 2008), with KN-62. Drp1 is also activated by nitrosylation (Cho et al, 2009). As it had been reported previously 8 (Barsoum et al, 2006; Cho et al, 2009), here we also showed that inhibition of the nitric oxide synthase with 7-Nitroindazole (7-Ni) blocked the mitochondrial fragmentation to the same extent as the mutant Drp1-K38A or mdivi-1 (Fig. 2E). These experiments indicate that NO-mediated Drp1 activation plays a key role in mitochondrial fragmentation during excitotoxicity, although other mechanisms may also be involved. Mfn2 intervenes in an irreversible late phase of mitochondrial fragmentation Previous studies have shown that glutamate and nitric oxide mediated mitochondrial fragmentation are amenable to restoration once the stimulus has ceased (Barsoum et al, 2006; Rintoul et al, 2003). Thereby, we decided to test the reversibility of mitochondrial morphology in our system. We applied NMDA for 1 hour and then washed out the NMDA and returned to fresh media. We observed that the mitochondrial tubular morphology was recovered 90 minutes after replacing the media but that after 180 minutes the mitochondria fragmented once again (Fig. 3A). The events that triggered the decline in Mfn2 levels began during the first hour of NMDA exposure since subsequent washout of the agonist did not block the reduction in Mfn2 (Fig. 3B). Because the delayed phase of mitochondrial fragmentation correlates with the reduction in Mfn2 protein levels (Fig. 1A, Fig. 2B and Fig. 3A, B) we reasoned that the reduction in Mfn2 levels in excitotoxicity intervenes in late mitochondrial fission. We knocked down Mfn2 using shRNA, which targets its sequence (shMfn2). Importantly, shMfn2 reduced Mfn2 protein levels by around 50%, resembling the reduction observed in excitotoxicity (Fig. 3C). As expected, knockdown of Mfn2 was sufficient to cause mitochondrial fragmentation (Fig. 3D). Another two shRNAs targeting Mfn2 at different sequences also showed mitochondrial fragmentation (Fig. E3A and B). 9 Conversely, exogenous expression of Mfn2 significantly blocked the late phase of mitochondrial fragmentation after NMDA washout (Fig. 3E and F). All of this indicates that excitotoxicity promotes mitochondrial fragmentation by at least two mechanisms, a fast mechanism relying on Drp1 activation that lasts while the insult is present, and a late mechanism dependent on the reduction in Mfn2 expression that takes place hours after the insult is generated and persists independently of the removal of the insult. Reduced Mfn2 expression causes mitochondrial dysfunction and altered Ca In addition to its mitochondrial fusion activity, Mfn2 has unique properties which are not shared with Mfn1, such as activating mitochondrial oxidative metabolism or tethering of mitochondria with the ER regulating Ca2+ homeostasis (Bach et al, 2003; Baloh et al, 2007; de Brito & Scorrano, 2008a; Pich et al, 2005). Since mitochondrial dysfunction and altered Ca2+ homeostasis are hallmarks of excitotoxicity we wondered how decreased levels of Mfn2 could affect mitochondrial function. Respirometric assays showed decreased respiratory control ratio (RCR) and spare respiratory capacity (SRC) in Mfn2 KD neurons (Fig. 4A, B). Determination of mitochondrial membrane potential showed that mitochondria in Mfn2 KD neurons had 15% lower mitochondrial membrane potential than control transfected cells (Fig. 4C), in concordance with what has been observed in other cell types (Bach et al, 2003; Soriano et al, 2006a). In agreement with lower SRC, when neurons were challenged with subtoxic doses of NMDA (15 M) mitochondrial membrane potential loss was greatly enhanced in Mfn2 KD neurons (Fig. 4C). Concomitantly, when the culture medium was changed to a medium with pyruvate but without glucose just before the NMDA application (to rely 10 10 solely on mitochondrial metabolism) ATP levels decreased significantly in Mfn2 KD neurons (Fig 4D). All together these results indicate an impaired mitochondrial function in the Mfn2 KD neurons. Because mitochondria play a key role in buffering the increase in cytosolic Ca2+ produced during excitotoxicity, which depends on proper mitochondrial membrane potential (Nicholls, 2009), we reasoned that Ca2+ homeostasis may be impaired in Mfn2 KD neurons. Under basal conditions Mfn2 KD neurons have a slightly lower (9.9 ±3.52%) mitochondrial Ca2+ concentration than control neurons (Fig. 4E, F). The application of NMDA produced a lower increase of the mitochondrial Ca2+ in the Mfn2 KD neurons than in the control neurons (Fig. 4E, F). Oppositely, the level of cytoplasmic Ca2+ was moderately higher in the Mfn2 KD neurons and NMDA application produced a bigger increase in the Mfn2 KD neurons (Fig 4G, 4H, E4A). Because Rhod-2 localization to mitochondria is voltage sensitive and lower mitochondrial membrane potential could lead to reduced Rhod-2 fluorescence we also analyzed cytoplasmic Ca2+ and we found that, oppositely to mitochondrial Ca2+, cytoplasmic Ca2+ was moderately higher in Mfn2 KD neurons and NMDA application produced bigger increase in Mfn2 KD neurons (Fig 4G, 4H, E4A). Reduced Mfn2 expression causes mitochondrial dysfunction and altered Ca Increase in cytoplasmic Ca2+ was due to reduced mitochondrial Ca2+ uptake since depolarization of mitochondria with CCCP, hence abolishment of mitochondrial Ca2+ uptake, produced the same increase in NMDA mediated rise in cytoplasmic Ca2+ in control or Mfn2 KD neurons (Fig. E4B). Together, these data support the notion that a reduction in Mfn2 causes mitochondrial malfunction, which causes disturbances in Ca2+ homeostasis. Mfn2 reduction intervenes in delayed excitotoxic death. 11 11 Given that reduced levels of Mfn2 had a great impact on mitochondrial membrane potential and Ca2+ handling when neurons were treated with low doses of NMDA, next we investigated whether the reduction in Mfn2 could have consequences for cell viability. We showed that this was indeed the case, as knockdown of Mfn2 sensitized neurons to subtoxic doses of NMDA (Fig. 5A). Another two shRNAs targeting Mfn2 at different sequences also showed sensitization to subtoxic doses of NMDA, ruling out the possibility of an off-target effect (Fig. E5A). Next, we overexpressed Mfn2 and observed an almost two fold protection to the NMDA-induced neuronal death (Fig. 5B).The protection was not due to the restoration of mitochondrial morphology since overexpression of Mfn1 did not show significant protection to NMDA (Fig. E5B). Shortly after exposure to glutamate, a subpopulation of neurons died by necrosis. Surviving neurons can recover or transiently and undergo delayed cell death, depending on mitochondrial function (Ankarcrona et al, 1995; Luetjens et al, 2000). We studied how exogenous expression of Mfn2 could affect the different phases of excitotoxic cell death. We determined cell death after an hour treatment with NMDA (30 M) followed by the wash out of NMDA and allowed the neurons to recover for different length of time. After 1.5 hours recovery (i.e. 2.5 h from the onset of the insult), nearly 40% of the neurons died with no protection by Mfn2 expression. From then on, and correlating with endogenous Mfn2 reduction, cell death in the neurons that were transfected with the control vector increased progressively but Mfn2 expression completely blocked the delayed cell death (Fig. 5C). Given that several mechanism are responsible for the NMDA-mediated cell death, to Given that several mechanism are responsible for the NMDA-mediated cell death, to better characterize the mechanism by which reduction of Mfn2 causes delayed excitotoxicity we used NMDA at 15 M, which only causes death when Mfn2 is knocked down (Fig 5A). Analysis of the NAD+/NADH ratio as a readout of the 12 metabolic status of the neuron did not indicate that an energetic collapse was responsible for the neuronal death when Mfn2 expression was reduced (Fig. E5C). Evidences indicate that opening of the permeability transition pore (mPTP) and necrotic death occurs during the early phase of excitotoxicity as opposed to apoptosis that occurs in delayed excitotoxicity (Dirnagl et al, 1999). Mfn2 reduction intervenes in delayed excitotoxic death. Consequently, inhibition of the mPTP with CsA failed to protect Mfn2 KD neurons to low doses of NMDA (15 M; Fig. E5D). During delayed excitotoxic cell death Bax translocates to mitochondria to produce the mitochondrial outer membrane permeabilization (MOMP) (D'Orsi et al, 2012; Shelat et al, 2013; Xiang et al, 1998). Bax is known to interact with Mfn2 (Brooks et al, 2007; Karbowski et al, 2006) and Mfn2 interferes with Bax activation (Neuspiel et al, 2005; Sugioka et al, 2004). Bax siRNA blocked the NMDA-dependent neuronal death in Mfn2 KD neurons (Fig 5D). In addition, overexpression of the anti- apoptotic Bcl-xL protein also blocked the neuronal death (Fig. 5E). In agreement with the reported activation of calpain downstream of Bax activation (D'Orsi et al, 2012), by measuring cleavage of spectrin (calpain substrate) we observed an enhanced calpain activation in Mfn2 KD neurons (Fig. E5E). According to the role of Blc-2 family proteins regulating mitochondrial dynamics by using siBax and Bcl-xL overexpression partially prevented the delayed mitochondrial fragmentation after NMDA application (Fig. E5F, G). N di d h h i f Mf 2 ff d B l i Next, we studied whether exogenous expression of Mfn2 affected Bax translocation to mitochondria in excitotoxicity. We observed that in GFP-Bax expressing neurons Bax was mainly cytoplasmic and that NMDA (30 M) application resulted in Bax translocation to mitochondria, forming the typical foci on the tips of the mitochondria (Karbowski et al, 2002; Yuan et al, 2006); Fig. 5F). Bax translocation to mitochondria was progressive after NMDA treatment and Mfn2 expressing neurons showed less 13 mitochondrial Bax than globin (control) expressing neurons (Fig. 5F, G). As expected, NMDA-mediated delayed cytochrome c release was blocked by Mfn2 expression in a similar pattern to mitochondrial Bax localization (Fig. 5H, I). All these data data indicate that the late reduction of Mfn2 levels impairs mitochondrial function and enhances delayed excitotoxic death by promoting Bax recruitment to mitochondria. Mfn2 is regulated at the transcriptional level in excitotoxicity Next we investigated the mechanism by which Mfn2 expression is reduced in excitotoxicity. It is well known that Mfn2 is degraded by the proteasome in a Parkin dependent manner (Chan et al, 2011; Tanaka et al, 2010). We wondered whether the decrease in Mfn2 levels in excitotoxicity was due to its proteasomal degradation. Surprisingly, we found that pretreatment of neurons with the proteasome inhibitor MG- 132 did not abolish the NMDA-dependent reduction in Mfn2 (Fig. 6A, B) even when the concentration of MG-132 used (10 M) promoted the accumulation of ubiquitinated proteins and effectively blocked the CCCP-mediated degradation of Mfn2 (Fig. E6A, B). Therefore we can conclude that excitotoxic dependent downregulation of Mfn2 is not dependent on proteasomal degradation. Excitotoxicity causes changes in transcriptional programs (Cook et al, 2012; Zhang et al, 2007), which could be consistent with the slow effect of NMDA on Mfn2 expression. We investigated whether Mfn2 reduction was due to changes in its gene expression. Mfn2 gene expression showed an initial increase during the first hour after NMDA application, probably a defensive response to malfunctioning mitochondria. 14 14 Mfn2 gene expression started to decrease during the second hour, reaching a plateau after 4 hours that remained steady for up to 8 hours of treatment and correlated with the time course of protein expression (Fig. 6C). Neurons subjected to OGD also showed downregulation of Mfn2 mRNA expression (Fig. E6C).The repression of Mfn2 expression is specific since mitochondrial Mfn1 and cytoplasmic SESN2 expression were not affected by NMDA treatment (Fig. 6C). These data indicate that Mfn2 gene expression is downregulated during excitotoxicity, but do not rule out the possibility that other proteases could participate in the excitotoxicity mediated reduction in Mfn2. To verify that a transcriptional change is the main mechanism by which Mfn2 expression is reduced in excitotoxicity, we used the transcriptional inhibitor actinomycin D or the translational inhibitor cycloheximide. As expected, the use of these inhibitors reduced Mfn2 protein levels but additional application of NMDA did not produce a further reduction in Mfn2, as would be expected if a proteolytic process was responsible for NMDA-mediated Mfn2 downregulation (Fig. 6 D, E). Thus, all these sets of experiments clearly indicate that the main mechanism by which NMDA mediates Mfn2 downregulation is at the transcriptional level rather than being a proteolytic process. Excitotoxicity mediated Mfn2 downregulation depends on MEF2 ROS is an important regulator of cell signaling (Holmstrom & Finkel, 2014). We first examined whether mitochondrial ROS produced in excitotoxicity could mediate Mfn2 downregulation. The use of general scavenger trolox, or apocynin, an inhibitor of NADPH oxidase, which is the major source of ROS in excitotoxicity (Brennan et al, 2009), failed to block NMDA-mediated Mfn2 downregulation (Fig. E7A). 15 15 MEF2(A–D) are transcription factors that play an important role in neuronal development and viability (Flavell et al, 2008; Mao et al, 1999). During excitotoxicity MEF2s are cleaved by caspases generating DNA binding domains without the transactivation domain, which acts as a dominant negative interfering form (Li et al, 2001; Okamoto et al, 2002). Time-course analysis of protein extracts of cortical neurons exposed to NMDA, subjected to OGD or brains from rats that have been subjected to ischemia showed a calcium dependent reduction of MEF2A in a pattern that correlated well with Mfn2 expression (Fig. 7A, B and Fig. E7B, C). This raises the possibility that NMDA-dependent cleavage of MEF2s may be responsible for Mfn2 downregulation in excitotoxicity. Neurons expressing the DNA binding domain of MEF2 without the transactivation domain (MEF2-DN) showed reduced expression of Mfn2 protein and mRNA (Fig. 7C). Consistent with the reduced expression of Mfn2, MEF2-DN transduced neurons contained fragmented mitochondria whose morphology was restored by expressing exogenous Mfn2 (Fig. 7D, E). Mitochondria of MEF2-DN expressing neurons showed reduced mitochondrial membrane potential that could be raised by overexpression of Mfn2 (Fig. 7F). MEF2-DN expression caused neuronal death when doses of NMDA at the threshold of toxicity (15 M) were applied, but co- expression of Mfn2 completely blocked neuronal death (Fig. 7G, H). All these results demonstrate that Mfn2 expression is regulated by MEF2 but do not answer the question of whether MEF2 degradation is the main factor mediating Mfn2 downregulation in excitotoxicity. Thus, we analyzed the levels of Mfn2 mRNA in neurons transduced with AAV coding MEF2-DN and observed that Mfn2 downregulation with respect to its control (GFP transduced) neurons was not additively enhanced when NMDA was applied, suggesting that they act in a common pathway (Fig. 7C; note that the AAV transduction efficiency was around 75-90%, thus the results underestimate the actual 16 16 MEF2-DN-dependent repression). Together, these data demonstrate a crucial role of MEF2 in the excitotoxicity-dependent downregulation of Mfn2. MEF2A directly regulates basal Mfn2 expression in neurons Next, we investigated whether MEF2 could directly regulate Mfn2 expression. In promoter reporter assays, MEF2-DN repressed the activity of human Mfn2 promoter in neurons but did not repress the activity of SESN2 promoter, consistent with the unchanged expression of SESN2 in excitotoxicity (Fig. 8A). The effect of MEF2-DN on the Mfn2 promoter was neuron specific since it did not affect the promoter activity in 10T1/2 fibroblasts, a cell line with far less expression of MEF2A than neurons (Fig. 8A and Fig. E8A). These results are consistent with a prevalent role of MEF2 in the regulation of basal Mfn2 expression in neurons. Next we determined the cis-elements involved in the effects of MEF2A on the transcriptional activity of the Mfn2 promoter. In agreement with the low expression of MEF2A in 10T1/2 cells, over-expression of MEF2A strongly activated the Mfn2 promoter (Fig. 8B). Deletion analysis of the Mfn2 promoter identified the MEF2A activated region as being between -1332 and -668 relative to the transcription start site (Fig. 8B). The MEF2 transcription factors bind DNA in A/T rich sequences (Black & Olson, 1998). The sequence -1332/-862 contains two putative MEF2 binding sites (Fig. E8B). In order to show the functional role of these putative MEF2 binding sites we disrupted the A/T rich sequence, introducing C and G by directed mutagenesis (Fig. E8B). Mutation of BOX 1 did not modify the MEF2A induced promoter activation but mutation of BOX 2 cancelled the effect of MEF2A (Fig. 8C). These results indicated 17 17 that BOX 2 is the cis-element required for MEF2A mediated activation of the Mfn2 promoter. Since MEF2A was able to trans-activated the Mfn2 promoter, EMSA experiments were performed in order to ascertain whether MEF2A was bound to BOX 2. A DNA fragment encompassing BOX 2 was radioactively labeled, and incubated with nuclear extracts of HeLa cells overexpressing MEF2A. MEF2A bound BOX 2 containing probe, inducing the typical double band mobility shift (Santalucia et al, 2001), but did not bind the mutated probe (Fig. 8D). The retarded bands were competed with a 25- or 100-fold excess of unlabeled oligonucleotide probe or 100-fold excess of a probe of the Glut4 promoter that has previously been shown to bind MEF2 (Santalucia et al, 2001), whereas the cold mutated probe was unable to compete with the retarded bands. In addition, an antibody against MEF2 supershifted the complex (Fig. 8E). Discussion Mitochondria are dynamic organelles that continuously fuse and divide. Changes in mitochondrial dynamics have profound effects on mitochondrial function and therefore mitochondrial viability. Here, we have studied mitochondrial dynamics in excitotoxicity. Our findings show that most of the mitochondrial fragmentation occurs within the first hour following the excitotoxic insult. This early phase depends partially on Drp1mitochondrial translocation and lasts as long as the stimulus is present. Four hours after initiation of the insult, and regardless of its removal, delayed mitochondrial fragmentation that correlates with the reduction in Mfn2 protein levels due to its transcriptional downregulation occurs. Loss of Mfn2 impairs mitochondrial function and participates in delayed excitotoxic damage. MEF2A directly regulates basal Mfn2 expression in neurons Bioinformatic analysis of 2.5 kb of the rat Mfn2 promoter identified that there were four putative MEF2 binding sites in the region spanning -2313/-1983 (Fig. E8C). We designed primers to amplify within this region and performed ChIP with anti-MEF2A antibody in rat cortical neurons unstimulated or stimulated with NMDA for 4 hours. We found that under basal conditions MEF2A was bound to this region but after NMDA stimulation there was no enrichment of this region with the chromatin immunoprecipitate (Fig. 8F). These observations support the hypothesis that MEF2A regulates basal transcription of Mfn2 in neurons and as a consequence of MEF2A degradation Mfn2 transcription is downregulated in excitotoxicity (Fig. 9). 18 Mechanisms of mitochondrial fragmentation in excitotoxicity Drp1 is subjected to several posttranslational modifications that regulate its fission activity. Neuronal depolarization activates CaMKI, which phosphorylates Drp1 at Ser 600, promoting mitochondrial fragmentation that is reversible once neurons repolarize (Han et al, 2008). The reversibility of Drp1-mediated mitochondrial fragmentation has also been observed in pathological conditions. Mitochondrial fragmentation induced by NO donors is a rapid and reversible process (Barsoum et al, 2006). Ca2+ influx through the NMDA receptor produces NOS activation, which has been implicated in nitrosative stress and cell death (Sattler et al, 1999). Nitrosative stress causes S-nitrosylation of Drp1 at Cys644, which enhances mitochondrial fission (Cho et al, 2009). In agreement with previous reports (Barsoum et al, 2006), we show that NOS inhibition partially blocks NMDA-mediated mitochondrial fragmentation. The degree of inhibition of the mitochondrial fragmentation achieved by NOS inhibitors is similar to that achieved 19 19 using genetic and pharmacological inhibitors of Drp1. That suggests the involvement of a mechanism other than Drp1 in the early phase of mitochondrial fragmentation. Opa1 is cleaved in a mitochondrial membrane potential dependent manner, promoting mitochondrial fission (Griparic et al, 2007; Ishihara et al, 2006; Song et al, 2007), but we were unable to detect changes in the proportion of short and long forms of Opa1 in excitotoxicity, either in vitro or in vivo. Another possibility is that excitotoxicity could promote the modification of lipids, which has been shown to affect mitochondrial dynamics (Choi et al, 2006). The possibility that as yet unknown posttranscriptional modifications inhibit the fusion machinery in excitotoxic conditions cannot be ruled out either. Our data also indicate that Mfn2 intervenes in the delayed phase of mitochondrial fragmentation. Cultured rat primary cortical neurons exposed to excitotoxic doses of NMDA or subjected to OGD, and in vivo middle cerebral artery occlusion in rats show reduced expression of Mfn2 but no changes in Mfn1 or Opa1. During the preparation of the manuscript two independent studies confirmed the downregulation of Mfn2 in excitotoxicity. Primary rat cortical neurons exposed to 3 hours of OGD also produced a reduction in Mfn2 protein with no changes in Opa1 and an increase in Mfn1 expression (Wappler et al, 2013). In an in vivo study, a reduction in Mfn2, and also of Opa1 expression, was observed in mice subjected to MCAO (Kumari et al, 2012). Dependence of neurons on Mfn2 Mfn1 can compensate for some but not all of the functions of Mfn2. Analysis of Mfn2 and Mfn1 knockout mice indicates that they have both redundant and distinct functions (Chen et al, 2003; Chen et al, 2007). Unlike Mfn1, a lack of or defects in Mfn2 have a 20 great impact on neuronal viability. Mfn2 mutations have been found to cause the dominantly inherited neurological disease Charcot-Marie-Tooth type 2A (CMT2A) (Zuchner et al, 2004). Defects in mitochondrial mobility and fusion have been proposed as the mechanism by which Mfn2 mutations cause CMT2A but this has not been fully established (Baloh et al, 2007). Mfn2 is required for postnatal development of the cerebellum (Chen et al, 2007). Conditional dopaminergic Mfn2 knockout neurons show that Mfn2, but not Mfn1, is essential for striatal projections and proper nigrostriatal circuit function (Lee et al, 2012; Pham et al, 2012). As a consequence of excitotoxicity-mediated Ca2+ overload neurons die initially by necrosis, surviving neurons can undergo delayed death in a manner dependent on the mitochondrial function (Ankarcrona et al, 1995; D'Orsi et al, 2012; Luetjens et al, 2000). We show here that, unlike Mfn1 expression, exogenous expression of Mfn2 protects neurons against NMDA-induced cell death. Mfn2 expression interferes with Bax recruitment to mitochondria and subsequent release of cytochrome c, as has been observed in other cell types (Neuspiel et al, 2005; Sugioka et al, 2004). The precise mechanism by which Mfn2 interferes with Bax activation is unknown. One possibility is that loss of Mfn2 facilitates OMM remodeling by Drp1 that has been shown to stimulate Bax oligomerization (Montessuit et al, 2010). Mfn2 interacts with different members of the Bcl-2 family, both pro- and anti-apoptotic (Suen et al, 2008). It cannot be ruled out the possibility that by changing Mfn2 levels it affects the equilibrium of interactions between pro- and anti-apoptotic Bcl-2 proteins to favor Bax assembly and MOMP formation. The relation between autophagy and cell death is controversial (Baehrecke, 2005; Galluzzi et al, 2012). Although in most known cases autophagy constitutes a cytoprotective response activated by dying cells in an attempt to cope with stress 21 (Galluzzi et al, 2012), cell death with and by autophagy has been reported in focal cerebral ischemia (Puyal et al, 2013; Puyal et al, 2009). Mfn2 KO fibroblasts show reduced autophagy in response to ER stress (Muñoz et al, 2013). Dependence of neurons on Mfn2 Interestingly, it has been reported that phosphorylated Mfn2 by PINK1 acts as a receptor for Parkin (Chen & Dorn, 2013) which is recruited to damaged mitochondria to promote mitophagy (Chen & Dorn, 2013; Narendra et al, 2008). How the reduction of Mfn2 in excitotoxicity affects mitophagy and autophagy-mediated cell death awaits further investigations. Mfn2 is transcriptionally downregulated in excitotoxicity During recent years several groups have established the role of the ubiquitin/proteasomal system in stress-mediated Mfn2 degradation. Mitochondrial depolarization promotes Mfn2 ubiquitinylation and proteasomal degradation in a Parkin-dependent manner (Chan et al, 2011; Tanaka et al, 2010). In stress conditions JNK phosphorylates Mfn2 at Ser 27 which recruits the E3 ubiquiting ligase Huwe 1, leading to its ubiquitination and degradation (Leboucher et al, 2012). Although excitotoxicity causes both mitochondrial depolarization and JNK activation (Borsello et al, 2003; Nicholls, 2009; Soriano et al, 2008; Soriano et al, 2006b), the reduction in Mfn2 protein levels was not blocked by inhibiting the proteasome. We found that the main mechanism implicated in Mfn2 reduction acts at the transcriptional level and does not seem to depend on enhanced proteolysis since inhibiting transcription or translation does not further reduce Mfn2 levels. Interestingly, microarray analysis has showed more than 50% reduction in Mfn2 mRNA in the penumbra of rats subjected to MCAO (Ramos-Cejudo et al, 2012). We have shown that transcription factor MEF2 regulates basal Mfn2 transcription in neurons and that excitotoxicity-mediated MEF2 degradation (Li et al, 2001; Okamoto et al, 2002) results in transcriptional downregulation of Mfn2 22 (Fig. 8). MEF2 transcription factors play a pivotal role in brain development, synapse development and neuronal survival. The genetic program regulated by MEF2 that controls synaptic remodeling has been characterized (Flavell et al, 2008) but although many studies have pointed to the importance of MEF2 in supporting neuronal viability (Li et al, 2001; Mao et al, 1999; Okamoto et al, 2002), the precise target genes are poorly defined. Here we demonstrate that Mfn2 is a novel MEF2 target gene that can mediate its prosurvival function. Mitochondrial dynamics in neurodegeneration Mitochondrial dysfunction and excitotoxicity are common features of adult-onset neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases, some of them occurring primarily in the absence of genetic linkage. Alterations in mitochondrial dynamics have been reported in all these diseases (Itoh et al, 2013). Mfn2 is dynamically regulated, thus its missregulation could intervene in the pathogenesis of neurodegenerative disorders. Further studies are required to clarify the role of Mfn2 in the progression of chronic late-onset neurodegenerative disorders. In this study we have shown that neurons with reduced Mfn2 have dysfunctional mitochondria, altered Ca2+ homeostasis that could sensitize neurons to additional insults. In our excitotoxicity model, Mfn2 expression protects against delayed exicitotoxic cell death by interfering with Bax recruitment to mitochondria. This suggests that Mfn2 downregulation could determine the fate of neurons in the penumbra area, the most clinically relevant therapeutic target against ischemic stroke (Lo, 2008). Thereby, Mfn2 is a potential therapeutic target against excitotoxicity in acute episodes and chronic neurodegenerative diseases. 23 23 Focal ischemia model All animal procedures were authorized by the veterinary office of the Canton of Vaud and were in accordance with the directives of the Swiss Academy of Medical Science. After anesthesia (2.5% isoflurane), middle cerebral artery (MCA) occlusion was performed in 12-day-old (P12) male Sprague-Dawley rats as previously described (Vaslin et al, 2009). Briefly, first, the main (cortical) branch of the left MCA was electrocoagulated just below its bifurcation into the parietal and frontal branches. Then, the left common carotid artery was transiently occluded with a clamp for 90 minutes. Rat pups were maintained at 37°C in the induction chamber (2.5% isoflurane) while the left common carotid artery was clamped. The arterial clamp was then removed and the rat pups were transferred back to their mother until sacrifice. Rat pups were decapitated and the brains were removed in PBS containing 1 mmol/L MgCl2 on ice. The cortex were dissected and collected in protein hypotonic lysis buffer (20 mmol/L HEPES, pH 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl2, 2.5 mmol/L EGTA, 0.1 mmol/L dithiothreitol, 50 mmol/L NaF, 1 mmol/L Na3VO4, 1% Triton X-100, and a protease inhibitor cocktail (Roche)). Tissues were homogenized and sonicated, and protein concentration was determined using the Bradford assay. Cell culture, transfection and determination of cell death Cortical neurons from E21 Sprague Dawley rats were cultured as described previously (Soriano et al, 2008). Experiments were performed after a culturing period of 10–11 days during which cortical neurons develop a rich network of processes, express functional NMDA-type and AMPA/kainate-type glutamate receptors, and form synaptic contacts. Prior to stimulations and transfections, neurons were transferred from growth medium to a medium containing 10% MEM (Invitrogen), 90% salt-glucose-glycine (SGG) medium (SGG: 114 mM NaCl, 0.219 % NaHCO3, 5.292 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 1 mM glycine, 30 mM glucose, 0.5 mM sodium pyruvate, 0.1% phenol red; osmolarity 325 mosm/l). Transfections were performed with Lipofectamine 2000 (Invitrogen). For cell death determination neurons transfected with GFP plus the indicated expression vectors were treated with NMDA for 6 hours and fixed. At this time point damaged neurons show pyknotic nuclei but GFP signal has not been lost yet. Nuclei were stained with DAPI and cell death was determined by counting the number of DAPI-stained pyknotic nuclei as a percentage of the total transfected neurons. NMDA-dependent cell death was calculated subtracting basal cell death to the cell death after NMDA treatment. 10T1/2 and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in subconfluent cultures. Transfections were performed with either Lipofectamine 2000 (Invitrogen) or Fugene (Roche). For Bax knock down experiments 25 ng of Rat Bax (24887) siRNA-SMART pool (Thermo scientific) containing a pool of 4 siRNAs targeting rat Bax were transfected. 24 24 Oxygen and Glucose deprivation. Neurons were transferred from TMo, washed once in a glucose-free, balanced salt solution (SGG): 114 mM NaCl, 0.219% NaHCO3, 5.292 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 1 mM glycine, 0.5 mM sodium pyruvate, 0.1% phenol red; which had previously been degassed by flushing the solution with 95% N2-5% CO2 for 5 min. Cells were placed in degassed glucose-free SGG and put in a modular incubator chamber, which was flushed with 95% N2-5% CO2 for 4 min at a flow rate of 20 L/min, 25 according to manufacturer’s instructions (Stemcell technologies, Grenoble, France), in order to fully expel any remaining oxygen within the chamber. The chambered cells were then left in OGD at 37°C for 1 h, before being returned to normoxic conditions and glucose-containing media (TMo). No OGD control cells were placed in SGG and maintained in normoxic conditions for 1 h before also being returned to TMo. All cells were left in TMo until protein and RNA isolation for the time point indicated. Plasmids and virus generation The following plasmids have been described previously: the -1982/+45 Mfn2- Luciferase vector and its 5’ deletion constructs (Sorianello et al, 2012), SESN2-Luc (Papadia et al, 2008), and mtRFP (Legros et al, 2002). HA-Mfn2 was subcloned into the pEF vector at the BamHI/XbaI sites. The MEF2A expression vector was a gift from Pilar Ruiz-Lozano (Stanford University, USA), GFP-Drp1 was a gift from AM van der Bliek (UCLA, USA), Drp1-K38A-myc was Addgene plasmid 26049, deposited by Dr. Chan, and Mfn2-myc was Addgene plasmid 23213, deposited by Dr. Chan (Chen et al, 2003)). The vectors used to construct and package recombinant adeno-associated viruses (rAAVs) were provided by Dr. Bading (U. Heidelberg, Germany (Zhang et al, 2007)). For construction of pAAV-MEF2-DN the first 252 bp of the mouse MEF2D cDNA (a gift from E. Olson; U. Texas South Western, USA) were amplified using the primers: forward 5’- ATA GGA TCC ATG GGG CGA AAG AAG ATA CAA ATC ACA CGC ATA ATG GAT G -3’ and reverse 5’- ATA AAG CTT TCA CAG ATC TTC TTC AGA AAT AAG TTT TTG TTA GGA GGG CCT CGT TTG AAA ATA AAA TC - 3’. The amplified product contains sequences that produce BamHI and HindIII restriction sites at the 5’ and 3’ respectively (italics) and also for inserting a myc tag 3’. The amplified product contains sequences that produce BamHI and HindIII restriction sites at the 5’ and 3’ respectively (italics) and also for inserting a myc tag 26 26 into the C terminus (bold). GFP in the rAAV-GFP vector was removed by BamHI/HindIII digestion and the MEF2-DN PCR product was cloned into the rAAV vector to express the first 84 amino acids of the N terminus of MEF2, which contains the DNA binding domain but not the transactivation domain. rAAV for shRNA expression contains the U6 promoter for shRNA expression and a CMV/chicken beta- actin hybrid promoter driving hrGFP expression. rAAV-shRNA targeting Mfn2 were made by swapping the sh-sc sequence of rAAV-sh-sc (gift from H. Bading) for the following sequences of the rat Mfn2 into the BamHI and HindIII sites: shMfn2: 5’- AGA GGG CCT TCA AGC GCC AGT-3’, shMfn2.2: 5’- GGG AAG AGC ACC GTG ATC AAT-3’, shMfn2.3: 5’- TCC TCA AGG TTT ATA AGA ATG-3’. All newly generated constructs were confirmed by sequencing. Neurons were infected with rAAV at DIV4. Site-directed mutagenesis Site-directed mutagenesis was performed using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions. The mutated sequences are shown in Fig. E8B. Plasmids and virus generation Infection efficiencies were determined at DIV 10-11 by analyzing GFP fluorescence or immunocytochemical analysis; they ranged from 80 to 90% of the viable neurons. RNA isolation, RT-PCR and qPCR RNA was isolated using an RNA extraction kit (Life Technologies). For qPCR, cDNA was synthesized from RNA using the SuperScript® III First-Strand Synthesis SuperMix (Life Technologies) according to the manufacturer’s instructions. qPCR was performed in a StepOne Real-Time PCR System (Applied Biosystem) using GoTaq QPCR Master Mix (Promega) according to the manufacturer’s instructions. The primers used were: Mfn2 -F: 5’- ATG TCA AAG GGT ACC TGT CCA-3’, -R: 5’- CAA TCC CAG ATG GCA GAA CTT-3’; SESN2 -F: 5’- GGA TTA TAC CTG GGA AGA CC -3, -R: 5’- CGC AGT GGA TGT AGT TCC -3’; Mfn1 –F: 5’-CAA ACT GCA GCC ACC AAG T-3’, -R: 5’- GTT GGC ACA GTC GAG CAA-3’; 18S -F: 5’-GTG GAG CGA TTT GTC TGG TT-3’, -R: 5’-CAA GCT TAT GAC CCG CAC TT-3’. Expression of the gene of interest was normalized to 18S, a commonly used control. Luciferase assay Firefly luciferase-based reporter gene was transfected along with a Renilla expression vector (pTK-RL; Promega), and also, where relevant, MEF2-DN expression vector. Forty hours after transfection luciferase assays were performed using the Dual Glo assay kit (Promega) with Firefly luciferase-based reporter gene activity normalized to the Renilla control (pTK-RL plasmid) in all cases. 27 27 Western blotting and antibodies Total cell lysates were boiled at 100°C for 5 min in 1.5x sample buffer (1.5 M Tris pH 6.8; Glycerol 15%; SDS 3%; β-mercaptoethanol 7.5%; bromophenol blue 0.0375%). Gel electrophoresis was performed using 9% polyacrylamide gels. The gels were blotted onto PVDF membranes, which were then blocked for 1 hour at room temperature with 5% (w/v) non-fat dried milk in PBS with 0.1% Tween 20. The membranes were then incubated at 4°C overnight with the primary antibodies diluted in blocking solution: anti-Mfn2 (1:2000; Abcam), Mfn1 (1:250, Santa Cruz), Opa1 (1:1000; BD Biosciences), Drp1 (1:1000, BD Biosciences), MEF2A (1:750, Santa Cruz), Jun (1:1000, BD Biosciences), Actin (1:10000, Sigma) and Porin (1:100000, Abcam). For visualization of Western blots, HRP-based secondary antibodies were used followed by chemiluminescent detection on Kodak X-Omat film. Western blots were analyzed by 28 28 digitally scanning the blots, followed by densitometric analysis (ImageJ). All analyses involved normalizing to a loading control, Actin and Porin. Measurement of cytoplasmic Ca2+ with Indo-1. AAV-sh-sc- or AAV-sh-Mfn2-transduced neurons were loaded with 5 M of the ratiometric Ca2+ indicator Indo-1 for 30 minutes at 37ºC in SGG medium without phenol red. After two washes with PBS neurons were incubated for 15 additional minutes in order to allow de-esterification of Indo-1. Measurements were made on Infinite M200PRO (TECAN). Neurons were excited at 350 nm and the ratio of the emitted fluorescence values at 405 (Indo-1 Ca2+ bound) and 485 nm (Indo-1 Ca2+ free) was used as an index of [Ca2+]. was used as an index of [Ca2+]. Imaging studies Cells were visualized using a TCS SP2 Leica confocal laser scanning microscope (Leica Lasertechnick GmbH, Mannheim, Germany) adapted to an inverted Leitz DMIRBE microscope at 37ºC in a controlled 5% CO2 atmosphere (Life Imaging Services). Pictures were acquired using a 63X (1.32 NA) Leitz Plan-Apochromatic objective. Images were analyzed using ImageJ software (Rasband, W.S., 1997-2012). To quantify mitochondrial membrane potential neurons were loaded with 20 nM tetramethylrhodamine methylester (TMRM; Sigma) in SGG medium without phenol red. Transfected cells were identified by co-transfecting GFP expression plasmid. Single cells were monitored, TMRM was excited at 540 nm and emission was measured using a 570 nm filter. The mitochondrial membrane potential was compared to that observed in surrounding untransfected neurons and subsequently expressed as a percentage of that observed in untransfected cells before NMDA stimulation. For mitochondrial Ca2+ analysis, neurons were loaded with 5 M Rhod-2 (Life Technologies) for 30 minutes at 4 ºC followed for extensive washout of the dye and incubation for 20 additional minutes with SGG without phenol red at 37 ºC. Single cell were monitored. Rhod-2 was excited at 540 nm and emission was measured using a 570 nm filter. Cytoplasmic Ca2+ was monitored with Fluo-4 (Life Technologies). Neurons were loaded 5 M Fluo-4 for 30 minutes at 37 ºC, after 2 washes with PBS were incubate for additional 15 minutes and excited at 488 nm and emission captured with a 516 filter. 29 29 For mitochondrial morphology analysis neurons were transfected with mitochondrially targeted RFP. After treatment neurons were fixed, and nuclei were stained with DAPI. The number of live neurons with tubular or fragmented mitochondria was counted. Drp1 or Bax localization was determined by transfecting cortical neurons with GFP- Drp1 or Bax-GFP, respectively, and mtRFP. Forty-eight hours after transfection neurons were treated when appropriate with 30 M NMDA and fixed. For Cytochrome c inmunofluorescence, neurons were fixed with 4% parafolmaldehyde, permeabilized, blocked and incubated over night at 4 ºC with anti-cytochrome c antibody (Millipore). Antibody binding was visualized using biotinylated secondary antibody/Cy3-conjugated streptavidin. ATP measurements. Thirty minutes before NMDA addition, neurons were incubated with medium without glucose and with pyruvate. After 30 minutes of NMDA treatment (15 M) ATP was 30 measured using the ATPlite Luminiscence Assay System (Perkin-Elmer) on Infinite M200PRO (TECAN) . measured using the ATPlite Luminiscence Assay System (Perkin-Elmer) on Infinite M200PRO (TECAN) . Chromatin immunoprecipitation (ChIP) Two 35 mm dishes were used for each treatment (approx 4.5 x 106 cells). Medium was removed and treated cells were washed with pre-warmed medium and incubated for 10 minutes at room temperature with 1% para-formaldehyde in pre-warmed medium, to crosslink proteins to DNA. The reaction was stopped by adding glycine to a final concentration of 125 mM for 5 min. Cells were washed twice with ice-cold PBS and harvested on ice in swelling buffer (25% HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP40 plus protease inhibitor cocktail set III (Calbiochem)). Nuclei were isolated using a douncer and centrifuging at 1500 g for 5 minutes. Nuclei were resuspended in 200 µl sonication buffer (50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X100, 0.25% NaDeoxicolate, 0.1% SDS plus protease inhibitors). The nuclei were sonicated using a Diagenode Bioruptor (Liege, full power 30 s on, 30 s off, in an ice bath for 20 min) to produce fragments < 500 bp. Sonicated chromatin was precleared with protein-A agarose beads/salmon sperm DNA for 1 h at 4°C with agitation, beads were collected by centrifugation and supernatants were collected and subjected to immunoprecipitation. Eight micrograms of anti-MEF2A (Santa Cruz) or anti-Rabbit IgG (control, Sigma) was added for overnight incubation at 4ºC with agitation. After chromatin immunoprecipitation, DNA was purified using Qiagen DNA purification columns. Input (1% of total immunoprecipitated) and immunoprecipitated DNA were subjected to qPCR analysis with primers amplifying the Mfn2 promoter (- 2204/-2136 of the rat Mfn2 promoter) 5’-TGG AGA TGG AAT TCA AGT TGG-3’ forward and 5’-TGGTCACAAAATGGCTCAGT-3’ reverse. As negative controls we Two 35 mm dishes were used for each treatment (approx 4.5 x 106 cells). Medium was removed and treated cells were washed with pre-warmed medium and incubated for 10 minutes at room temperature with 1% para-formaldehyde in pre-warmed medium, to crosslink proteins to DNA. The reaction was stopped by adding glycine to a final concentration of 125 mM for 5 min. Cells were washed twice with ice-cold PBS and harvested on ice in swelling buffer (25% HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP40 plus protease inhibitor cocktail set III (Calbiochem)). Nuclei were isolated using a douncer and centrifuging at 1500 g for 5 minutes. NAD+/NADH measurements. NAD+ and NADH levels were measured individually taking advantage of the different stabilities of the different forms in acidic or basic buffer. We employed NAD+/NADH- Glo assay (Promega) following manufacturer’s instructions. Oxygen consumption measurements. Cortical neurons were plated in SeaHorse Bioscience XF24 plates and transduced with AAV producing shRNA-scr or shRNA- Mfn2. Seahorse Bioscience XF24 extracellular flux analyzer was used to measure oxygen consumption in these cells. The instrument was calibrated the day before the experiment, following the manufacturer’s instructions. On the day of the experiment, the injection ports on the sensor cartridge were loaded with 1.25 µM oligomycin (oli; complex V inhibitor) to distinguish the percentage of oxygen consumption devoted to ATP synthesis and the percentage of oxygen consumption needed to overcome the natural proton leak across the inner mitochondrial membrane, 3µM FCCP to calculate the "spare" respiratory capacity of cells, 1 µM rotenone (complex I inhibitor) and 1 µM antimycin A (complex III inhibitor) to calculate the remaining respiration due to residual oxygen consumption (rox). During the sensor calibration, neurons remained in a 37 °C incubator without CO2 in 700 μl of artificial cerebrospinal fluid (aCSF) assay medium (120 mMNaCl, 3.5 mMKCl, 1.3 mM CaCl2, 0.4 mM KH2PO4, 1 mM MgCl2, 15 mM glucose, 1.0 mM pyruvate and 4 mg/ml fattyacid free bovine serum album, pH 7.2.). Plates were immediately placed into the calibrated Seahorse XF24 flux analyzer for mitochondrial bioenergetic analysis. OCR was normalized for total protein/well and calculated as OCR – OCRroxto subtract non-mitochondrial respiration. Spare respiratory 31 31 capacity was calculated as OCRCCCP/OCRrutine. Respiratory control ratio was calculated as OCRCCCP/OCRoli Chromatin immunoprecipitation (ChIP) Nuclei were resuspended in 200 µl sonication buffer (50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X100, 0.25% NaDeoxicolate, 0.1% SDS plus protease inhibitors). The nuclei were sonicated using a Diagenode Bioruptor (Liege, full power 30 s on, 30 s off, in an ice bath for 20 min) to produce fragments < 500 bp. Sonicated chromatin was precleared with protein-A agarose beads/salmon sperm DNA for 1 h at 4°C with agitation, beads were collected by centrifugation and supernatants were collected and subjected to immunoprecipitation. Eight micrograms of anti-MEF2A (Santa Cruz) or anti-Rabbit IgG (control, Sigma) was added for overnight incubation at 4ºC with agitation. After chromatin immunoprecipitation, DNA was purified using Qiagen DNA purification columns. Input (1% of total immunoprecipitated) and immunoprecipitated DNA were subjected to qPCR analysis with primers amplifying the Mfn2 promoter (- 2204/-2136 of the rat Mfn2 promoter) 5’-TGG AGA TGG AAT TCA AGT TGG-3’ forward and 5’-TGGTCACAAAATGGCTCAGT-3’ reverse. As negative controls we 32 32 used primers for amplification of the actin gene 5’-AGC CAT GTA CGT AGC CAT CC-3’ forward and 5’-CTC TCA GCT GTG GTG GTG AA-3’ reverse. Electrophoretic mobility-shift assays HeLa cells were transfected with expression vector for MEF2A and 48 hours later, nuclear extracts were prepared as described previously (Santalucia et al, 2001). Radiolabeled double strand oligonucleotide probe containing the human Mfn2 promoter sequence (containing the MEF2 binding site: 5’-ATT TTT GTA TTT TTA GTA CAG- 3’ (MEF2wt), the mutated MEF2 binding site (in bold), which differs from the wild type in the same three nucleotides used in the luciferase assay: 5’-ATT TTT GGA TCC TTA GTA CAG-3’ (MEF2mut), and the MEF2 binding site in the Glut4 promoter: 5’- CGT GGG AGC TAA AAA TAG CCA-3’ (MEF2Glut4) was incubated with 10 μg nuclear extract in a final volume of 20 μl and electrophoresis was performed as described previously (Santalucia et al, 2001). Dried gel was exposed to Kodak film. Competitor mutated oligonucleotide differed from wild-type EMSA sequence by the same base substitution used in the functional experiments. For supershift assays, MEF2 antibody was added to the corresponding binding reactions after incubation with the radiolabeled probe, and incubated for a further 10 minutes at room temperature before loading onto gels. Statistical analysis Statistical testing involved two-tailed student T-tests. For any multiple comparisons within data sets we used a one-way ANOVA followed by the Bonferroni post-hoc test. All data are presented as mean ± s.e.m. of at least three independent experiments (n). 33 33 Acknowledgments We thank G.E. Hardingham for critically reading the manuscript. This work was supported by the Fundació La Marató de TV3 (111210; FXS) and Spanish Ministerio de Ciencia e Innovación (SAF2011-30283; FXS). 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Science 297: 259-263 Yuan H, Gerencser AA, Liot G, Lipton SA, Ellisman M, Perkins GA, Bossy-Wetzel E (2006) Mitochondrial fission is an upstream and required event for bax foci formation in response to nitric oxide in cortical neurons. Cell Death Differ 14: 462-471 Zhang SJ, Steijaert MN, Lau D, Schutz G, Delucinge-Vivier C, Descombes P, Bading H (2007) Decoding NMDA receptor signaling: identification of genomic programs specifying neuronal survival and death. Arundine M, Tymianski M (2003) Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity. Cell Calcium 34: 325-337 Neuron 53: 549-562 Zuchner S, Mersiyanova IV, Muglia M, Bissar-Tadmouri N, Rochelle J, Dadali EL, Zappia M, Nelis E, Patitucci A, Senderek J, Parman Y, Evgrafov O, Jonghe PD, Takahashi Y, Tsuji S, Pericak-Vance MA, Quattrone A, Battologlu E, Polyakov AV, Timmerman V, Schroder JM, Vance JM (2004) Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A. Nat Genet 36: 449-451 Zuchner S, Mersiyanova IV, Muglia M, Bissar-Tadmouri N, Rochelle J, Dadali EL, Zappia M, Nelis E, Patitucci A, Senderek J, Parman Y, Evgrafov O, Jonghe PD, Takahashi Y, Tsuji S, Pericak-Vance MA, Quattrone A, Battologlu E, Polyakov AV, Timmerman V, Schroder JM, Vance JM (2004) Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A. Nat Genet 36: 449-451 43 Figure legends Figure 1. Mfn2 expression is reduced in excitotoxicity in vitro and in vivo. Western analysis of mitochondrial fusion/fission proteins. A) In vitro primary cortical neurons exposed to NMDA (30 M) for the indicated times and (B) densitometric analysis normalized to actin (n=3–6). C) In vivo brain extracts of rats subjected to MCAO plus 90 minutes of ipsilateral common carotid artery clamp followed by clamp release for the indicated times and (D) densitometric analysis normalized to actin (n=3–7). Mean ± s.e.m. in this and subsequent cases. p<0.05 compared to control, two-tailed T-test. Figure 2. Activation of Drp1 induces mitochondrial fragmentation. A) Representative images of NMDA-induced mitochondrial fragmentation. Neurons transfected with mitochondria targeted RFP (mtRFP) were treated with NMDA (30 M) for the indicated times. Scale bar = 20 m. B) Mitochondrial morphology analysis from (A). (n=3-8). p<0.05 compared to control, one-way ANOVA followed by Bonferroni post hoc test. C) NMDA induces increased Drp1 translocation to mitochondria. Neurons were transfected with plasmids encoding GFP-Drp1 and mtRFP. After 48 h neurons were stimulated with NMDA (30 M) for 1 hour or left unstimulated, fixed and visualized under a confocal microscope. Brightness and contrast has been adjusted in the merged image to visualize GFP signal in the control condition. Scale bar yellow = 20 m, white = 4 m. D) Representative images showing that genetic and pharmacological inhibitors of Drp1 block mitochondrial fragmentation. Neurons were transfected with plasmids encoding mtRFP and Drp1-K38A or control (globin). After 44 44 48 hours neurons were stimulated with NMDA (30 M) for 1 hour or they were pre- treated for 1 hour before NMDA stimulation with mdiv-1 (25 M) or 7-Nitroindazole ((7-Ni) at 5 M in Arg free medium) and mitochondrial morphology was analyzed (E). (n=3). Scale bar = 20 m. p<0.05, one-way ANOVA followed by Bonferroni post hoc test. Figure 3. Mfn2 intervenes in an irreversible delayed phase of mitochondrial Figure 3. Mfn2 intervenes in an irreversible delayed phase of mitochondrial fragmentation. A) Representative images and analysis of secondary mitochondrial fragmentation after NMDA wash-out. Neurons were transfected with mtRFP. After 48 h neurons were stimulated with NMDA (30 M) for 1 hour and washed out for the indicated times and mitochondrial morphology was analyzed (n=4–5). p<0.05, one- way ANOVA followed by Bonferroni post hoc test. Scale bar = 20 m. B) Mfn2 is reduced after NMDA wash-out. Western and densitometric analysis of neurons treated with NMDA (30 M) for 1 hour and washed out for 3 additional hours (n=4). p<0.05, two-tailed T-test. C) Representative western blot and densitometric analysis of neurons transduced with AAV producing shRNA against Mfn2 or scrambled (n=4). p<0.05, one-way ANOVA followed by Bonferroni post hoc test. D) Mfn2 knockdown causes mitochondrial fragmentation. Representative images and analysis of mitochondrial morphology of neurons transfected with plasmids shMfn2 or sh-sc and mtRFP (n=4). p<0.05, two-tailed T-test. Scale bar = 20 m. E) Mfn2 blocks the delayed phase of NMDA-induced mitochondrial fragmentation. Representative images of neurons transfected with plasmids encoding mtRFP and Mfn2 or globin (CT) were treated with NMDA (30 M) for 1 hour and washed out for 3 additional hours and mitochondrial morphology was analyzed (F). (n=3). p<0.05, one-way ANOVA followed by Bonferroni post hoc test. Scale bar = 20 m. 45 45 Figure 4 Mfn2 downregulation causes mitochondrial dysfunction and altered Ca2+ homeostasis. A) Respiratory control ratio and (B) spare respiratory capacity of neurons transduced with AAV expressing shRNA targeting Mfn2 (sh-Mfn2) or scrambled (sh- sc). (n=6) p<0.05, two-tailed T-test. C) Loss of mitochondrial membrane potential in neurons transfected with sh-Mfn2 or sh-sc and treated with NMDA (15 M; arrow) was determined by measuring TMRM fluorescence. The values were normalized to surrounding untransfected neurons (n=19–21 neurons analyzed in five independent experiments). p<0.05, two-tailed T-test. D) ATP levels were analyzed following 30 minutes NMDA (15 M) treatment to neurons transduced with AAV producing sh- Mfn2 or control sh-sc (in glucose free medium). (n= 4). p<0.05, two-tailed T-test. E) Determination of mitochondrial Ca2+ with Rhodamine-2 in cortical neurons transfected with plasmids producing sh-Mfn2 or sh-sc and treated with NMDA (15 M), the values were normalized to the signal of surrounding untransfected neurons (n=20 neurons from 5 independent experiments). F) Histograms show the average mitochondrial Ca2+ levels before and after NMDA application (15 M). Figure 3. Mfn2 intervenes in an irreversible delayed phase of mitochondrial p<0.05, one-way ANOVA followed by Bonferroni post hoc test. G) Determination of intracellular Ca2+ with Fluo-4 in cortical neurons transfected with plasmids producing sh-Mfn2 or sh-sc and treated with NMDA (15 M), the values were normalized to the signal of surrounding untransfected neurons (n=16 neurons from 4 independent experiments). H) Histograms show the average intracellular Ca2+ levels before and after NMDA application (15 M). p<0.05, one- way ANOVA followed by Bonferroni post hoc test. Figure 5. Mfn2 reduction intervenes in delayed excitotoxic death.A) Neurons Figure 4 Mfn2 downregulation causes mitochondrial dysfunction and altered Ca2+ homeostasis. A) Respiratory control ratio and (B) spare respiratory capacity of neurons transduced with AAV expressing shRNA targeting Mfn2 (sh-Mfn2) or scrambled (sh- sc). (n=6) p<0.05, two-tailed T-test. C) Loss of mitochondrial membrane potential in neurons transfected with sh-Mfn2 or sh-sc and treated with NMDA (15 M; arrow) was determined by measuring TMRM fluorescence. The values were normalized to surrounding untransfected neurons (n=19–21 neurons analyzed in five independent experiments). p<0.05, two-tailed T-test. D) ATP levels were analyzed following 30 minutes NMDA (15 M) treatment to neurons transduced with AAV producing sh- Mfn2 or control sh-sc (in glucose free medium). (n= 4). p<0.05, two-tailed T-test. E) Determination of mitochondrial Ca2+ with Rhodamine-2 in cortical neurons transfected with plasmids producing sh-Mfn2 or sh-sc and treated with NMDA (15 M), the values were normalized to the signal of surrounding untransfected neurons (n=20 neurons from 5 independent experiments). F) Histograms show the average mitochondrial Ca2+ levels before and after NMDA application (15 M). p<0.05, one-way ANOVA followed by Bonferroni post hoc test. G) Determination of intracellular Ca2+ with Fluo-4 in cortical neurons transfected with plasmids producing sh-Mfn2 or sh-sc and treated with NMDA (15 M), the values were normalized to the signal of surrounding untransfected neurons (n=16 neurons from 4 independent experiments). H) Histograms show the average intracellular Ca2+ levels before and after NMDA application (15 M). p<0.05, one- way ANOVA followed by Bonferroni post hoc test. Figure 5. Mfn2 reduction intervenes in delayed excitotoxic death.A) Neurons transfected with plasmids expressing sh-Mfn2 or sh-sc were treated with subtoxic doses Figure 5. Mfn2 reduction intervenes in delayed excitotoxic death.A) Neurons transfected with plasmids expressing sh-Mfn2 or sh-sc were treated with subtoxic doses Figure 5. Mfn2 reduction intervenes in delayed excitotoxic death.A) Neurons transfected with plasmids expressing sh-Mfn2 or sh-sc were treated with subtoxic doses 46 46 of NMDA (15 M) for 6 hours. Death was analyzed by fixing cells, DAPI-staining and counting pyknotic nuclei of the transfected neurons (n=5). p<0.05, two-tailed T-test. B) Analysis and representative images of cortical neurons transfected with plasmids expressing Mfn2 or control (globin). After 48 h neurons were exposed to NMDA (30 M) for 6 hours. NMDA dependent neuronal death was analyzed (n=8). p<0.05,two- tailed T-test. Arrowhead points to the transfected neuron. Scale bar = 20 m. C) Cortical neurons expressing Mfn2 or control plasmid (globin) were treated with 30 M NMDA for 1 hour followed for extensive washout and allowed to recover for the indicated times. Cell death was analyzed by fixing cells, DAPI-staining and counting pyknotic nuclei of the transfected neurons (n=5). p<0.05, one-way ANOVA followed by Bonferroni post hoc test. Representative images and cell death analysis of neurons transfected with a plasmid producing sh-Mfn2 plus (D) siRNA CT or targeting Bax, or (E) plasmid control or expressing Bcl-xL and treated with NMDA (15 M) for 6 hour. (n=8). p<0.05, two-tailed T-test. Arrowhead points to the transfected neuron. Scale bar = 20 m. F) Representative images of neurons expressing GFP-Bax and mtRFP plus control or Mfn2 expression vector and treated with NMDA (30 M) for 1 hour followed for extensive washout and allowed to recover for the 3 hours. Scale bar = 10 m. G) Analysis of mitochondrial Bax localization of neurons transfected as in (F) and allowed to recover after NMDA treatment (30 M) for the indicated times. (n= 3-5). p<0.05, one-way ANOVA followed by Bonferroni post hoc test. H) Cytochrome c inmunofluorescence of neurons transfected with GFP plus control or Mfn2 expressing plasmid and treated as in (G). Arrowhead points to the transfected neuron, asterisk highlight cells with diffuse or lost cytochrome c. Scale bar = 20 m. I) Analysis of data from (H). (n= 5). p<0.05, one-way ANOVA followed by Bonferroni post hoc test. 47 47 Figure 6. Mfn2 is regulated at transcriptional level in excitotoxicity. Cortical neurons with or without pre-incubation with proteasome inhibitor MG-132 (10 M) were treated for 4 hours with NMDA (30 M) and Mfn2 levels were analyzed by western blot. A) Representative western and B) densitometric analysis (n=7). p<0.05, one-way ANOVA followed by Bonferroni post hoc test. Figure 5. Mfn2 reduction intervenes in delayed excitotoxic death.A) Neurons transfected with plasmids expressing sh-Mfn2 or sh-sc were treated with subtoxic doses C) Cortical neurons were treated with NMDA (30 M) for the indicated times and mRNA expression was determined by real time qPCR. (n=4). p<0.05, two-tailed T-test. Cortical neurons were treated with transcriptional inhibitor actinomycin D, translational inhibitor cycloheximide and NMDA (30 M) for 4 hours as indicated and Mfn2 protein expression was analyzed by western blot. D) Representative western and E) densitometric analysis (n=4). p<0.05, two-tailed T-test. Figure 6. Mfn2 is regulated at transcriptional level in excitotoxicity. Cortical neurons with or without pre-incubation with proteasome inhibitor MG-132 (10 M) were treated for 4 hours with NMDA (30 M) and Mfn2 levels were analyzed by western blot. A) Representative western and B) densitometric analysis (n=7). p<0.05, one-way ANOVA followed by Bonferroni post hoc test. C) Cortical neurons were treated with NMDA (30 M) for the indicated times and mRNA expression was determined by real time qPCR. (n=4). p<0.05, two-tailed T-test. Cortical neurons were treated with transcriptional inhibitor actinomycin D, translational inhibitor cycloheximide and NMDA (30 M) for 4 hours as indicated and Mfn2 protein expression was analyzed by western blot. D) Representative western and E) densitometric analysis (n=4). p<0.05, two-tailed T-test. Figure 7. Excitotoxicity-mediated Mfn2 downregulation depends on MEF2. Time course of excitotoxicity dependent MEF degradation (A) in cortical neurons in vitro treated with NMDA (30 M) for 4 hours and (B) brain extracts of rats subjected to MCAO plus 90 minutes of ipsilateral common carotid artery clamp followed by clamp release for the indicated times. C) Cortical neurons transduced with AAV expressing MEF2-DN or GFP (control) were treated with NMDA (30 M) for 4 hours and Mfn2 mRNA and protein expression was determined (n=5). p<0.05, one-way ANOVA followed by Bonferroni post hoc test. D) Mitochondrial morphology in cortical neurons transfected with the indicated expression vectors (n=4–5). p<0.05, one-way ANOVA followed by Bonferroni post hoc test. E) Representative images from (D). F) Mitochondrial membrane potential of neurons expressing the indicated vectors relative 48 to surrounding untransfected neurons (n=9-10 cells from three independent experiments). p<0.05, two-tailed T-test. G) Cortical neurons were transfected with the indicated expression vectors. After 48 h were treated with subtoxic doses of NMDA (15 M) for 6 hours and cell death was analyzed. (n= 8) p<0.05, one-way ANOVA followed by Bonferroni post hoc test. H) Representative images from (G). Arrowhead points to the transfected neuron. Scale bar = 20 m. A Figure 1 Figure 1 Figure 8. MEF2A directly regulates basal Mfn2 expression in neurons. A) Figure 8. MEF2A directly regulates basal Mfn2 expression in neurons. A) Luciferase-based reporter of Mfn2 promoter in cortical neurons (left) or 10T1/2 cell line (right) and SESN2 promoter in cortical neurons (center) in control or over-expressing MEF2-DN (n=3-9). p<0.05 compared to control, two-tailed T-test. B) Deletion analysis of a luciferase-based reporter of the Mfn2 promoter in 10T1/2 cells co- expressed with control or MEF2 plasmids (n=3). p<0.05, two-tailed T-test. C) Effect of putative MEF2 binding site mutations on MEF2-dependent induction of the Mfn2 promoter in 10T1/2 cells (n=3). p<0.05 compared to control, two-tailed T-test. D) EMSA performed with nuclear extracts of HeLa cells overexpressing MEF2 and radiolabeled probe encoding BOX 2 from Mfn2 promoter. Retardation complexes are indicated with arrows. Excess of cold oligonucleotide, mutated oligonucleotide or oligonucleotide of the Glut4 gene that contains a MEF2 binding site was used to compete. Radiolabeled probe containing mutated BOX 2 did not produce retardation complexes. E) Supershift was performed using MEF2 polyclonal antibody. Pre-immune serum was used as a negative control. indicates nonspecific binding of the probe to MEF2 antibody. F) ChIP on cortical neurons untreated or treated with NMDA (30 M) 49 49 for 4 hours using the indicated antibodies (n=4). p<0.05, one-way ANOVA followed by Bonferroni post hoc test. Figure 9. Proposed model for the changes in mitochondrial morphology and vulnerability in excitotoxic conditions. Under physiological conditions MEF2 binds Mfn2 promoter and regulates its basal gene expression resulting in mainly tubular mitochondria. In excitotoxic conditions Drp1 translocates to mitochondria and mediates rapid, reversible mitochondrial fission. An increase in cytosolic Ca2+ produces MEF2 degradation and Mfn2 gene expression is consequently downregulated, producing a delayed long term effect on mitochondrial morphology. Reduced expression of Mfn2 impairs mitochondrial function, which causes dysregulation of Ca2+ homeostasis, facilitates Bax translocation to mitochondria, release of cytochrome c and enhances delayed excitotoxic death. 50 Figure 1 Mfn2 Mfn1 CT 1 2 4 8 NMDA (h) A 98 98 Actin Porin 98 98 50 36 Opa1 Drp1 Protein/Actin (A.U.) B Mfn2 Porin Drp1 Opa1 Mfn1 Actin CT 0 2 6 24 h after clamp release C 98 98 98 98 50 36 0 1 2 4 8 (h) * Protein/Actin (A.U.) D CT 0 2 6 24 (h) * * * g 0 0.25 0.5 0.75 1 1.25 1.5 Mfn2 Mfn1 Opa1 Drp1 0 0.25 0.5 0.75 1 1.25 1.5 1.75 Mfn2 Mfn1 Opa1 Drp1 Protein/Actin (A.U.) B 0 1 2 4 8 (h) * * 0 0.25 0.5 0.75 1 1.25 1.5 Mfn2 Mfn1 Opa1 Drp1 CT 1 2 4 8 NMDA (h) 98 98 98 98 50 36 Protein/Actin (A.U.) B 0 1 2 4 8 (h) * * 0 0.25 0.5 0.75 1 1.25 1.5 Mfn2 Mfn1 Opa1 Drp1 B A fn2 orin rp1 pa1 fn1 ctin CT 0 2 6 24 h after clamp release 98 98 98 98 50 36 Protein/Actin (A.U.) D CT 0 2 6 24 (h) * * * 0 0.25 0.5 0.75 1 1.25 1.5 1.75 Mfn2 Mfn1 Opa1 Drp1 D Figure 2 A CT 30 min. 1 h 2 h 4h NMDA 0 0.5 1 2 4 hours % Cells with tubular mitochondria * * N.S. 0 20 40 60 80 B mitoRFP GFP-Drp1 Merged Control NMDA (1 h) C * % Cells with tubular mitochondria Drp1-K38A CT 7-Ni mdivi-1 0 10 20 30 40 50 60 70 80 - NM CT NMDA CT 7-Ni mdivi-1 Drp1-K38A D E A A CT 30 min. 1 h 2 h 4h NMDA 0 0.5 1 2 4 hours % Cells with tubular mitochondria * * N.S. 0 20 40 60 80 B mitoRFP GFP-Drp1 Merged Control NMDA (1 h) C * mitochondria 50 60 70 80 CT NMDA CT Drp1-K38A D E A CT 30 min. 1 h 2 h 4h NMDA NMDA CT 1 h 2 h 4h 0 0.5 1 2 4 hours % Cells with tubular mitochondria * * N.S. 0 20 40 60 80 B mitoRFP GFP-Drp1 Control NMDA (1 h) C C B 0 0.5 1 2 4 hours % Cells with tubular mitocho * * N.S. Figure 1 0 20 40 60 mitoRFP GFP-Drp1 Merged * % Cells with tubular mitochondria Drp1-K38A CT 7-Ni mdivi-1 0 10 20 30 40 50 60 70 80 - NMD CT NMDA CT 7-Ni mdivi-1 Drp1-K38A D E Merged * % Cells with tubular mitochondria Drp1-K38A CT 7-Ni mdivi-1 0 10 20 30 40 50 60 70 80 - NMDA CT NMDA CT 7-Ni mdivi-1 Drp1-K38A D E D E 0 1 2 3 4 time (h) NMDA Wash out R0 R1.5 R3 CT R1.5 R0 R3 A % Cells with tubular mitochondria CT 0 1.5 3 Recovery (h) after 1h NMDA B C Mfn2 sh-sc sh-Mfn2 CT Ac n Porin sh-sc sh-Mfn2 CT 0 0,5 1 1,5 Mfn2/Actin (A.U.) * * N.S. 36 50 96 D * E r mitochondria * F 60 80 CT sh-sc sh-Mfn2 0 25 50 75 sh-sc sh-Mfn2 % Cells with tubular mitochondria 0 25 50 75 * N.S. * * 0 0.25 0.5 0.75 1 1.25 Mfn2 Actin Porin 96 36 50 CT NMDA Figure 3 * 0 1 2 3 4 time (h) NMDA Wash out R0 R1.5 R3 CT R1.5 R0 R3 A B 0 0.25 0.5 0.75 1 1.25 Mfn2 Actin Porin 96 36 50 CT NMDA Figure 3 * 0 1 2 3 4 time (h) NMDA Wash out R0 R1.5 R3 CT R1.5 R0 R3 A B 0.2 0 0.7 1.2 Figure 3 0 1 2 3 4 time (h) NMDA Wash out R0 R1.5 R3 B 1. ure 3 0 1 2 3 4 time (h) NMDA Wash out R0 R1.5 R3 CT R1.5 R0 R3 A % Cells with tubular mitochondria CT 0 1.5 3 Recovery (h) after 1h NMDA B C Mfn2 sh-sc sh-Mfn2 CT Ac n Porin sh-sc sh-Mfn2 CT 0 0,5 1 1,5 Mfn2/Actin (A.U.) * * N.S. 36 50 96 D * CT R3 E % Cells with tubular mitochondria CT Mfn2 * F 0 20 40 60 80 CT NMDA+ 3h recov sh-sc sh-Mfn2 0 25 50 75 sh-sc sh-Mfn2 % Cells with tubular mitochondria 0 25 50 75 * N.S. Figure 1 36 50 96 E mitochondria F 6 8 % Cells with tubular m C % Cells with tubular mitochondria CT Mfn2 * F 0 20 40 60 80 CT NMDA+ 3h recov F sh-sc sh-Mfn2 CT 0 0,5 Mfn2/Actin CT Mfn2 +R3 Mfn2 R3 E % Cells with tubular mitochondria CT Mfn2 * 0 20 40 60 80 CT NMDA+ 3h recov E CT RCR (A.U) sh-sc sh-Mfn2 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2 sh-sc sh-Mfn2 SRC (A.U) * * A B Figure 4 0 5 10 15 20 min. TMRM fuorescence (A.U.) 0 0.25 0.5 0.75 1 1.25 sh-sc sh-Mfn2 15 µM NMDA * C N.S. * ATP (A.U.) sh-sc sh-Mfn2 15 µM D 0.0 0.5 1.0 1.5 2.0 sh-sc sh-Mfn2 15 µM NMDA 0 1 2 3 4 5 6 7 8 min. E Relative Rhod-2 fuorescence (mito Ca2+ ) 0 0.2 0.4 0.6 0.8 1 1.2 CT NMDA * * * * F 3 4 sh-sc sh-Mfn2 15 µM NMDA G Fluo-4 e (cyt Ca2+) 15 µM 0 5 1.0 1.5 2.0 - NMDA elative Rhod-2 scence (mito Ca2+ ) RCR (A.U) sh-sc sh-Mfn2 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2 sh-sc sh-Mfn2 SRC (A.U) * * A B g 0 5 10 15 20 min. TMRM fuorescence (A.U.) 0 0.25 0.5 0.75 1 1.25 sh-sc sh-Mfn2 15 µM NMDA * C B RCR (A.U) sh-sc sh-Mfn2 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2 sh-sc sh-Mfn2 SRC (A.U) * * 0 5 10 15 20 TMRM fuorescence (A.U.) 0 0.25 0.5 0.75 1 1.25 15 µM NMDA N.S. * ATP (A.U.) sh-sc sh-Mfn2 15 µM D 0.0 0.5 1.0 1.5 2.0 sh-sc sh-Mfn2 15 µM NMDA 0 1 2 3 4 5 6 7 8 min. E Relative Rhod-2 fuorescence (mito Ca2+ ) 0 0.2 0.4 0.6 0.8 1 1.2 CT NMDA * * * * sh-sc sh-Mfn2 F 0 1 2 3 4 sh-sc sh-Mfn2 15 µM NMDA 0 1 2 3 4 5 6 7 8 min. G Relative Fluo-4 fuorescence (cyt Ca2+) sh-sc sh-Mfn2 * * * H 15 µM 15 µM 0.0 0.5 1.0 1.5 2.0 - NMDA 0 1 2 3 4 - NMDA * Relative Rhod-2 fuorescence (mito Ca2+ ) Relative Fluo-4 fuorescence (cyt Ca2+) N.S. Figure 1 * * 0 0.25 0.5 0.75 1 1.25 Mfn2 Actin Porin 96 36 50 CT NMDA Figure 3 * B B A A NMDA Wash out 0 1 2 3 4 time (h) R0 R1.5 R3 CT R1.5 R0 R3 % Cells with tubular mitochondria CT 0 1.5 3 Recovery (h) after 1h NMDA C Mfn2 sh-sc sh-Mfn2 CT Ac n Porin sh-sc sh-Mfn2 CT 0 0,5 1 1,5 Mfn2/Actin (A.U.) * * N.S. 36 50 96 D * CT Mfn2 +R3 Mfn2 R3 E % Cells with tubular mitochondria CT Mfn2 * F 0 20 40 60 80 CT NMDA+ 3h recov sh-sc sh-Mfn2 0 25 50 75 sh-sc sh-Mfn2 % Cells with tubular mitochondria 0 25 50 75 * N.S. * * 0 0.25 0.5 0.75 1 1.25 CT NMDA * CT R3 % Cells with tubular mitochondria CT 0 1.5 3 Recovery (h) after 1h NMDA 0 25 50 75 * N.S. * * % Cells with tubular mitochondria CT 0 1.5 3 Recovery (h) after 1h NMDA C Mfn2 sh-sc sh-Mfn2 CT Ac n 50 96 D * sh-sc sh-Mfn2 0 25 50 75 sh-sc h-Mfn2 Cells with tubular mitochondria 0 25 50 75 * N.S. * * % Cells with tubular mitochondria CT 0 1.5 3 Recovery (h) after 1h NMDA C Mfn2 sh-sc sh-Mfn2 CT Ac n Porin sh-sc sh-Mfn2 CT 0 0,5 1 1,5 Mfn2/Actin (A.U.) * * N.S. 36 50 96 D * CT R3 E % Cells with tubular mitochondria * F 20 40 60 80 CT NMDA 3h rec sh-sc sh-Mfn2 0 25 50 75 sh-sc sh-Mfn2 % Cells with tubular mitochondria 0 25 50 75 * N.S. * * D * sh-sc sh-Mfn2 0 25 50 75 sh-sc sh-Mfn2 % Cells with tubular mitochondria D after 1h NMDA C Mfn2 sh-sc sh-Mfn2 CT Ac n Porin sh-sc sh-Mfn2 CT 0 0,5 1 1,5 Mfn2/Actin (A.U.) * * N.S. Figure 1 * ATP (A.U.) sh-sc sh-Mfn2 15 µM D 0.0 0.5 1.0 1.5 2.0 sh-sc sh-Mfn2 15 µM NMDA 0 1 2 3 4 5 6 7 8 min. E Relative Rhod-2 fuorescence (mito Ca2+ ) 0 0.2 0.4 0.6 0.8 1 1.2 CT NMDA E D * * * * sh-sc sh-Mfn2 F 0 1 2 3 4 sh-sc sh-Mfn2 15 µM NMDA 0 1 2 3 4 5 6 7 8 min. G Relative Fluo-4 fuorescence (cyt Ca2+) sh-sc sh-Mfn2 * * * H 15 µM 15 µM 0.0 0.5 1.0 1.5 2.0 - NMDA 0 1 2 3 4 - NMDA * Relative Rhod-2 fuorescence (mito Ca2+ ) Relative Fluo-4 fuorescence (cyt Ca2+) * * * * sh-sc sh-Mfn2 F 0 1 2 3 4 sh-sc sh-Mfn2 15 µM NMDA 0 1 2 3 4 5 6 7 8 min G Relative Fluo-4 fuorescence (cyt Ca2+) H 15 µM 0.0 0.5 1.0 1.5 2.0 - NMDA Relative Rhod-2 fuorescence (mito Ca2+ ) F 0 1 2 3 4 sh-sc sh-Mfn2 15 µM NMDA 0 1 2 3 4 5 6 7 8 min. G Relative Fluo-4 fuorescence (cyt Ca2+) G H 0 20 40 60 CT NMDA N.S. * 15 µM % Cell death 0 20 40 60 * sh-sc sh-Mfn2 Mfn2 CT % NMDA induced cell death _ NMDA NMDA _ CT Mfn2 GFP DAPI Figure 5 A B % NMDA induced cell death C 0 10 20 30 40 50 CT Mfn2 1.5 3 5 N.S. * * * * N.S. N.S. CT Mfn2 _ NMDA F GFP-Bax/mtRFP 25 50 75 100 CT Mfn2 % Mitochondrial Bax G * * N.S. * % Cytosolic cyt c * * 20 40 60 CT Mfn2 * GFP DAPI Cyt c _ _ NMDA CT Mfn2 I H * 0 10 20 30 40 - NMDA % Cell death * N.S. siBax siCT D 15 µM NMDA NMDA _ _ sh-Mfn2 + siCT sh-Mfn2 + siBax 0 10 20 30 40 - NMDA 15 µM % Cell death N.S. * CT Bcl-xL E GFP DAPI NMDA NMDA _ _ sh-Mfn2 sh-Mfn2 + Bcl-xL GFP DAPI (h) after 1 h NMDA 0 20 40 60 CT NMDA N.S. Figure 1 * 15 µM % Cell death 0 20 40 60 * sh-sc sh-Mfn2 Mfn2 CT % NMDA induced cell death _ NMDA NMDA _ CT Mfn2 GFP DAPI A B B A 0 0 sh-sc sh-Mfn2 Mfn2 CT % NMD NMDA Mfn2 % NMDA induced cell death C 0 10 20 30 40 50 CT Mfn2 1.5 3 5 N.S. * * * * N.S. N.S. CT Mfn2 _ NMDA F GFP-Bax/mtRFP 0 25 50 75 100 CT Mfn2 % Mitochondrial Bax G 3 5 (h) after 1 h NMDA * * N.S. * % Cytosolic cyt c 3 5 (h) after 1 h NMDA * * 0 20 40 60 CT Mfn2 * * GFP DAPI Cyt c _ _ NMDA NMDA CT Mfn2 I H * 0 10 20 30 40 - NMDA % Cell death * N.S. siBax siCT D 15 µM NMDA NMDA _ _ sh-Mfn2 + siCT sh-Mfn2 + siBax 0 10 20 30 40 - NMDA 15 µM % Cell death N.S. * CT Bcl-xL E GFP DAPI NMDA NMDA _ _ sh-Mfn2 sh-Mfn2 + Bcl-xL GFP DAPI (h) after 1 h NMDA % NMDA induced cell death C 0 10 20 30 40 50 CT Mfn2 1.5 3 5 N.S. * * * * N.S. N.S. CT Mfn2 _ NMDA F GFP-Bax/mtRFP 75 100 Bax G * * N.S. * c * * 60 GFP DAPI Cyt c _ CT I H 0 10 20 30 40 - NMDA % Cell death * N.S. siBax siCT D 15 µM NMDA NMDA _ _ sh-Mfn2 + siCT sh-Mfn2 + siBax 0 10 20 30 40 - NMDA 15 µM % Cell death N.S. * CT Bcl-xL E GFP DAPI NMDA NMDA _ _ sh-Mfn2 sh-Mfn2 + Bcl-xL GFP DAPI (h) after 1 h NMDA NMDA induced cell death C 10 20 30 40 50 CT Mfn2 N.S. * * * * N.S. N.S. 0 10 20 30 40 - NMDA % Cell death * N.S. siBax siCT D 15 µM NMDA NMDA _ _ sh-Mfn2 + siCT sh-Mfn2 + siBax GFP DAPI % NMDA induced cell death C 0 10 20 30 40 50 CT Mfn2 1.5 3 5 N.S. * * * * N.S. N.S. F 0 10 20 30 40 - NMDA % Cell death * N.S. Figure 1 siBax siCT D 15 µM NMDA NMDA _ _ sh-Mfn2 + siCT sh-Mfn2 + siBax 30 40 h N S * E GFP DAPI _ sh-Mfn2 GFP DAPI (h) after 1 h NMDA D D sh-Mfn2 + siBax % N 0 1.5 3 5 CT Mfn2 _ NMDA F s s 0 10 20 30 40 - NMDA 15 µM % Cell death N.S. * CT Bcl-xL E GFP DAPI NMDA NMDA _ _ sh-Mfn2 sh-Mfn2 + Bcl-xL (h) after 1 h NMDA E F sh-Mfn2 + Bcl-xL 0 25 50 75 100 CT Mfn2 % Mitochondrial Bax G 3 5 (h) after 1 h NMDA * * N.S. * % Cytosolic cyt c 3 5 (h) after 1 h NMDA * * 0 20 40 60 CT Mfn2 * * GFP DAPI Cyt c _ _ NMDA NMDA CT Mfn2 I H * H G + * + + + A C D - + - 96 36 50 CT NMDA MG-132 Mfn2 Porin Actin NMDA CT ActD CHX RNA/18S (A.U.) 0 1 2 4 8 (h) * * 0 0.2 0.4 0.6 0.8 1 1.2 - NMDA B CT MG-132 Mfn2/Actin (A.U.) N.S. 0 0.5 1 1.5 2 2.5 Mfn2 Mfn1 SESN2 0.8 1.0 1.2 - (A.U.) E N.S. * N.S. * * Figure 6 + A - + - 96 36 50 CT NMDA MG-132 Mfn2 Porin Actin 0 0.2 0.4 0.6 0.8 1 1.2 - NMDA B CT MG-132 Mfn2/Actin (A.U.) N.S. * * B A * C RNA/18S (A.U.) 0 1 2 4 8 (h) * * 0 0.5 1 1.5 2 2.5 Mfn2 Mfn1 SESN2 C * C RNA/18S (A.U.) 0 1 2 4 8 (h) * * 0 0.5 1 1.5 2 2.5 Mfn2 Mfn1 SESN2 - + - + - + D 36 50 96 Mfn2 Actin Porin NMDA CT ActD CHX 0.0 0.2 0.4 0.6 0.8 1.0 1.2 - NMDA CT ActD CHX Mfn2/Actin (A.U.) E N.S. * N.S. E D A B h after clamp release NMDA (h) 50 50 50 50 CT 0.25 0.5 1 2 4 8 CT 0 2 6 24 Actin MEF2A Actin MEF2A B A A B h after clamp release NMDA (h) 50 50 50 50 CT 0.25 0.5 1 2 4 8 CT 0 2 6 24 Actin MEF2A Actin MEF2A * N.S. N.S. Figure 1 C Mfn2/18S RNA (A.U.) 50 98 36 CT MEF2-DN Actin Mfn2 Porin NMDA - + - + 0 0.2 0.4 0.6 0.8 1 1.2 - NMDA CT MEF2-DN % Cells with tubular mitochondria * 0 20 40 60 80 100 CT Mfn2 D CT MEF2-DN CT CT Mfn2 E D C E E CT G H F CT Mfn2 MEF2-DN MEF2-DN + Mfn2 * ψ (A.U.) CT MEF2-DN MEF2-DN + Mfn2 % NMDA-induced cell death * -5 0 5 10 15 20 25 Mfn2 CT MEF2-DN Mfn2 Mfn2 + MEF2-DN GFP DAPI GFP DAPI _ NMDA 0 0.25 0.5 0.75 1 1.25 Luciferase Activity (A.U.) CT MEF2-DN CT MEF2-DN CT MEF2-DN Mfn2 promoter (neurons) Mfn2 promoter (10T1/2) SESN2 promoter (neurons) 0 2 4 6 MEF2 CT +45 +45 -54 -682 -1332 -1982 Luc * * -1982 BOX 1 2 Luciferase Activity (A.U.) * 0 2 4 MEF2 CT MEF2: FP + + + + + + - FP+ + 32P-Probe: -907/-887 Mutated Fold cold probe Wild type : - - 25 100 - - - - - - - Mutated : - - - - 25 100 - - - - - Glut4 Enh.: - - - - - - 100 - - - - MEF2: - + + + Super Shift * * * Figure 8 A D C B E Luciferase Activity (A.U.) 3 3.5 0 0.5 1 1.5 0 0.5 1 1.5 0 0.5 1 1.5 F Luciferase Activity (A.U.) CT MEF2-DN Mfn2 promoter (neurons) * Figure 8 A 0 0.5 1 1.5 Figure 8 Figure 8 Luciferase Activity (A.U.) CT MEF2-DN CT MEF2-DN CT MEF2-DN Mfn2 promoter (neurons) Mfn2 promoter (10T1/2) SESN2 promoter (neurons) * Figure 8 A 0 0.5 1 1.5 0 0.5 1 1.5 0 0.5 1 1.5 A CT MEF2-DN CT MEF2-DN Mfn2 promoter (10T1/2) SESN2 promoter (neurons) 0 0.5 1 1.5 0 0.5 1 1.5 Lucif CT MEF2-DN CT MEF2-DN CT MEF2-DN 0 2 4 6 MEF2 CT +45 +45 -54 -682 -1332 -1982 Luc * * -1982 BOX 1 2 Luciferase Activity (A.U.) 0 2 4 MEF2 CT MEF2: FP + + + + + + - FP+ + 32P-Probe: -907/-887 Mutated Fold cold probe Wild type : - - 25 100 - - - - - - - Mutated : - - - - 25 100 - - - - - Glut4 Enh.: - - - - - - 100 - - - - MEF2: - + + + * Shift Super-Shift Ab: MEF2 - MEF2 IgG NMDA - + - IP: MEF2A IgG IgG fold enrichement * * * N.S. Figure 1 C Mfn2/18S RNA (A.U.) 50 98 36 CT MEF2-DN Actin Mfn2 Porin NMDA - + - + 0 0.2 0.4 0.6 0.8 1 1.2 - NMDA CT MEF2-DN % Cells with tubular mitochondria * 0 20 40 60 80 100 CT Mfn2 D CT MEF2-DN CT MEF2-DN CT Mfn2 E 50 50 50 50 Actin MEF2A Actin MEF2A * N.S. N.S. C Mfn2/18S RNA (A.U.) 50 98 36 CT MEF2-DN Actin Mfn2 Porin NMDA - + - + 0 0.2 0.4 0.6 0.8 1 1.2 - NMDA CT MEF2-DN % Cells with tubular mitochondria * 0 20 40 60 80 100 CT Mfn2 D CT MEF2-DN CT MEF2-DN CT Mfn2 E CT Mfn2 MEF2-DN MEF2-DN + Mfn2 * ψ (A.U.) CT MEF2-DN MEF2-DN + Mfn2 % NMDA-induced cell death F * -5 0 5 10 15 20 25 G Mfn2 CT MEF2-DN Mfn2 Mfn2 + MEF2-DN GFP DAPI GFP DAPI _ NMDA 0 0.25 0.5 0.75 1 1.25 H Actin Actin * N.S. N.S. C Mfn2/18S RNA (A.U.) 50 98 36 CT MEF2-DN Actin Mfn2 Porin NMDA - + - + 0 0.2 0.4 0.6 0.8 1 1.2 - NMDA CT MEF2-DN % Cells with tubular mitochondria * 0 20 40 60 80 100 CT Mfn2 D CT MEF2-DN CT MEF2-DN CT Mfn2 E F G GFP DAPI GFP DAPI _ NMDA 1.25 H % Cells with tubular mitochondria * 0 20 40 60 80 100 CT Mfn2 D CT MEF2-DN * N.S. N.S. Figure 1 Amplifed region: D C B E Luciferase Activity (A.U.) 0 0.5 1 1.5 2 2.5 3 3.5 Mfn2 prom Ac!n 0 0 0 F 0 2 4 6 MEF2 CT +45 +45 -54 -682 -1332 -1982 Luc * * -1982 BOX 1 2 Luciferase Activity (A U ) 0 2 4 MEF2 CT MEF2: FP + + + + + + - FP+ + 32P-Probe: -907/-887 Mutated Fold cold probe Wild type : - - 25 100 - - - - - - - Mutated : - - - - 25 100 - - - - - Glut4 Enh.: - - - - - - 100 - - - - * * D C B Luciferase Activity (A.U.) 0 2 4 6 MEF2 CT +45 -54 -682 -1332 -1982 Luc * * B Luciferase Activity (A U ) 0 2 4 6 MEF2 CT +45 -54 -682 -1332 -1982 Luc * * MEF2: FP + + + + + + - FP+ + 32P-Probe: -907/-887 Mutated D B Luciferase Activity (A U ) MEF2: FP + + + + + + - FP+ + 32P-Probe: -907/-887 Mutated Fold cold probe Wild type : - - 25 100 - - - - - - - Mutated : - - - - 25 100 - - - - - Glut4 Enh.: - - - - - - 100 - - - - D B D Mutated 6 0 Luciferase Activity (A.U.) C 0 MEF2: - + + + * Shift Super-Shift Ab: MEF2 - MEF2 IgG NMDA - + - IP: MEF2A IgG IgG fold enrichement * N.S. Amplifed region: E 0 0.5 1 1.5 2 2.5 3 3.5 Mfn2 prom Ac!n F MEF2: - + + + * Shift Super-Shift Ab: MEF2 - MEF2 IgG E NMDA - + - IP: MEF2A IgG IgG fold enrichement * N.S. Figure 1 Amplifed region: 0 0.5 1 1.5 2 2.5 3 3.5 Mfn2 prom Ac!n F F E Ab: MEF2 - MEF2 IgG Figure 9 Glutamate Excitotoxic conditions Nucleus Drp1 NMDAR Cell death [Ca2+] Mfn2 R Mfn2 C N MEF2 MEF2 degradation ΔΨm Bax recruitment Cyt c release Mitochondrial fragmentation Nucleus p65 NF- kB MEF2 ΔΨm Mfn2 C N NMDAR Survival Physiological conditions R Mfn2 [Ca2+] Glutamate Glutamate Excitotoxic conditions Nucleus Nucleus p65 NF- kB MEF2 ΔΨm Mfn2 C N Drp1 NMDAR NMDAR Survival Cell death Physiological conditions [Ca2+] Mfn2 R R Mfn2 Mfn2 C N [Ca2+] MEF2 MEF2 degradation Glutamate ΔΨm Bax recruitment Cyt c release Mitochondrial fragmentation
https://openalex.org/W4388509905	https://www.remap.ugto.mx/index.php/remap/article/download/374/287	es		Periodos electorales como incentivos para la deuda pública estatal: Una revisión desde la relación ejecutivo-legislativo en México en el periodo 2012-2020	Revista mexicana de análisis político y administración pública (en línea)/Revista mexicana de análisis político y administración pública	2,022	cc-by	7,613	Revista Mexicana de Análisis Político y Administración Pública Volumen X, número 2, julio-diciembre 2021 pp. 142-157 Periodos electorales como incentivos para la deuda pública estatal: Una revisión desde la relación ejecutivo-legislativo en México en el periodo 2012-2020 Electoral periods as incentive for state public debt: A revision from the legislative-executive interaction in Mexico from 2012 to 2020 Mario Joel Ramírez Hernández Resumen Los procesos electorales en México son detonantes de comportamientos políticos que rompen la rutina. La deuda pública en las entidades federativas ha probado en años recientes ser un indicador que debe su comportamiento al ambiente político que la rodea. Asimismo, el gasto público se ha convertido en el objetivo del oportunismo político durante los periodos electorales. Este escrito pretende detectar si las condiciones políticas y legislativas durante los periodos electorales convierten al endeudamiento público en un blanco para que los gobernadores obtengan recursos públicos con los que puedan asegurarse la consecuente elección, así como la postura de sus legislaturas ante dichas acciones. Palabras clave: Finanzas públicas estatales, legislaturas locales, deuda pública estatal, gobierno estatal. Abstract 142 Election processes in Mexico regularly detonate political behaviors out of the routine. In recent years, state debt has proven to be an index attached to the surrounding political environment. As such, public spent has become target of political opportunism during election periods. This text intends to detect if political and legislative circumstances during election periods turn public debt into a target so governors can get public resources with which they can assure the next election for their party, such as the stances of their congresses around these actions. Key words: Public finances, local congresses, public state debt, state governments Introducción Autores como Sánchez (2017) han demostrado que las condiciones políticas y electorales son poderosas determinantes para el funcionamiento presupuestal de los gobiernos estatales. Temas como el presupuesto y el gasto dependen de las oportunidades políticas y electorales que los funcionarios públicos tengan al alcance. Es válido preguntarse dónde radica tal relación, para lo cual se plantean los siguientes puntos: Recibido: 1 de agosto de 2021 Aceptado: 15 de octubre de 2021 Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 • Aumentar el gasto en un año electoral para destinarlo a obras públicas es una manera en que un gobernador puede mejorar la imagen de su gestión y así aumentar las probabilidades de que su partido gane la elección consiguiente.1 • Un gobernador es más efectivo cuando tiene mayoría partidista en el congreso local, por lo tanto, no solo le interesará aumentar el gasto en años electorales al término de su gestión, sino en aquellas en que se jueguen los curules de su legislatura. • Armesto y Olmeda (2016) Clarifican que un gobernador con menor pluralidad al interior de su congreso será más efectivo en manejar las operaciones presupuestales de su entidad, lo que por consecuencia le dará mayor discrecionalidad en la búsqueda de mejorar las probabilidades electorales de su partido. • López Lara (2017) establece que las condiciones políticas de los poderes de cada entidad federativa son determinantes del poder de acción de cada gobernador. Esto implica que aunque tengan funcionamientos diferentes, cada gobernador hará el esfuerzo por crear o mantener dichas condiciones a través de los procesos electorales. ¿Qué alcances financieros tiene, entonces, la dependencia del gasto público estatal en relación con los ciclos electorales? Ibarra Salazar (2013) y Merino (2010) analizan la poca capacidad con la que cuentan las entidades federativas para conseguir recursos de manera independiente de las transferencias federales. Desde la instauración de la Ley Federal de Coordinación Fiscal el Estado mexicano centró sus miras en la centralización tanto del gasto como de la recaudación fiscal en todo el país, dejando a los gobernadores como meros instrumentos para la materialización de la voluntad del gobierno federal. Con el paso de los años y varias reformas a la Constitución los gobernadores han ido recuperando su capacidad de gasto, no así en el caso de la capacidad de recaudación. En una búsqueda de aumentar su capacidad de recaudación los gobernadores han recurrido al endeudamiento como alternativa, como lo plantea Reyes Tepach (2012). La dependencia de los ejecutivos estatales hacia las transferencias alcanzó su cúspide en 1998 con la creación del ramo 33, lo que dejó las finanzas públicas estatales en manos de la federación. Esto colocó a los gobiernos estatales a merced de la agenda de los presidentes en turno, forzándolos a un constante juego de negociación para mejorar sus finanzas. Para hacer contrapeso a esta pérdida de autonomía en la discrecionalidad fiscal, las entidades decidieron recurrir al endeudamiento. Los recursos que entren por esta vía deben destinarse a obras públicas, sin embargo no tienen etiqueta que los limite a un gasto particular dentro de dicho rubro. La hipótesis con la que trabajará este estudio es la siguiente: Los gobernadores con gobierno unificado, mínima diversidad legislativa y mayor estabilidad política electoral son más proclives a incurrir en el endeudamiento público con fines electorales. Siendo así que un gobernador con un menor número de obstáculos y el contexto político a su favor tiene mayor poder de acción discrecional y tendrá como prioridad tanto mantener el estatus de poder de su gestión como lograr que su partido gane la siguiente elección. Los gobernadores tienen prioridad para que su partido gane las elecciones del ejecutivo estatal y las de su legislatura por las siguientes razones: • Si bien no existe la reelección para gobernadores, conseguir que su partido gane las siguientes elecciones a la gubernatura implica un logro para su carrera dentro del partido al que pertenecen. Provocar las condiciones para que el partido repita gestión en la entidad puede abrir las puertas al gobernador saliente para nuevos cargos administrativos o jerarquías altas dentro de su partido. • Mantener una mayoría dentro de su legislatura es de gran utilidad, sirve para que puedan Ramírez Rodríguez y Erquizio Espinal (2012) abordan el ciclo electoral oportunista en el que los gobernadores intentan mejorar el panorama económico durante periodos electorales para dar la apariencia de un manejo económico óptimo. Sin embargo, las estrategias y proyectos a los que se destinan tales recursos suelen carecer de seguimiento cuando han logrado la meta electoral. 1 Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 143 conservar su poder discrecional y seguir generando condiciones favorables en periodos electorales que resulten en victorias. Un gobernador sin mayoría se ve limitado ante la necesidad de integrar los intereses de los partidos mayoritarios en la legislatura y por tanto su agenda deja de estar centrada en las prioridades de su partido. Las variables con las que trabajará este estudio son: • Independientes • Tipo de gobierno. Sánchez (2017) y Patrón (2014) dejaron claro que las capacidades de los gobernadores están ligadas a tener o no mayoría dentro de su legislatura, siendo así que un gobernador cuenta con mayor poder discrecional cuando tiene mayoría. Esta tipificación del gobierno de acuerdo con la presencia o ausencia de mayoría en el congreso conforma un rasgo imprescindible del ambiente político. • Pluralidad política de la legislatura. A través del número efectivo de partidos en una legislatura (NEPL) se puede conocer el grado de pluralidad política existente. Una legislatura más plural tiende a generar comportamientos más inestables dada la cantidad de agendas partidistas en juego, así como a complicar la libertad con la que los gobernadores ejercen su agenda. • Estabilidad política de las gubernaturas. La estabilidad se puede medir a través de contar las alternancias que ha experimentado el ejecutivo local. Una entidad con pocas alternancias puede dar un seguimiento más puntual a su agenda, mientras que la imposición de nuevas prioridades y el abandono de proyectos previos será más frecuente cuando la alternancia se vuelva común. • Diversidad en la estabilidad política. Esta variable se medirá contando la cantidad de partidos que han ocupado la gubernatura en el periodo de estudio. Una alternancia entre dos partidos permite un seguimiento más estable a los programas propuestos que una alternancia entre 3 o más. • Dependiente • Deuda pública estatal. Para abordar esta variable se revisarán las cifras de deuda de las 32 entidades federativas en el periodo estudiado en peso mexicano constante, considerando la inflación al año 2021. 144 A través de una correlación entre variables dentro del lapso de estudio se revisará la incidencia del comportamiento de las variables independientes paralelo al de la dependiente. Así se comprobará qué tanto del contexto y la situación política y legislativa tienen alcance en la utilización de endeudamiento con fines electorales. Deudas estatales y su comportamiento Más de medio siglo de presidencialismo no desaparece sin dejar cicatrices, tal es el caso del Estado mexicano, que vivió una ocupación partidista hegemónica durante más de 70 años. Durante esa etapa los gobernadores contaban con alta capacidad de acción pues debían ejecutar la voluntad presidencial libre de obstáculos. Loaeza (2010) establece que gran cantidad de las interacciones entre el presidente y los gobernadores eran de naturaleza tradicional y extra constitucional, por lo que no estaban debidamente institucionalizados. En ausencia de un poder y agenda centralizados, eso sumado a la creciente pluralización partidista en los ejecutivos estatales, los gobernadores quedaron libres de ataduras políticas, lo que obligó al presidente a negociar con ellos para obtener su cooperación por primera vez en la historia. Hernández Trillo (2010) describe que durante el presidencialismo mexicano, se buscó que la capacidad de gasto y recaudación se concentrara en el gobierno federal, relegando a los gobernadores a meros ejecutores de la agenda. Sin embargo, tras la crisis fiscal de la década Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 de 1980 se hizo evidente que la capacidad de gasto debía regresar a manos de los gobernadores pues el gobierno federal se vio incapaz de ejecutar todo desde la centralidad. Gastos como la educación, infraestructura social, y seguridad pública fueron asignados a las entidades federativas y municipios. Los recursos para dichos fines venían etiquetados y fuera de lo obtenido a través de los departamentos de tránsito las entidades se quedaron carentes de poder recaudatorio. Es hasta 2011 que inicia una nueva etapa en que los gobernadores acudieron a una nueva fuente de ingresos: la deuda pública. El evento detonante ocurrió con el gobierno del estado de Coahuila, el cual generó controversia al esconder una deuda pública de 32 mil millones de pesos2. Si bien el evento estuvo envuelto en controversias debido al mal uso de los procesos para aprobación de deuda, marcó un antes y un después en el interés de los gobernadores en el endeudamiento. Gráfica 1 Endeudamiento de las entidades federativas en México en el periodo 2012-2020 en millones de pesos 145 Fuente: Elaboración propia con Datos de INEGI ajustados a inflación 2021 Al realizar una comparativa de la cifra nacional de deuda pública estatal entre el cierre de 2006 y el de 20123 se observa que hubo un aumento en la cifra total: • 2006 - 496,456,911,426 mxn • 2012 - 612,106,943,366 mxn En este primer momento es notorio que los gobernadores incurrieron con mayor grado en el endeudamiento en este lapso de 6 años. Esta etapa fue el inicio de un nuevo perfil crediticio para las entidades federativas. Pues marcó un inicio general en que los gobernadores recurrieron más a la deuda como fuente de ingreso. Sin embargo contrario a lo que se puede suponer, este cambio tampoco supuso una espiral fuera de control en que la deuda crezca desmedidamente. Cuando se revisan las cifras ajustadas a la inflación anual se encuentra una nueva etapa de estabilidad. 2 La narrativa correspondiente al evento se puede revisar en Ramírez (2014). El evento fue notorio no solamente por poner de moda las macro deudas de las entidades federativas sino por originarse en contextos polémicos y manejo indebido de los procesos públicos involucrados. Entidades como Veracruz y Estado de México también fueron notorios por la polémica alrededor de sus endeudamientos. 3 Como se mencionó en la introducción, las cifras de deuda pública se presentan calculando la inflación al año 2021. Esto con la finalidad de evitar la apariencia de que el endeudamiento va en aumento. Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 Si bien hay aumentos por encima de los demás en 2012 y 2015, la generalidad del comportamiento del endeudamiento de las entidades federativas apunta más a la estabilidad que al descontrol creciente. Los eventos de 2011 entonces pueden verse más como la detonación de un nuevo panorama con nuevas reglas y nuevos niveles de manejo crediticio que como el inicio del caos. El comportamiento individual no es el mismo que el colectivo, las entidades federativas pueden exigir diferentes conductas en su camino al endeudamiento. El perfil crediticio de un estado altamente industrializado como Nuevo León o Estado de México no es el mismo que el de uno turístico como Quintana Roo o de mayor arraigo a las tradiciones como Oaxaca. El solo endeudamiento y su reacción ante el contexto histórico es insuficiente para entender el fenómeno al que se enfrenta este estudio. En el siguiente apartado se hará una revisión de los aportes teóricos necesarios para justificar lo que podemos esperar de la revisión teórica. Influencia de las alineaciones legislativas locales en el poder presupuestal de los gobernadores 146 En esta sección se realizará una revisión de los textos recientes que han abordado la relación financiera entre poderes locales en materia de finanzas, cuál es el alcance del poder del gobernador y qué papel juegan sus legislaturas con las facultades al alcance. Gutiérrez Cuéllar (2016) se hace la pregunta ¿qué características locales explican las diferencias de poder de los gobernadores en México? Esto teniendo en consideración que no en todas las entidades federativas existen las mismas prerrogativas que permite a los ejecutivos estatales una gama de facultades estandarizadas. Por el contrario, cada caso es un escenario distinto en el que incluso las mismas capacidades tienen alcances diferentes. Un descubrimiento que realiza es que la división de poderes falla en la distribución del poder a nivel local, pues los gobernadores pueden ser poderosos aún en presencia de un poder legislativo que vigile sus acciones. Para llegar a estos resultados se realizó un índice que pretende medir el poder de los gobernadores a lo largo de las 32 entidades federativas por medio de un índice generado a partir de las siguientes variables en encuestas: • El gobernador era el principal líder de su partido. • El gobernador era el principal líder de su partido, pero había más dirigentes importantes. • Era uno más entre los principales dirigentes del partido. • Era un líder partidista menos importante que otros. • El gobernador no tenía partido propio. Basado en este índice, Chiapas (-3.17) resulta la entidad con el gobernador más débil mientras que Puebla (1.7) tiene al más poderoso. En el estudio se comprueba que la pertenencia partidista de un gobernador es capaz de influir en el nivel poder que éste detente. Sin embargo, la autora demuestra que esto depende que el gobernador tenga mayoría en su respectivo congreso, lo que es el caso de algunos mandatarios que pertenecen al PRI. No es lo mismo con gobernadores de otros dos partidos. Analizando ese índice junto a la variable de alternancia política se demostró que ésta no es relevante para determinar el índice de poder del gobernador. Si se toma en cuenta el poder de los gobernadores pensando en fenómenos como el patronazgo es tentador preguntarse si ¿la hegemonía partidista a nivel estatal inhibe el patronazgo o lo incentiva? Armesto y Olmeda (2016) en otro trabajo abordan dos hipótesis opuestas basadas en este cuestionamiento: • A menor pluralidad política, se incentiva el patronazgo político en el gobierno de las entidades federativas. • A mayor pluralidad política, se constriñe el patronazgo político en el gobierno de las entidades federativas. La primera hipótesis tiene su fundamento en la idea de que cuando hay un solo partido mayoritario en un congreso local, es más fácil asignar a los miembros del partido un cargo Revista Mexicana Revista Mexicana de Análisis dePolítico AnálisisyPolítico Administración y Administración Pública, Universidad Pública, Universidad de Guanajuato, de Guanajuato, Volumen Volumen X, número X,2,número julio-diciembre 2, julio-diciembre 2021 2021 público, por lo que el interés de éstos crecerá. En este caso el partido mayoritario tiene más alcances y más cargos por llenar así que existe una gran cantidad de beneficios por repartir. La segunda hipótesis se fundamenta en que cuando hay más partidos ocupando un gran número de escaños en el congreso local, buscarán obtener beneficios a los que el partido en el ejecutivo federal tenga acceso. Por esta razón la negociación se vuelve una prioridad para los partidos minoritarios que pueden entregar su lealtad a cambio de los cargos que el ejecutivo estatal pueda otorgarles. En los resultados se obtuvo que el gobierno dividido se asocia positivamente pero no es significativo con respecto del patronazgo. En gobiernos del PRI el patronazgo es más alto, comprobado por la relación positiva y significativa entre la variable de movilización partidista interna y el gasto en salarios. El gasto particularista en salarios disminuye en años electorales de manera significativa, esto debido a que la variable de los ciclos electorales y su efecto en el gasto no resulta significativa ni positiva después de la regresión. El número efectivo de partidos legislativos (NEPL) hacia el interior de un parlamento ha sido de utilidad en varios estudios, debido a su capacidad para determinar el margen de pluralidad efectiva con el que tiene lugar la interacción política legislativa a nivel local. Esto ha resultado importante gracias a la noción que plantea de que no todo partido con escaños en un parlamento tiene una participación trascendente. Armesto (2015) recurre a esta variable con el interés de corroborar que en presencia de un escenario de pluralidad política es posible afectar las labores de un gobernador. Para ello plantea como hipótesis que esta pluralidad logra dos cosas: • La constricción del poder ejecutivo en términos de gasto, pues al no tener de su lado a su respectivo congreso se verá obligado a negociar antes de hacer realidad cualquier iniciativa que proponga. • La reducción del gasto particularista de parte del estado, pues al haber más de un interés con voz y voto en la política de gasto, los recursos se destinarán a una mayor cantidad de campos. Sin embargo aclara que para ambas hipótesis puedan comprobarse es necesario un marco institucional fuerte y sólido que permita a los congresos la capacidad de actuar y ejercer sus funciones como contrapesos que limiten y revisen el trabajo de su respectivo poder ejecutivo. En ausencia de estas condiciones, no es posible que un congreso pueda ejercer sus funciones y por tanto ninguna hipótesis se comprueba. Como resultado se obtuvo que en gobiernos de mayoría, a mayor cantidad de partidos políticos aumenta la estabilidad de la deuda durante las dos legislaturas dentro del mismo sexenio. Se deduce que a mayor necesidad de negociación con el congreso para aprobar una deuda, mayor es la necesidad de mantener una visión de endeudamiento a largo plazo. Por el contrario, en gobiernos de mayoría absoluta en el congreso, la cifra de deuda suele crecer en el año previo a las campañas electorales, pues se cierne mayor presión sobre el partido gobernante para mantener su popularidad y quedarse en cargo durante un periodo más. Si bien los marcos legales y/o normativos otorgan a los congresos un papel de contrapeso que sirva para contener o frenar los actos del poder ejecutivo, la recurrencia hacia la pasividad los priva de esta facultad. En resumidas cuentas, un congreso solamente puede considerarse como un contrapeso legislativo en la medida en la que eche mano de sus facultades constitucionales para intervenir en cualquier propuesta o iniciativa del ejecutivo. Probablemente la parte del trabajo de Patrón (2014) que mejor resume la función del poder legislativo en materia de finanzas públicas es la siguiente: “La participación del Poder Legislativo se enfoca, entonces, a limitar los abusos de poder del Ejecutivo sobre la utilización de los recursos públicos” (Patrón, 2014: 90). De acuerdo con este trabajo, la iniciativa de un congreso para retar las propuestas de su poder Revista Mexicana Revista Mexicana de Análisis dePolítico AnálisisyPolítico Administración y Administración Pública, Universidad Pública, Universidad de Guanajuato, de Guanajuato, Volumen Volumen X, número X,2,número julio-diciembre 2, julio-diciembre 2021 2021 147 ejecutivo depende de factores como la disciplina partidista. En este caso, la facción partidista del gobierno hacia el interior del pleno depende de cuánto poder detente dentro de su propio partido político. Se dan casos en que los gobernadores no gozan de influencia o popularidad entre los miembros de su partido, por tanto se pierden los incentivos para que exista disciplina partidista. En cambio, cuando se trata de un político posicionado y con un buen historial dentro de su propio partido, su facción en el congreso se comportará con mucha mayor inclinación para colaborar con sus ideas e iniciativas. Los estudios de caso corresponden a ocho entidades federativas, dentro de las cuales se utilizan las dos últimas legislaturas al momento de la publicación y se analizan los alcances de las propuestas realizadas por sus congresos locales y sus índices de aprobación a lo largo de ambos periodos. Las entidades estudiadas son: Distrito Federal, Guanajuato, Jalisco, Nayarit, Sonora, Sinaloa, Veracruz y Yucatán. Cuando se realiza el índice de aceptación de propuestas provenientes del Ejecutivo en todos los casos (con la excepción de una legislatura en Guanajuato) se tiene una aprobación de la mayoría de las propuestas siempre que el estatus del gobierno sea unificado. Asimismo, cuando el gobierno está dividido, el porcentaje de propuestas aprobadas por el Ejecutivo no alcanza más allá del 35%. Existen cuatro escenarios, de acuerdo con el autor, que propician la participación activa de las legislaturas con respecto a las iniciativas del ejecutivo: • En presencia de un sistema multipartidista en el que no exista una mayoría correspondiente a un solo partido dentro del pleno. • Los partidos políticos padecen fragmentaciones y no están unidos en su interior. De esta manera no hay cohesión ni una agenda unificada qué seguir. • Cuando los partidos del pleno son ajenos a unidades externas como el poder ejecutivo o comités internos. • Cuando las comisiones legislativas son independientes, permanentes y paralelas a otras unidades administrativas de control interno. 148 Esta perspectiva sobre el alcance del poder de los gobernadores, así como del sometimiento de sus respectivos congresos basado en el tipo de gobierno que presentan, se expone en el trabajo de Sánchez (2017). A un nivel más profundo y específico analiza el papel y el lugar de los congresos estatales en México. En su trabajo busca entender cuáles son los factores que incentivan la alineación de un congreso local con su respectivo gobernador en cuanto a la aprobación presupuestal anual. El autor hace énfasis en que un amplio poder constitucional es más notorio que uno partidista, pues le permite imponerse en todos los escenarios e ignorar cualquier aportación de parte de la oposición en su respectivo congreso. Sánchez (2017) asegura que no es posible asegurar que el estatus de un gobierno como dividido o unificado sea suficiente para determinar su capacidad en el dominio de un poder sobre otro. Existen más variables que deben tenerse en consideración como lo es la conformación y afiliación partidista de los funcionarios en cargo. En el caso de México, esta variable sí influye. El ejercicio de la presión en contra del gobierno estatal también ocupa un lugar importante en las interacciones entre poderes. El autor habla de cómo los diputados del partido del gobernador recurren a mecanismos oficiales como la auditoría estatal para actuar en contra de los miembros de la oposición. A su vez, la oposición también llega a recurrir a grupos sociales par que ejerzan presión en contra del gobernador para hacer llegar sus intereses a la agenda estatal en la planeación del programa de egresos. En el panorama general se percibe que la oposición dentro del congreso local funciona bajo la prioridad de una búsqueda ciega de recursos, sin mucha importancia en cuál sea su origen o destino. Esto se suma a que los diputados ejercen su cargo carentes de información acerca de las legislaciones y capacidades técnicas que les permitan hacer una labor más completa y mejor fundamentada. Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 En trabajo de Sánchez (2017) realiza un análisis de cinco entidades federativas para analizar el fenómeno de la lucha por el presupuesto entre legislaturas y ejecutivo estatal. Para tal efecto su análisis combina las variables presencia de alternancia estatal y tipo de gobierno (unificado o dividido). Los casos que se analizan son los siguientes: • Estado de México: gobierno sin alternancia, unificado y dividido. • Jalisco: gobierno con alternancia: unificado y dividido. • Zacatecas: gobierno con alternancia: unificado y dividido. Dos casos desviados: • Sinaloa • Tamaulipas En estos casos se hace un análisis de las revisiones realizadas al presupuesto por parte de la legislatura y de esta manera se pretende corroborar qué tanto afecta a esta labor si un gobierno es dividido o unificado. Se concluye que un congreso estará en favor del presupuesto de su gobernador siempre que la mayoría pertenezca al mismo partido y que esté cerrada la puerta a la búsqueda de recursos que puedan utilizar para su propio distrito. En el momento en el que esta última posibilidad queda abierta, los diputados locales verán más por su zona que por su lealtad partidista, en cuyo caso el estatus del gobierno como unificado o dividido queda relegado a segundo plano. Esto se corrobora en los casos de Jalisco y Zacatecas, entidades donde este principio se mantuvo constante y donde siempre que hubo gobierno unificado las revisiones al presupuesto disminuían, mientras que en gobierno dividido éstas aumentaron. En los dos casos tuvo lugar que la cooperación entre partidos fue un elemento guía para el análisis. Al respecto, los testimonios de antiguos diputados declararon que se les otorgaba poco tiempo para revisar el presupuesto y se buscaba que lo realizaran ciegamente. El caso del Estado de México se corroboró que tiene un peso importante la opción de buscar de recursos para beneficio del respectivo distrito de los diputados, de forma que en su primera etapa de gobierno unificado las revisiones al presupuesto fueron mínimas. En su posterior etapa de gobierno dividido aumentaron en gran medida, sin embargo en su tercera etapa, en la que se volvió al gobierno unificado, las revisiones se mantuvieron constantes. Esto fue gracias a la creación de programas que les permitían obtener recursos de uso distrital. Flamand (2006) realiza un estudio en el que busca comprobar que la competencia política en el gobierno vertical incentiva las relaciones fiscales entre niveles de gobierno. Su hipótesis gira en torno a que cuando los gobiernos subnacionales pertenecen a un partido diferente al del gobierno federal, logran verse beneficiados por un aumento en las transferencias federales de recursos tanto condicionados como no condicionados. En este trabajo se habla de la importancia de la alineación política y cómo la diferencia entre los intereses de los partidos determina el tipo de relación fiscal existente entre federación y entidades. También integra variables como la capacidad de un gobierno federal para controlar a sus niveles inferiores o la capacidad de un presidente o gobernador para controlar a otros miembros de su partido. Esto debido a que pueden darse casos en que los gobiernos federales cuentan con poder institucional para ejercer su control sin oposición significativa, en cuyo caso la presencia de otros poderes o niveles de gobierno no implica problemas para ejecutar sus iniciativas. Por otro lado, la disciplina hacia el interior de un partido es una herramienta básica para la alineación de intereses y acciones sin complicaciones. Se concluye que a partir de estas variables aplicadas al caso mexicano, un gobierno estatal de oposición al gobierno federal cuenta con mayor probabilidad de recibir más recursos Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 149 de la federación. De acuerdo con la necesidad de que la federación negocie con estos gobiernos subnacionales, debe ceder para conseguir su apoyo en otros temas políticos, y los recursos transferidos son el blanco favorito de los gobernadores a cambio de simpatía para el Presidente. En cambio, cuando un gobierno estatal pertenece al mismo partido que el federal, no hay necesidad de negociación, y no hay que cederles ninguna clase de incentivo a cambio de su apoyo. Cruce de variables y presentación de resultados A continuación, se presenta el comportamiento de las variables analizadas tanto a nivel individual como en modo correlacional. Primero se verá cada una y después se cruzarán entre sí para revisar los patrones de comportamiento observables. Número efectivo de partidos legislativos (NEPL) A lo largo del periodo de estudio en promedio las legislaturas locales en México mantuvieron un promedio de 3 partidos efectivos. Esto representa una baja fragmentación y una alta facilidad para el logro de acuerdos. Que exista cierta estabilidad en las cifras de endeudamiento puede deberse a que en casos de gobierno dividido el gobernador o el partido mayoritario tienen pocas fuerzas opositoras con presencia considerable con quienes pactar para lograr apoyo en sus iniciativas. El promedio es representativo, y es posible darse cuenta con solo notar el rango, pues las legislaturas con más partidos efectivos tienen 4 mientras que el menor número de partidos es de 2. 150 Fuente: Elaboración Propia Otro apoyo para la representatividad del promedio es que más de la mayoría absoluta de las entidades federativas mantienen un promedio de 3 partidos efectivos. El caso mexicano puede parecer caótico e inestable a nivel coyuntura, sin embargo la siempre presente estabilidad en los procesos legislativos estatales se debe a que no se enfrenta a un verdadero escenario de alta fragmentación. Sí existe un alto grado de representación política, no así un alto número de partidos efectivos. Número de alternancias y número de partidos en la gubernatura En materia de alternancias en la gubernatura se observó un periodo de mediana estabilidad, pues en promedio cada entidad enfrentó una alternancia en el periodo. Pensar esto desglosando Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 el valor del promedio implica que hubo entidades que no enfrentaron ninguna alternancia mientras que hubo otras con dos o más. En cuanto al número de partidos que ocuparon lugar en la gubernatura encontramos que en su mayoría hay partidos sin alternancia o con una sola. Esto revela un alto grado de estabilidad política en cuanto a cambios de partido en el ejecutivo local. En el panorama global quedan los siguientes resultados: Fuente: Elaboración propia Al realizar el cruce hay que señalar que las entidades federativas que tuvieron dos alternancias eligieron partidos diferentes en cada elección. Esto apunta a que si una entidad federativa es inestable en término de alternancias también lo será en término del número de partidos en la gubernatura. Sin embargo, esta inestabilidad se limita a un mínimo de casos. En el panorama general la estabilidad política de los estados mexicanos es alta, mantienen su preferencia por un partido político y en caso de cambiarlo por otro, se mantienen con esa nueva alternativa. Tipo de gobierno Para el tipo de gobierno se presentó una variedad de resultados con comportamientos que podrían parecer difíciles de interpretar a primera vista. Por tal motivo se realizó la siguiente tipificación que ofrezca a éste y otros proyectos de la misma naturaleza una alternativa para tal fin: • Entidad estable: Aquella con más momentos de gobierno unificado que dividido. • Entidad inestable: Aquella con más momentos de gobierno dividido que unificado. • Entidad intermedia: Aquella con igual número de ambos tipos de gobierno. Esto debido a que un escenario con gobierno unificado implica menos negociación y la toma ágil de decisiones, mientras que uno con gobierno dividido requiere la integración de otras agendas, periodos más arduos de negociación y tomas de decisión con menos enfoque. Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 151 Aplicando esta clasificación al caso de cada entidad federativa nos arroja la siguiente alineación: Fuente: Elaboración propia En un 50% las entidades federativas en México mantienen un perfil de estabilidad, sumando las cinco que tienen estabilidad intermedia se puede confirmar que el panorama mexicano es mayoritariamente estable. Casi un 33% de los gobiernos estatales son políticamente inestables, lo que significa que este perfil tiene una presencia que merece atención en periodos venideros, pues un aumento podría tener consecuencias para el manejo crediticio estatal. El que México sea mayoritariamente estable no debe distraer de que la porción inestable podría arrojar información interesante en términos de su comportamiento. Cruce de variables ¿cómo impacta la inestabilidad y los periodos electorales en la deuda pública? 152 Para analizar la influencia de los periodos electorales en el endeudamiento de las entidades federativas lo primero es hacer una clasificación del comportamiento crediticio de las entidades federativas en años electorales. Para ello se creó el siguiente orden, el cual sigue un criterio similar al del tipo de gobierno: • Tendencia alta al endeudamiento. Cuando la deuda crece en la mayoría de los años electorales. • Tendencia intermedia al endeudamiento. Cuando hay crecimiento y disminución en igual número de años. • Sin tendencia al endeudamiento. Cuando la deuda disminuye o se mantiene igual en la mayoría de los años electorales. De acuerdo con dicha clasificación las entidades quedan en el siguiente orden: Fuente: Elaboración propia Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 Las entidades se decantan por igual hacia endeudarse en años electorales como a no hacerlo. Hay que clarificar que no todas las entidades federativas se endeudan al mismo ritmo. Un aumento en la deuda pública puede ser pequeño, por eso hay que ser minuciosos al analizarlo. Pensando en ello se realizará el siguiente filtro. Solo se considerará un aumento de deuda de alto perfil cuando éste sea a partir de mil millones de pesos. Asimismo se integrarán las variables previamente analizadas para detectar patrones de comportamiento en el perfil de las entidades con alto perfil crediticio en años electorales. 153 Fuente: Elaboración propia Haciendo observación de la tabla 5 se llega a las siguientes deducciones: • La presencia de aumentos de deuda de alto perfil en años electorales sucede primordialmente en entidades federativas con una propensión alta a a endeudarse en esas fechas. Aunque no sea en todas las ocasiones, al menos en una aumentan los créditos para aprovechar los periodos de elecciones. • No existe relación con la presencia de gobiernos unificados o divididos, por lo que el endeudamiento de alto perfil en periodos electorales puede venir de gobernadores con apoyo mayoritario en el congreso o sin él. • Las alternancias y el número de partidos que han ocupado la gubernatura muestran tendencia hacia la estabilidad. La deuda de alto perfil en años electorales puede suceder en presencia Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 de pocas alternancias y siempre que éstas sean con partidos que regresan al cargo después de un periodo de ausencia. • La pluralidad en la legislatura es un factor clave. Cuando hay un alto número de NEPL habrá endeudamiento de alto perfil en años electorales. Esto se puede deber a que en presencia de alta competencia política los gobernadores buscarán generar recursos para mejorar su imagen pues saben que su partido puede perder la administración. Para complementar se revisará si existe un patrón en el caso de las entidades federativas con endeudamiento de perfil bajo. Las observaciones basadas en la tabla 6 se enlistan a continuación: • La tendencia al endeudamiento es baja, complementando la información de la tabla 5. Una entidad federativa se endeudará con un alto perfil cuando sea tendenciosa a endeudarse con regularidad en periodos electorales. • La estabilidad basada en el tipo de gobierno no representa una determinante en el nivel de endeudamiento en años electorales, por lo que se soporta la idea de que un gobierno estatal puede ser unificado o dividido sin que afecte la probabilidad de endeudarse cuando se acerquen los comicios. • La estabilidad basada en el número de alternancias y el número de partidos que han ocupado la gubernatura también se inclina hacia la estabilidad. El sistema de partidos en México si bien es pluralista no alcanza un grado de fragmentación alto, por lo que esta estabilidad puede permitir o restringir endeudamientos de alto perfil con mayor facilidad. • El NEPL fue inferior que en la tabla 5, sin embargo no con un margen amplio (promedio de 3.1 y 2.8 respectivamente). Al igual que con la variable anterior se puede concluir que la tendencia a endeudarse con alto o bajo perfil se debe a la estabilidad de la pluralidad partidista mexicana y la agilidad que permite para la toma de decisiones. 154 Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 Fuente: Elaboración propia Conclusiones La teoría revisada fue clara en varios puntos, como que la pluralidad política legislativa genera dificultad en la toma de decisiones y la presencia de gobiernos divididos obstaculiza la discrecionalidad en los gobernadores. El caso mexicano ha resultado particular por demostrar que en sus condiciones la teoría no siempre ha acertado. ¿Significa esto que la teoría es insuficiente o que quizás estamos viendo el caso mexicano con mayor inestabilidad de la que en realidad padece? Este estudio se decanta por la segunda opción. El que pueda existir endeudamiento público estatal de alto o bajo perfil indiferente de las condiciones como la estabilidad política o el tipo de gobierno reside en que la pluralidad política sigue sin ser alta en el caso de las legislaturas locales. Existe una gran variedad de partidos políticos en el escenario legislativo mexicano, sin embargo pocas fuerzas con verdadero poder de veto. Como una primera conclusión los periodos electorales pueden fungir como incentivos para el endeudamiento estatal de alto o bajo perfil pues incluso los gobernadores sin mayoría legislativa tienen pocas fuerzas partidistas que enfrentar para lograr acuerdos. Esto debido a que el veto legislativo estatal está concentrado en un mínimo de partidos efectivos. Esta fortaleza para promover o limitar el endeudamiento público está ligada a la agenda que cada partido en la gubernatura mantenga. La pluralidad se mantiene a un nivel que cualquier gobernador puede manejar para sacar adelante su agenda presupuestal. Sin embargo, esta poca pluralidad también significa que en escenarios de gobierno dividido la oposición tendrá pocos obstáculos para rechazar el presupuesto del gobernador. Esto nos lleva a la siguiente conclusión, el que un gobernador tenga la facilidad para endeudar a su entidad no le garantiza una reelección para su partido. Las entidades enfrentaron escenarios de alta o baja estabilidad basada en la alternancia sin que hubiera algún efecto en la propensión a endeudarse en años electorales. La diversidad en las agendas presupuestales de los gobernadores es amplia. En un ambiente de poca pluralidad es sencillo que las refuercen con poco esfuerzo. El porqué de sus acciones puede residir en otros factores como su gestión o las influencias del partido al que pertenezcan. Es notorio que las legislaturas, a pesar de tener un veto considerable siguen manteniendo regímenes de funcionamiento basados en la sumisión. Las legislaturas reactivas parecen seguir siendo la normalidad en el campo político estatal en México. Al haber un panorama con una pluralidad efectiva al mínimo cuentan con mejores herramientas para discutir los proyectos crediticios de los gobernadores, ventaja de la que no siempre eligen sacar provecho. Esto se puede conectar con lo arriba mencionado, que entienden que el alto o bajo endeudamiento cercano a los periodos electorales no garantiza que el partido del gobernador gane la siguiente elección. La coyuntura de los casos individuales también podría ser un factor que ayude a explicar el alto o bajo aprovechamiento de los gobernadores en años electorales para endeudarse. El caso de Guanajuato es notorio por su constancia como gobierno unificado, en su agenda no mantienen el endeudamiento como una prioridad electoral, probablemente debido a que Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021 155 confían en la lealtad ciudadana para seguir ganando elecciones o que recurren a otros medios para mejorar su imagen con fines electorales. El caso de Tlaxcala también llamó la atención pues tuvieron una deuda intermitente que solo aparecía en años electorales (2012 y 2015) y desaparecía al año siguiente. Esto aunado a un escenario de gobierno dividido sin alternancias puede tratarse de un caso en que un gobernador sin respaldo legislativo se vuelve dependiente de medios alternativos como la deuda para desarrollar obras públicas que le garanticen la victoria del siguiente comicios. Hay que considerar que las características que envolvieron ambos casos no constituyeron una generalidad, pues otras entidades en condiciones similares presentaron comportamientos muy diferentes. Esto significa una casi completa imposibilidad para predecir el comportamiento crediticio de las entidades federativas basado en variables que no ahonden con mayor especificidad en el contexto individual de cada entidad federativa. Cuestiones como la cultura de la sumisión legislativa hacia su gobernador o la actitud reactiva más que una propositiva parecen estar enraizadas en la costumbre y la tradición más que en las condiciones políticas. Esto no necesariamente supone algo malo, pues parece que herramientas necesarias como la alternancia y la rendición de cuentas han encontrado su camino sin la necesidad de legislaturas activas que monitoreen y presionen las acciones de los ejecutivos estatales. A manera de cierre queda clara la complejidad del funcionamiento legislativo local en México y que no puede ser medido o predicho limitando su estudio al campo general. Sin embargo, acercamientos globales son lo que permite empezar a comprender los pormenores del campo de estudio al que nos estamos enfrentando. Resta priorizar la necesidad de que más estudios aborden el tema para contribuir a la creación de un corpus conceptual que permita entender los fenómenos del nivel estatal mexicano pues cada vez es más prominente. Referencias 156 Armesto, A. (2015) “Competencia política y gasto particularista de los gobiernos subnacionales en México y Argentina”, en Latin American Research Review, 50(1), 160-183. Armesto, A. y Olmeda, J. (2016) “Estrategias de interacción ejecutivo-legislativo y patronazgo político subnacional en México”, en En N.Loza e I.Méndez (Coords.), Poderes y democracias. La política subnacional en México. México: FLACSO- IEDF, pp. 93-112 Flamand, L. (2006) “El juego de la distribución de recursos en un sistema federal. La influencia del gobierno dividido verticalmente en la asignación de fondos federales a los estados mexicanos”, en Política y gobierno No. 13(2), México. CIDE, pp. 315359. Gutiérrez Cuéllar, Paola C. (2016) “Poder político subnacional: fortalezas de los gobernadores en México 2001-2012”, en N. Loza e I. Méndez (Coords.) Poderes y democracias: La política subnacional en México. México. FLACSO, pp. 71-92 Hernández Trillo, Fausto (2010) “Las finanzas públicas en el México posrevolucionario”, en Kuntz Ficker, Sandra (2010) Historia económica general de México: De la Colonia a nuestros días. México. El Colegio de México. Ibarra Salazar, Jorge, Entorno político y dependencia financiera de los estados mexicanos, Gestión y Política Pública, vol. XXII, núm. 1, 2013, pp. 3-44 Loaeza, Soledad (2010) “La metamorfosis del Estado: del jacobinismo centralizador a la fragmentación democrática”, en Loaeza, Soledad y Prud ́homme Jean Francois (Coords.) Instituciones y proceso políticos. Los grandes problemas de México Vol. XIV. México. El Colegio de México, pp. 23-70 López Lara, Álvaro F. (2016) “Gobernadores proactivos y legislaturas reactivas. Una Revista Mexicana Revista Mexicana de Análisis dePolítico AnálisisyPolítico Administración y Administración Pública, Universidad Pública, Universidad de Guanajuato, de Guanajuato, Volumen Volumen X, número X,2,número julio-diciembre 2, julio-diciembre 2021 2021 comparación entre el juicio de los expertos y el comportamiento legislativo”, en N. Loza e I. Méndez (Coords.) Poderes y democracias: La política subnacional en México. México. FLACSO, pp. 131-160. Merino, Mauricio (2010) “Nuevo federalismo, nuevos conflictos” en Loaeza, Soledad y Prud ́homme Jean Francois (Coords.) Instituciones y proceso políticos. Los grandes problemas de México Vol. XIV. México. El Colegio de México, pp. 487-530. Patrón Sánchez, Fernando (2014) Congresos estatales en México: una revisión a partir de la revisión legislativa y la aprobación presupuestal. México. Universidad de Guanajuato, Editorial Fontamara. Ramírez Hernández, Mario Joel (2014) Finanzas estatales e información financiera en la prensa de México: un análisis de cobertura, calidad y precisión informativa en el periodo 2011. Toluca, México. El Colegio Mexiquense. Ramírez Rodríguez, Roberto; Erquizio espinal, Alfredo, Análisis del ciclo político electoral a partir de variables de gasto público por entidad federativa en México, 1993-2009, Paradigma económico. Revista de economía regional y sectorial, vol. 4, núm. 2, julio- diciembre, 2012, pp. 5-27. Reyes Tepach, M. (2012) La deuda pública de las entidades federativas explicada desde la perspectiva del federalismo fiscal mexicano. México. Subdirección de Análisis Económico de la Cámara de Diputados, Dirección General de Servicios de Documentación, Información y Análisis. Sánchez Martínez, José Said (2017) Los congresos subnacionales y la política de gasto en México: El ejercicio de la función presupuestaria, Instituto Electoral del Estado de México, Toluca, México. Mario Joel Ramírez Hernández: Doctor en ciencias sociales con especialidad en ciencias políticas, maestro en ciencias sociales con especialidad en desarrollo municipal. Especialista en gobiernos estatales, legislaturas locales y deuda pública estatal. Profesor en la Universidad de Guanajuato y Universidad De La Salle Bajío. Correo electrónico: marioj.consultor@gmail. com. ORCID: 0000-0002-8157-8378 157 Revista Mexicana de Análisis Político y Administración Pública, Universidad de Guanajuato, Volumen X, número 2, julio-diciembre 2021
https://openalex.org/W2899735875	https://link.springer.com/content/pdf/10.1007%2F978-3-662-60756-5_13.pdf	English	null	Protection of Animals Through Human Rights: The Case-Law of the European Court of Human Rights	Beiträge zum ausländischen öffentlichen Recht und Völkerrecht	2,020	cc-by	13,117	Chapter 13 Protection of Animals Through Human Rights: The Case-Law of the European Court of Human Rights Tom Sparks Abstract The chapter discusses the potential of a human rights framework to contribute to the growth and development of global animal law. It takes as example the jurisprudence of the European Court of Human Rights, and examine the major trends in the Court’s judgments and admissibility decisions that directly or indirectly concern the rights or welfare of animals. It is concluded that the Court is not indifferent to the welfare of animals, but that animal welfare is instrumentalised: it is understood not as a good in itself, but is instead valued for its implications for human welfare and rights. The chapter then considers the obstacles that the anthro- pocentrism of the human rights idea and the instrumentalisation of animal concerns present to the use of human rights frameworks to further the development of global animal law, as well as the opportunities that exist in the meeting of these paradigms. It concludes that although the telos of human rights law is different from that of animal law, nevertheless there exist many overlapping concerns within which mutually beneﬁcial interactions are possible. T. Sparks () Max Planck Institute for Comparative Public Law and International Law, Heidelberg, Germany e-mail: sparks@mpil.de 15 T. Sparks () Max Planck Institute for Comparative Public Law and International Law, Heidelberg, Germany e-mail: sparks@mpil.de © The Author(s) 2020 A. Peters (ed.), Studies in Global Animal Law, Beiträge zum ausländischen öffentlichen Recht und Völkerrecht 290, https://doi.org/10.1007/978-3-662-60756-5_13 2A small number of cases fall into neither category, and are brieﬂy mentioned in section three. 1The relevant cases are, in chronological order, ECHR, Steel and Others v. UK, Chamber Judgment of 23 September 1998, Application No. 24838/94; ECHR, Chassagnou and Others v. France, Grand Chamber Judgment of 29 April 1999, Applications Nos. 25088/94, 28331/95, and 28443/95; ECHR, Bladet Tromsø and Stensaas v. Norway, Grand Chamber Judgment of 20 May 1999, Application No. 21980/93; ECHR, Hashman and Harrup v. UK, Grand Chamber Judgment of 25 November 1999, Application No. 25594/94; ECHR, Geert Drieman and Others v. Norway, Third Section Decision on Admissibility of 4 May 2000, Application No. 33678/96; ECHR, Cha’are Shalom Ve Tsedek v. France, Grand Chamber Judgment of 27 June 2000, Application No. 27417/95; ECHR, Verein gegen Tierfabriken v. Switzerland, Second Section Judgment of 28 June 2001, Application No. 24699/94 (VgT No. I); ECHR, Kyrtatos v. Greece, First Section Judgment of 22 May 2003, Application No. 41666/98; ECHR, Piippo v. Sweden, Second Section Partial Decision on Admissibility of 7 December 2004, Application No. 70518/01; ECHR, Steel and Morris v. UK, Fourth Section Judgment of 15 February 2005, Application No. 68416/01; ECHR, Piippo v. Sweden, Second Section Decision on Admissibility of 21 March 2006, Applica- tion No. 70518/01; ECHR, Schneider v. Luxembourg, Second Section Judgment of 10 July 2007, Application No. 2113/04; ECHR, Baudinière and Vauzelle v. France, Third Section Decision on Admissibility of 6 December 2007, Application Nos. 25708/03 and 25719/03; ECHR, Nilsson v Sweden, Third Section Decision on Admissibility of 26 February 2008, Application No. 11811/05; ECHR, Verein gegen Tierfabriken Schweiz v. Switzerland (No.2), Grand Chamber Judgment of 30 June 2009, Application No. 32772/02 (VgT No. II); ECHR, Friend and Others v. United Kingdom, Fourth Section Decision on Admissibility of 24 November 2009, Application Nos. 16072/06 and 27809/08; ECHR, Jakóbski v. Poland, Fourth Section Judgment of 7 December 2010, Application No. 18429/06; ECHR, Berü v. Turkey, Second Section Judgment of 11 January 2011, Application No. 47304/07; ECHR, Georgel and Georgeta Stoicescu v. Romania, Third Section Judgment of 26 July 2011, Application No. 9718/03; ECHR, ASPAS and Lasgrezas v. France, Fifth Section Judgment of 22 September 2011, Application No. 29953/08; ECHR, Herrmann v. Germany, Grand Chamber Judgment of 26 June 2012, Application No. 9300/07; ECHR, Chabauty v. France, Grand Chamber Judgment of 4 October 2012, Application No. 57412/ 08; ECHR, PETA Deutschland v. Germany, Fifth Section Judgment of 8 November 2012, Appli- cation No. 43481/09; ECHR, Animal Defenders International (ADI) v. UK, Grand Chamber Judgment of 22 April 2013, Application No. 48876/08; ECHR, Tierbefreier e.V. v. Germany, Fifth Section Judgment of 16 January 2014, Application No. 45192/09. 2 2 The Hunting Cases In 1998, the ﬁrst hunting case came before the Court. Steel and Others concerned a series of individuals arrested for the English common law offence of breach of the peace for acts of protest, and who had been subject to binding over orders.3 The protest in the case of the ﬁrst applicant was the disruption of a grouse shooting party and, following her refusal to accept a binding over order, she was jailed for 28 days.4 The Court examined the complaint under the article 5 prohibition on arbitrary deprivation of liberty and as an interference with the applicant’s right to free expression (article 10). In ﬁnding no violation, it noted that Ms Steel had been subjected to ‘serious interferences with the exercise of her right to freedom of expression’,5 but balanced this against the ‘obstruction’ of the ‘lawful pastime’ of the hunting party and the ‘risk of disorder’ arising therefrom,6 as well as the ‘importance in a democratic society of maintaining the rule of law and the authority of the judiciary’.7 It therefore held that her arrest and detention were not dispropor- tionate interferences with her convention rights.8 In 1998, the ﬁrst hunting case came before the Court. Steel and Others concerned a series of individuals arrested for the English common law offence of breach of the peace for acts of protest, and who had been subject to binding over orders.3 The protest in the case of the ﬁrst applicant was the disruption of a grouse shooting party and, following her refusal to accept a binding over order, she was jailed for 28 days.4 The Court examined the complaint under the article 5 prohibition on arbitrary deprivation of liberty and as an interference with the applicant’s right to free expression (article 10). 13 Protection of Animals Through Human Rights: The Case-Law of the European Court. . . 155 13 Protection of Animals Through Human Rights: The Case-Law of the European Court. . . 1 155 2 The Hunting Cases In ﬁnding no violation, it noted that Ms Steel had been subjected to ‘serious interferences with the exercise of her right to freedom of expression’,5 but balanced this against the ‘obstruction’ of the ‘lawful pastime’ of the hunting party and the ‘risk of disorder’ arising therefrom,6 as well as the ‘importance in a democratic society of maintaining the rule of law and the authority of the judiciary’.7 It therefore held that her arrest and detention were not dispropor- tionate interferences with her convention rights.8 However, in subsequent cases, the Court has asserted that a moral conviction against hunting is capable of attracting Convention protection,9 that animal welfare is a matter of public interest,10 and that no Convention protection of the right to hunt exists.11 Nevertheless, it remains a mixed practice. 3ECHR, Steel and Others v. UK (n. 1), paras. 6-24. 4Ibid., para. 13. 5Ibid., para. 103. 6Ibid. 7Ibid., para. 107. 8Ibid. 9ECHR, Chassagnou v. France (n. 1), para. 114; see also ECHR, Schneider v. Luxembourg (n. 1), para. 80; ECHR, Herrmann v. Germany (n. 1), para. 80. 10ECHR, Bladet Tromsø and Stensaas (n. 1), paras. 63-64, 73; see also ECHR, Steel and Morris v. UK (n. 1), para. 88; ECHR, PETA Deutschland v. Germany (n. 1), para. 47; ECHR, VgT No. II (n. 1), para. 92. 11ECHR, Chassagnou v. France (n. 1), para. 113; see also ECHR, Nilsson v. Sweden (n. 1), 11. 9ECHR, Chassagnou v. France (n. 1), para. 114; see also ECHR, Schneider v. Luxembourg (n. 1) para. 80; ECHR, Herrmann v. Germany (n. 1), para. 80. 11ECHR, Chassagnou v. France (n. 1), para. 113; see also ECHR, Nilsson v. Sweden (n. 1), 11. Ibid. 9ECHR, Chassagnou v. France (n. 1), para. 114; see also ECHR, Schneider v. Luxembourg (n. 1), para. 80; ECHR, Herrmann v. Germany (n. 1), para. 80. 10ECHR, Bladet Tromsø and Stensaas (n. 1), paras. 63-64, 73; see also ECHR, Steel and Morris v. UK (n. 1), para. 88; ECHR, PETA Deutschland v. Germany (n. 1), para. 47; ECHR, VgT No. II (n. 1), para. 92. 11ECHR, Chassagnou v. France (n. 1), para. 113; see also ECHR, Nilsson v. Sweden (n. 1), 11. 3ECHR, Steel and Others v. UK (n. 1), paras. 6-24. 4Ibid 13 10ECHR, Bladet Tromsø and Stensaas (n. 1), paras. 63-64, 73; see also ECHR, Steel and Morri v. UK (n. 1), para. 88; ECHR, PETA Deutschland v. Germany (n. 1), para. 47; ECHR, VgT No. I (n. 1), para. 92. 1 Introduction The institutionalisation of human rights under the regional human rights frameworks has given legal force to an idea already rich in transformative potential. Human rights have become a vital tool in efforts to achieve change both for individuals, and across legal systems. To seek, though, to harness the potential of human rights institutions and discourse to advance animal welfare and animal rights may appear farfetched: one could be forgiven for a certain scepticism concerning the availability of space for animal concerns in this deliberately human-centred area of law and policy. Nevertheless, there are indications of certain modest advances which offer 153 © The Author(s) 2020 A. Peters (ed.), Studies in Global Animal Law, Beiträge zum ausländischen öffentlichen Recht und Völkerrecht 290, https://doi.org/10.1007/978-3-662-60756-5_13 154 T. Sparks opportunities for animal welfare and rights concerns to be considered within the framework of human rights. This chapter will consider the case-law of the European Court of Human Rights (ECtHR) in the ﬁeld of animal law, and will identify the broad themes and trends within that jurisprudence. The ECtHR has only rarely considered questions either directly or tangentially relevant to the protection of animal rights, but there are nonetheless almost thirty relevant judgments and admissibility decisions,1 which will be divided thematically (Sects. 2 and 3).2 Section 4 will then consider the current legal and conceptual barriers to more effective animal protection under the Conven- tion, and will make some tentative remarks on the potential of the ECHR and its Court (as well as human rights frameworks more broadly) to contribute to the development of global standards on animal welfare. 7Ibid., para. 107. 2.1 Hunting Under Article 1 of Protocol 1 The 1999 case of Chassagnou v. France concerned ten applicants, each of whom owned land in areas regulated by the Loi Verdeille. Under that law, all landowners whose holdings are below a certain threshold are required to pool their lands for the purposes of creating an area within which members of the relevant municipal T. Sparks 156 hunting association (ACCA) may freely hunt. The landowners whose property forms a part of the hunting area are automatically members of the local ACCA.12 The applicants in the case were all ethically opposed to hunting, and made unsuccessful applications to have their properties removed from the hunting areas, and themselves released from membership of the ACCAs.13 The Court found violations of article 11 (freedom of association) and article 1 of Protocol 1 (protection of property) taken separately, and also found violations of each of these provisions when read in conjunction with the protection from discrimination in the application of the con- vention (article 14). The Court accepted that the imposition of ACCA membership and the require- ment to permit hunting on the applicants’ land pursued a legitimate aim (it commented that ‘it is undoubtedly in the general interest to avoid unregulated hunting and encourage the rational management of game stocks’).14 Nevertheless, it recognised that the applicants’ ethical objections were relevant to the assessment of the proportionality of the interference. In relation to article 1 of Protocol 1 it noted that the Government’s characterisation of membership of the ACCA as ‘compensa- tion’ for the loss of the exclusive right to hunt (or, as the case may be, to choose not to hunt) over one’s land ‘is valuable only in so far as all the landowners concerned are hunters or accept hunting.’15 It consequently found that ‘[c]ompelling small landowners to transfer hunting rights over their land so that others can make use of them in a way which is totally incompatible with their beliefs imposes a dispropor- tionate burden’.16 Perhaps even more telling was the Court’s 2012 judgment in Chabauty v. 12ECHR, Chassagnou v. France (n. 1), paras. 13-15, 46. 13Ibid., paras. 16-18, 23-24, 28-30. 14Ibid., para. 79. 15Ibid., para. 82. 16Ibid., para. 85. The Court has subsequently conﬁrmed Chassagnou in ECHR, Schneider v. Luxembourg (n. 1); and ECHR, Herrmann v. Germany (n. 1). 17ECHR, Piippo v. Sweden (Second Decision, 2006) (n. 1); ECHR, Nilsson v. Sweden (n. 1); ECHR, Baudinière and Vauzelle v. France (n. 1); and ECHR, Chabauty v. France (n. 1). 18Ibid., paras. 12-17. 19ECHR, Chassagnou v. France (n. 1); ECHR, Schneider v. Luxembourg (n. 1); ECHR, Herrmann v. Germany (n. 1). 19ECHR, Chassagnou v. France (n. 1); ECHR, Schneider v. Luxembourg (n. 1); ECHR, Herrmann v. Germany (n. 1). 12ECHR, Chassagnou v. France (n. 1), paras. 13-15, 46. 14Ibid., para. 79. 12ECHR, Chassagnou v. France (n. 1), paras. 13-15, 46. 13Ibid., paras. 16-18, 23-24, 28-30. 13Ibid., paras. 16-18, 23-24, 28-30. p 21Çoban notes that the Court’s general approach to article 1 of Protocol 1 has been to ‘favour[] the public interest rather than individual rights.’ Çoban, Protection of Property Rights 2004, 257. 22ECHR, Chassagnou v. France (n. 1), para. 103, 117; ECHR, Schneider v. Luxembourg (n. 1), paras. 82-83. 23ECHR, Steel and Others v. UK (n. 1); ECHR, Drieman and Others v. Norway (n. 1). 24ECHR, Steel and Others v. UK (n. 1), paras. 6-13. See also above, sec. 2.1. 25The Court decided that it was not necessary to consider the application of article 11 because the complaint did not ‘raise[] any issues not already examined in the context of article 10’: ECHR, Steel and Others v. UK (n. 1), para. 113. 26Ibid., paras. 102-107. 27Ibid para 105 2.1 Hunting Under Article 1 of Protocol 1 France, the most recent in a line of cases brought under article 1 of Protocol 1 by hunters.17 The applicant challenged the inclusion of his land in the hunting area, but in this case because he wished privately to rent the right to hunt on his land.18 The Court found no violation of article 1 of Protocol 1, read in conjunction with article 14, and in so doing expressly distinguished the case from Chassagnou and its line19 as a result of the applicant’s (lack of) ethical objections to hunting. Absent the conﬂict of con- science, the decision on how hunting should be regulated fell within the state’s 157 Protection of Animals Through Human Rights: The Case-Law of the European Court. . . 15 13 margin of appreciation, and no disproportionate interference with the right to property was found.20 As the line of cases culminating in Chabauty shows, this is no reiﬁcation of the right to property. On the contrary, the Court has been inclined to give the state a wide margin of appreciation to regulate hunting.21 Nevertheless, ethical objections to hunting are sufﬁcient signiﬁcantly to narrow the margin of appreciation, and the Court has consistently held that national regulation of hunting must make provision for the rights of opponents of hunting to use their land in ways that accord with their beliefs. Though this is only a small step towards Convention support for animal concerns, it is nevertheless noteworthy both as a protection for animal rights activists, and because it recognises opposition to hunting (and perhaps by implica- tion concern for animals more broadly) as a politico-moral opinion capable of attracting ECHR protection. 20Ibid., paras. 41-50, 56-57. 2.2 Hunting Under Articles 10 and 11 Articles 10 and 11 have been invoked alongside the right to property as further grounds to ﬁnd the obligation to accept hunting interferes with the Convention rights of conscientious objectors,22 but have also twice been invoked in the separate context of anti-hunting protest.23 Here the Court has seemed more reluctant to grant protection: in neither case were the restrictions of the applicants’ acts of protest in defence of animal rights considered to be violations of the Convention. Articles 10 and 11 were ﬁrst invoked by an anti-hunting protester in Steel and Others v. UK.24 The ﬁrst applicant claimed violations of articles 10 and 11 of the Convention, but only article 10 was considered,25 and no violation was found.26 The Court emphasised that the applicant’s protest—which involved placing herself in front of the hunters to prevent them from ﬁring—‘created a danger of serious physical injury to herself and others’ and ‘risked culminating in disorder and violence.’27 It appears, therefore, to be the applicant’s direct action which justiﬁed her arrest and imprisonment, a conclusion reinforced by Geert Drieman and Others 27Ibid., para. 105. T. Sparks 158 v. Norway. Here the applicants were arrested and held on remand for actions taken to disrupt a whale hunt in Norway’s exclusive economic zone.28 The applicants claimed that their arrest and detention violated articles 10 and 11 of the Convention, and the Court accepted that these actions amounted to an interference. Nevertheless, it decided that the application was manifestly ill founded under article 35(3) of the Convention, and therefore inadmissible.29 The decision that the complaint in Drieman was manifestly ill founded and not worthy of further consideration is somewhat surprising, and seems to indicate a hostility to direct action as a form of protest. Though it was accepted by the Court that there had been an interference with articles 10 and 11, it considered it sufﬁ- ciently obvious that the state’s actions were proportionate that a more detailed assessment was manifestly unnecessary. Its reasoning supports two possible (non-exclusive) interpretations: that the applicants’ aims did not require protection in a democratic society; or that their methods were sufﬁciently outrageous that states cannot be required to tolerate such conduct in defence of the right to protest. The ﬁrst, it seems, played a role. The Court noted that the interference pursued the legitimate aim of ‘enforc[ing . . 33The Court has implied in a series of cases that there is a hierarchy within articles 10 and 11, wherein certain subjects (those that are “political” or in the “public interest”) will receive a higher level of protection than others. See ECHR, Sunday Times v. UK, Grand Chamber Judgment of 26 April 1979, Application No. 6538/74, 29-30; ECHR, Lingens v. Austria, Grand Chamber Judgment of 8 July 1986, Application No. 9815/82, paras. 34-47; ECHR, Thorgeirson v. Iceland, para. 60-70; Chamber Judgment of 25 June 1992, Application No. 13778/88, paras. 55-70; ECHR, Jersild v. Denmark, Grand Chamber Judgment of 23 September 1994, Application No. 15890/89, paras. 25-37; and contrast ECHR, Handyside v. UK, Grand Chamber Judgment of 7 December 1976, Application No. 5493/72, paras. 42-59; ECHR, Wingrove v. UK, Chamber Judgment of 25 November 1996, Application No. 17419/90, paras. 52-64, esp. 58; ECHR, Vereinigung Bildender Künstler v. Austria, First Section Judgment of 25 January 2007, Application No. 68354/01, paras. 26-39. 28ECHR, Drieman and Others v. Norway (n. 1), 2. 29Ibid., 10. 30Ibid., 9. 31Ibid., 10. 32Ibid. 33The Court has implied in a series of cases that there is a hierarchy within articles 10 and 11, wherein certain subjects (those that are “political” or in the “public interest”) will receive a higher level of protection than others. See ECHR, Sunday Times v. UK, Grand Chamber Judgment of 26 April 1979, Application No. 6538/74, 29-30; ECHR, Lingens v. Austria, Grand Chamber Judgment of 8 July 1986, Application No. 9815/82, paras. 34-47; ECHR, Thorgeirson v. Iceland, para. 60-70; Chamber Judgment of 25 June 1992, Application No. 13778/88, paras. 55-70; ECHR, Jersild v. Denmark, Grand Chamber Judgment of 23 September 1994, Application No. 15890/89, paras. 25-37; and contrast ECHR, Handyside v. UK, Grand Chamber Judgment of 7 December 1976, Application No. 5493/72, paras. 42-59; ECHR, Wingrove v. UK, Chamber Judgment of 25 November 1996, Application No. 17419/90, paras. 52-64, esp. 58; ECHR, Vereinigung Bildender Künstler v. Austria, First Section Judgment of 25 January 2007, Application No. 68354/01, paras. 26-39. 28ECHR, Drieman and Others v. Norway (n. 1), 2. 35Fenwick et al consider that the ﬁndings in Steel and Others and the cases that followed it demonstrate that direct action protests engage article 11 in principle: Helen Fenwick/Gavin Phillipson/Alexander Williams, Texts, Cases and Materials on Public Law and Human Rights (4th ed., Abingdon: Routledge 2017), 999. However, the Court has tended to apply a very wide margin of appreciation in such cases, characterising direct action as ‘reprehensible’, and implying that it cannot be considered wholly ‘peaceful’ even when no violent action is taken: Kudrevičius and Others v. Lithuania, Grand Chamber Judgment of 15 October 2015, Application No. 37553/05, paras. 173-174; see also Steel and Others (n. 1); G. v. Germany, Decision by the Commission on Admissibility of 6 March 1989, Application No. 13079/87; Lucas v. UK, Fourth Section Decision on Admissibility of 18 March 2003, Application No. 39013/02; Baracco v. France, Fifth Section Judgment of 5 March 2009, Application No. 31684/05. Nevertheless the Court has held that the margin it grants in such cases ‘although wide, is not unlimited’ (para. 86), and it has been willing to ﬁnd that a certain level of criminal sanction (in Taranenko three years’ imprisonment) is disproportionate to the aim of preventing illegal protest: ECHR, Taranenko v. Russia, First Section Judgment of 15 May 2014, Application No. 19554/05, paras.81-97. 36Ibid., 10. 34In this connection, it is particularly relevant that the Court observed that the protest had been taking place unimpeded for one month, and only when the protestors’ activities interfered with the hunt did the authorities take action. See ECHR, Drieman and Others v. Norway (n. 1), 10. 38ECHR, Kudrevičius and Others v. Lithuania (n. 35), paras. 142-184, esp. 169-175. 2.2 Hunting Under Articles 10 and 11 .] the rules protecting whaling’,30 and counterbalanced that remark with a ﬁnding that the protest ‘forc[ed] the whalers to abandon their lawful activity’.31 It noted, too, that the relevant conduct ‘could not enjoy the same privileged protection under the Convention as political speech or debate on questions of public interests or the peaceful demonstration of opinions on such matters’.32 Although this latter comment is more closely tied to the question of methods, taken together these statements indicate the Court’s opinion that the subject of the protest did not attract a high standard of protection.33 On the contrary, 31Ibid., 10. 32Ibid. 13 159 Protection of Animals Through Human Rights: The Case-Law of the European Court. . . the Court privileged the economic activity of the whalers over the protest of the animal rights activists.34 In its assessment of the methods, too, the Court seemed ill-disposed to the direct action of the protestors.35 The comment above contrasting the protestors’ actions with ‘peaceful demonstration of opinions’ on questions of public interest should be read alongside the characterisation of those actions as ‘a form of coercion’, and ‘an ultimatum’.36 Yet unlike in Steel and Others there was no suggestion that the applicants’ protests had created a danger to the whalers or to any other person37; unlike in Kudrevičius and Others the disruption caused to ‘activities lawfully carried out by others’ did not affect a large number of people, but instead only a small group38; and unlike in Taranenko v. Russia the protests did not result in violence.39 Nevertheless, the Court in Drieman directly contrasted its approach in cases involv- ing ‘the peaceful demonstration of opinions on [matters of public interest]’ (in which a narrow margin of appreciation is appropriate) with the facts before it, where it found that ‘[c]ontracting States must be allowed a wide margin of appreciation in their assessment of the necessity of taking measures to restrict such conduct.’40 34In this connection, it is particularly relevant that the Court observed that the protest had been taking place unimpeded for one month, and only when the protestors’ activities interfered with the hunt did the authorities take action. See ECHR, Drieman and Others v. Norway (n. 1), 10. 37ECHR, Steel v. Others (n. 1), para. 103. 39ECHR, Taranenko v. Russia (n. 35). 40Ibid 2.2 Hunting Under Articles 10 and 11 35Fenwick et al consider that the ﬁndings in Steel and Others and the cases that followed it demonstrate that direct action protests engage article 11 in principle: Helen Fenwick/Gavin Phillipson/Alexander Williams, Texts, Cases and Materials on Public Law and Human Rights (4th ed., Abingdon: Routledge 2017), 999. However, the Court has tended to apply a very wide margin of appreciation in such cases, characterising direct action as ‘reprehensible’, and implying that it cannot be considered wholly ‘peaceful’ even when no violent action is taken: Kudrevičius and Others v. Lithuania, Grand Chamber Judgment of 15 October 2015, Application No. 37553/05, paras. 173-174; see also Steel and Others (n. 1); G. v. Germany, Decision by the Commission on Admissibility of 6 March 1989, Application No. 13079/87; Lucas v. UK, Fourth Section Decision on Admissibility of 18 March 2003, Application No. 39013/02; Baracco v. France, Fifth Section Judgment of 5 March 2009, Application No. 31684/05. Nevertheless the Court has held that the margin it grants in such cases ‘although wide, is not unlimited’ (para. 86), and it has been willing to ﬁnd that a certain level of criminal sanction (in Taranenko three years’ imprisonment) is disproportionate to the aim of preventing illegal protest: ECHR, Taranenko v. Russia, First Section Judgment of 15 May 2014, Application No. 19554/05, paras.81-97. 36Ibid., 10. T. Sparks T. Sparks 160 50See, for example, the statement by the Court in Thorgeirson that the press has a ‘pre-eminent role [. . .] in a State governed by the rule of law’: ECHR, Thorgeirson v. Iceland (n. 33), para. 63. See further, among others, Sunday Times v. UK (n. 33), 33; ECHR, Lingens v. Austria (n. 33), para. 13; 41ECHR, Steel and Others (n. 1), paras. 112-113; ECHR, Drieman (n. 1), 7-10. 42ECHR, Bladet Tromsø and Stensaas (n. 1). 43ECHR, VgT Nos. I&II (n. 1); ECHR, Steel and Morris v. UK (n. 1); ECHR, PETA Deutschland v. Germany (n. 1); ECHR, ADI v. UK (n. 1); and ECHR, Tierbefreier e.V. v. Germany (n. 1). 44ECHR, Bladet Tromsø and Stensaas (n. 1), paras. 6-38. 45Ibid 59 41ECHR, Steel and Others (n. 1), paras. 112-113; ECHR, Drieman (n. 1), 7-10. 42ECHR, Bladet Tromsø and Stensaas (n. 1). 43ECHR, VgT Nos. I&II (n. 1); ECHR, Steel and Morris v. UK (n. 1); ECHR, PETA Deutschland v. Germany (n. 1); ECHR, ADI v. UK (n. 1); and ECHR, Tierbefreier e.V. v. Germany (n. 1). 44ECHR, Bladet Tromsø and Stensaas (n. 1), paras. 6-38. 45Ibid., para. 59. 46Ibid., para. 62. 47Ibid., para. 64. 48Ibid., para. 63. 49Ibid., para. 73. 50See, for example, the statement by the Court in Thorgeirson that the press has a ‘pre-eminent role [. . .] in a State governed by the rule of law’: ECHR, Thorgeirson v. Iceland (n. 33), para. 63. See further, among others, Sunday Times v. UK (n. 33), 33; ECHR, Lingens v. Austria (n. 33), para. 13; 3 Animal Welfare and Freedom of Speech Claims under article 10 in the context of hunting have come in parallel to article 11, in cases concerning protest. There, the articles were considered to raise the same issues.41 Freedom of speech has also been invoked separately from the freedom of association, however; both in relation to reporting on hunting,42 and publications by animal rights groups.43 The ﬁrst animal welfare case to raise article 10 outwith the context of protest was Bladet Tromsø and Stensaas v. Norway. The case was brought by the Bladet Tromsø newspaper and its editor, following a successful defamation suit against them for articles which reported allegations by a seal hunt inspector of cruel and illegal practices.44 Defamation proceedings were brought by the hunters concerned sequen- tially against the inspector, Bladet Tromsø and its editor, and (unsuccessfully) against several other media outlets. The Court began its assessment with its familiar assertion of the high importance of the press, and declared that ‘[i]n cases such as the present one the national margin of appreciation is circumscribed by the interest of democratic society in enabling the press to exercise its vital role of ‘public watch- dog’ in imparting information of serious public concern’.45 It held too that ‘in order to determine whether the interference was based on sufﬁcient reasons which ren- dered it ‘necessary’, regard must be had to the public-interest aspect of the case.’46 This it found to be high, referring to the ‘legitimate public concern’ with the subject matter,47 and noting that it was in actuality ‘of evident concern to the local, national and international public’.48 The Court’s strong declaration that animal welfare and the exposure of cruelty to animals is a legitimate matter of public interest is, in the author’s opinion, more signiﬁcant than the ultimate ﬁnding that article 10 had been violated in the circum- stances of the case.49 The Court has been loath to hold that restrictions on journalistic speech on matters of public interest can be justiﬁed except under circumstances of clear abuse, and has repeatedly held that the state’s margin of appreciation will be very narrow where the freedom of the press is concerned.50 That article 10 was 46Ibid., para. 62. 47Ibid., para. 64. 48Ibid., para. 63. 49Ibid., para. 73. Protection of Animals Through Human Rights: The Case-Law of the European Court. . . 3 Animal Welfare and Freedom of Speech 161 13 violated was an unsurprising conclusion, therefore, once the ﬁnding had been made that a debate on animal cruelty was a matter of public interest: it was the combination of public interest and journalistic speech which was important for the outcome. The same can be said of the subsequent article 10 cases concerning aspects of animal welfare that have come before the Court: for present purposes the ultimate decisions are of secondary importance, being determined primarily by factors not directly relevant to animal welfare. That is signiﬁcant in itself, however, insofar as it demonstrates that the role given to animal welfare concerns in article 10 cases is a narrow one, limited to determination of the appropriate standard of review. In the years following Bladet Tromsø and Stensaas the Court has ruled on the application of article 10 in relation to animal welfare in six cases, each of which concerned the legality of restrictions on communications by animal welfare groups either (as in VgT Nos. I&II and ADI v. UK) relating to the media available to them,51 or (as in Steel and Morris, PETA Deutschland and Tierbefreier e.V.) the content of those communications.52 In each case the Court reiterated its ﬁnding that animal welfare and animal rights are ‘topics of general concern’,53 and ‘questions of public interest’.54 Accordingly, it has repeatedly noted that the margin of appreciation due to states in determining to what extent such communications can be restricted is narrow,55 and has even gone so far as to suggest that the standard of protection appropriate to campaigning groups working on such matters is similar (although perhaps not identical) to that applicable to journalists.56 ECHR, Oberschlick v. Austria, Grand Chamber Judgment of 23 May 1991, Application No. 1162/ 85, para. 58; ECHR, Observer and Guardian v. UK, Grand Chamber Judgment of 26 November 1991, Application No. 13585/88, para. 59; ECHR, Jersild v. Denmark (n. 33), para. 31; ECHR, Goodwin v. UK, Grand Chamber Judgment of 27 March 1996, Application No. 17488/90, para. 39. These principles have been reafﬁrmed in the recent case of ECHR, Satakunnan Markkinapörssi Oy and Satamedia Oy v. Finland, Grand Chamber Judgment of 27 June 2017, Application No. 931/13, paras. 124-128. 51ECHR, VgT No. I (n. 1), paras. 8-23; VgT No. II (n. 1), paras. 12-27; ECHR, ADI v. UK (n. 1), paras. 8-33. 52ECHR, Steel and Morris v. UK (n. 1), paras. ECHR, Oberschlick v. Austria, Grand Chamber Judgment of 23 May 1991, Application No. 1162/ 85, para. 58; ECHR, Observer and Guardian v. UK, Grand Chamber Judgment of 26 November 1991, Application No. 13585/88, para. 59; ECHR, Jersild v. Denmark (n. 33), para. 31; ECHR, Goodwin v. UK, Grand Chamber Judgment of 27 March 1996, Application No. 17488/90, para. 39. These principles have been reafﬁrmed in the recent case of ECHR, Satakunnan Markkinapörssi Oy and Satamedia Oy v. Finland, Grand Chamber Judgment of 27 June 2017, Application No. 931/13, paras. 124-128. 55In VgT No. I the Court noted that ‘in the present case the extent of the margin of appreciation is reduced, since what is at state is not a given individual’s purely “commercial” interests, but [their] participation in a debate affecting the general interest’: ECHR, VgT No. I (n. 1), para. 71. See also ECHR, ADI v. UK (n. 1), para. 104. 56ECHR, Steel and Morris v. UK (n. 1), para. 89, [references omitted]. 54ECHR, PETA Deutschland v. Germany (n. 1), para. 47. See also ECHR, VgT No.II (n. 1) para. 92. ECHR, Oberschlick v. Austria, Grand Chamber Judgment of 23 May 1991, Application No. 1162/ 85, para. 58; ECHR, Observer and Guardian v. UK, Grand Chamber Judgment of 26 November 1991, Application No. 13585/88, para. 59; ECHR, Jersild v. Denmark (n. 33), para. 31; ECHR, Goodwin v. UK, Grand Chamber Judgment of 27 March 1996, Application No. 17488/90, para. 39. These principles have been reafﬁrmed in the recent case of ECHR, Satakunnan Markkinapörssi Oy and Satamedia Oy v. Finland, Grand Chamber Judgment of 27 June 2017, Application No. 931/13, paras. 124-128. 51ECHR, VgT No. I (n. 1), paras. 8-23; VgT No. II (n. 1), paras. 12-27; ECHR, ADI v. UK (n. 1), paras. 8-33. 52ECHR, Steel and Morris v. UK (n. 1), paras. 8-36; ECHR, PETA Deutschland v. Germany (n. 1), paras. 6-19; ECHR, Tierbefreier e.V. v. Germany (n. 1), paras. 5-21. 53ECHR, Steel and Morris v. UK (n. 1), para. 88. 54ECHR, PETA Deutschland v. Germany (n. 1), para. 47. See also ECHR, VgT No.II (n. 1), para. 92. 55In VgT No. I the Court noted that ‘in the present case the extent of the margin of appreciation is reduced, since what is at state is not a given individual’s purely “commercial” interests, but [their] participation in a debate affecting the general interest’: ECHR, VgT No. I (n. 1), para. 71. See also ECHR, ADI v. UK (n. 1), para. 104. 56ECHR, Steel and Morris v. UK (n. 1), para. 89, [references omitted]. 57ECHR, PETA Deutschland v. Germany (n. 1), para. 7. 58Ibid., para. 11. 59Ibid., paras. 17-18; see also Landgericht Berlin, Judgment of 18 March 2004, 27 O 207/04; Landgericht Berlin 22 April 2004, 27 O 207/04; Kammergericht, Judgment of 30 July 2004, 9 U 118/04; Kammergericht, Judgment of 27 August 2004, 9 U 118/04; Landesgericht Berlin, Judgment of 2 December 2004, 27 O 676/04; Kammergericht, Judgment of 25 November 2005, 9 U 15/05; Bundesverfassungsgericht, Judgment of 20 February 2009, 1 BvR 2266/04, 1 BvR 2620/05. 60ECHR, PETA Deutschland v. Germany (n. 1), para. 49. 61Ibid., para. 48. 62Ibid. 3 Animal Welfare and Freedom of Speech 8-36; ECHR, PETA Deutschland v. Germany (n. 1) paras. 6-19; ECHR, Tierbefreier e.V. v. Germany (n. 1), paras. 5-21. 53ECHR, Steel and Morris v. UK (n. 1), para. 88. 54ECHR, PETA Deutschland v. Germany (n. 1), para. 47. See also ECHR, VgT No.II (n. 1) para. 92. 55In VgT No. I the Court noted that ‘in the present case the extent of the margin of appreciation is reduced, since what is at state is not a given individual’s purely “commercial” interests, but [their] participation in a debate affecting the general interest’: ECHR, VgT No. I (n. 1), para. 71. See also ECHR, ADI v. UK (n. 1), para. 104. 56ECHR, Steel and Morris v. UK (n. 1), para. 89, [references omitted]. T. Sparks T. Sparks 162 4 Obstacles and Opportunities Nevertheless, it is abundantly clear from the Courts’ reasoning in these and other cases that what is protected is the interest that humans may feel in the welfare and suffering of animals, and not the welfare of animals as an end in itself. The distinction is illustrated particularly clearly in PETA Deutschland v. Germany. That case concerned an advertising campaign which juxtaposed images of mass farming methods with Nazi-era concentration camps, together with text which claimed similarities between the treatment of holocaust victims and treatment of animals in the modern meat industry. One such caption ran ‘where animals are concerned everyone becomes a Nazi’ (‘[w]o es um Tiere geht, wird jeder zum Nazi’).57 An injunction was requested by Jewish community leaders, and granted by the domestic Courts on the basis that ‘the debasement of concentration camp victims was [. . .] exploited in order to militate for better accommodation of laying hens and other animals.’58 Though the courts at all levels noted that the campaign was not malicious in the sense that PETA did not intend to minimise the suffering of holocaust victims nor to violate their human dignity, they nevertheless concluded that the comparison was ‘banalising’, and that ‘the Basic Law drew a clear distinc- tion between human life and dignity on the one side and the interests of animal protection on the other’. The injunction was therefore justiﬁed on the basis that the ‘content of the campaign affected the plaintiffs’ personality rights.’59 The ECtHR unanimously agreed. Though its judgment noted the particular context of Jews living in Germany and pointed out ‘that courts in other jurisdictions might address similar matters in a different way’,60 it accepted that the decision of the domestic courts was reasonable. In coming to that ﬁnding it agreed that the posters did not ‘aim to debase the depicted concentration camp inmates, as the pictures merely implied that the suffering inﬂicted on the depicted humans and animals was equal.’61 Nevertheless, and revealingly, it characterised the treatment of the concentration camp victims as ‘instrumentalisation’ in the ‘interests of animal protection’.62 This theme was taken up—and taken further—by Judge Zupančič in a concurring opinion joined by Judge Spielmann. There they asked 13 163 Protection of Animals Through Human Rights: The Case-Law of the European Court. 63Concurring Opinion of Judge Zupančič, Joined by Judge Spielmann, ECHR, PETA Deutschland v. Germany (n. 1), 16-18, at para. 5. 64Ibid., paras. 14-15. A similar argument in the academic sphere is made by Leslie Pickering Francis and Richard Norman, who argue that the term “animal liberation” ‘has the effect of trivializing [. . .] real liberation movements, putting them on a level with what cannot but appear as a bizarre exaggeration’: Francis/Norman, ‘Some Animals are More Equal than Others’ 1978, 527. Kymlicka and Donaldson respond powerfully to such arguments: Kymlicka/Donaldson, ‘Animal Rights, Multiculturalism, and the Left’ 2014, 116-135. A historical analysis of the human/animal dichotomy is given by Anna Becker in her contribution to this volume. 65See ECHR, Chassagnou v. France (n. 1), and the cases that followed it. 66ECHR, Tierbefreier e.V. v. Germany (n. 1). 67ECHR Cha’are Shalom (n. 1); ECHR, Jakóbski v. Poland (n. 1). 68ECHR Cha’are Shalom (n. 1). Importantly, though, the Court held here that the state retained a margin of appreciation to decide on what basis permits to slaughter animals in accordance with religious requirements (in this case the strict requirements to qualify as glatt kosher) would be granted in order to, among other things, enable it to protect public health and animal welfare (paras. 76-77, 84). Provided that meat prepared according to the requirements of one’s religion is available, article 9 does not extend to a right to slaughter one’s meat oneself (paras. 80-82). 69ECHR, Stoicescu v. Romania (n. 1). 63Concurring Opinion of Judge Zupančič, Joined by Judge Spielmann, ECHR, PETA Deutschland v. Germany (n. 1), 16-18, at para. 5. 64Ibid., paras. 14-15. A similar argument in the academic sphere is made by Leslie Pickering Francis and Richard Norman, who argue that the term “animal liberation” ‘has the effect of trivializing [. . .] real liberation movements, putting them on a level with what cannot but appear as a bizarre exaggeration’: Francis/Norman, ‘Some Animals are More Equal than Others’ 1978, 527. Kymlicka and Donaldson respond powerfully to such arguments: Kymlicka/Donaldson, ‘Animal Rights, Multiculturalism, and the Left’ 2014, 116-135. A historical analysis of the human/animal dichotomy is given by Anna Becker in her contribution to this volume. 65See ECHR, Chassagnou v. France (n. 1), and the cases that followed it. 67ECHR Cha’are Shalom (n. 1); ECHR, Jakóbski v. Poland (n. 1). 68ECHR Cha’are Shalom (n. 1). Importantly, though, the Court held here that the state retained a margin of appreciation to decide on what basis permits to slaughter animals in accordance with religious requirements (in this case the strict requirements to qualify as glatt kosher) would be granted in order to, among other things, enable it to protect public health and animal welfare (paras. 76-77, 84). Provided that meat prepared according to the requirements of one’s religion is available, article 9 does not extend to a right to slaughter one’s meat oneself (paras. 80-82). 69ECHR S i R i ( 1) 4 Obstacles and Opportunities [W]hether reasonable [people] could indeed or could not differ on the utterly disgraceful and unacceptable comparison between pigs on the one hand and the inmates of Auschwitz or some other concentration camp, on the other hand.63 [W]hen human beings in their utter suffering and indignity are, as here, compared to hens and pigs for the lesser purpose of protecting otherwise legitimate advancement of animal rights, we are no longer in the position to maintain that the human beings seen in these pictures are treated as an end in themselves. [. . .] If their image is so instrumentalised, little is left of their human dignity[.]64 Though these statements were seemingly too strongly put for the majority of the Court, the underlying reasoning appears to be the same. Certainly, Judge Zupančič is correct in pointing out that using the picture of the concentration camp victims simply as a comparator does not accord with the Kantian imperative that individuals be treated as ends in themselves, but it is worth pausing to consider whether a comparison of holocaust victims with—for example—victims of modern-day international crimes would have attracted the same condemnation. That would be no less of an instrumentalisation, but it seems clear that for Judges Zupančič and Spielmann (even if not for the domestic courts) the context of the comparison was a signiﬁcant part of the harm the campaign committed. It may be that it was not instrumentalisation per se that was objectionable, but rather instrumentalisation of the human in service of the animal. Nor is that conclusion—or its counterpart that the animal may be instrumentalised in service of the human—a surprising position for the Court to reach. It is almost unnecessary to say, for example, that the Court has no objection in principle to the use of animals as game,65 for medical experimentation,66 or for food.67 In some circumstances animal welfare concerns take second place to facilitation of religious practice.68 Given a direct conﬂict between animal life and human safety and wellbeing the Court unsurprisingly privileges human safety.69 The Court should 69ECHR, Stoicescu v. Romania (n. 1). T. 70ECHR, Khamtokhu and Aksenchik v. Russia, Grand Chamber Judgment of 24 January 2017, Application Nos. 60367/08 and 961/11, para. 73 [references omitted]. 71ECHR, Tyrer v. UK, Chamber Judgment of 25 April 1978, Application No. 5856/72, para. 31. 72ECHR, Khamtokhu and Aksenchik (n. 70), para. 73; see also ECHR, Selmoui v. France, Grand Chamber Judgment of 28 July 1999, Application No. 25803/94, para. 101. 73ECHR, Khamtokhu and Aksenchik (n. 70), para. 74 74For different perspectives on these questions see Cochrane, Animal Rights without Liberation: Applied Ethics and Human Obligations 2012; Taylor, ‘Whiter Rights? Animal Rights and the Rise of New Welfarism’ 1999, 27-41; Harrop, ‘Climate Change, Conservation and the Place for Wild Animal Welfare in International Law’ 2011, 441-462. Outside academia, compare the remit of the Animal Welfare Council (http://www.animalwelfarecouncil.org/?page_id¼9), with PETA (https:// www.peta.org/about-peta/faq/what-is-the-difference-between-animal-rights-and-animal-welfare/). 75Partly Concurring and Partly Dissenting Opinion of Judge Pinto de Albuquerque, ECHR, Herrmann v. Germany (n. 1), 32-49. Similar themes were also discussed in the earlier Partly Dissenting Opinion of Judge Zagrebelsky, ECHR, Kyrtatos v. Greece (n. 1), 14-15. 76Ibid., 32. 77Ibid. 78Ibid., 33 [references omitted]. 78Ibid., 33 [references omitted]. 75Partly Concurring and Partly Dissenting Opinion of Judge Pinto de Albuquerque, ECHR, Herrmann v. Germany (n. 1), 32-49. Similar themes were also discussed in the earlier Partly Dissenting Opinion of Judge Zagrebelsky, ECHR, Kyrtatos v. Greece (n. 1), 14-15. 76 4For different perspectives on these questions see Cochrane, Animal Rights without Liberation: pplied Ethics and Human Obligations 2012; Taylor, ‘Whiter Rights? Animal Rights and the Rise f New Welfarism’ 1999, 27-41; Harrop, ‘Climate Change, Conservation and the Place for Wild Animal Welfare in International Law’ 2011, 441-462. Outside academia, compare the remit of the Animal Welfare Council (http://www.animalwelfarecouncil.org/?page_id¼9), with PETA (https:// www.peta.org/about-peta/faq/what-is-the-difference-between-animal-rights-and-animal-welfare/). 70ECHR, Khamtokhu and Aksenchik v. Russia, Grand Chamber Judgment of 24 January 2017, Application Nos. 60367/08 and 961/11, para. 73 [references omitted]. 71ECHR, Tyrer v. UK, Chamber Judgment of 25 April 1978, Application No. 5856/72, para. 31. 72ECHR, Khamtokhu and Aksenchik (n. 70), para. 73; see also ECHR, Selmoui v. France, Grand Chamber Judgment of 28 July 1999, Application No. 25803/94, para. 101. 73ECHR Kh t kh d Ak hik ( 70) 74 p Animal Welfare Council (http://www.animalwelfarecouncil.org/?page_id¼9), with PETA (https:// www.peta.org/about-peta/faq/what-is-the-difference-between-animal-rights-and-animal-welfare/). 4 Obstacles and Opportunities Sparks 164 not be criticised for these positions: to adopt the contrary ﬁnding on any of these points would take the Court radically beyond the understanding of the rights involved prevalent in the Council of Europe states, and thus well beyond its remit to ‘interpret[the Convention] in the light of present-day conditions and of the ideas prevailing in democratic States’.70 The Court generally cites Tyrer v. UK as the source of this principle, which casts the Convention as a ‘living instrument’.71 Although this means that standards of protection may develop,72 the Court has also explicitly arrived at the corollary conclusion that it should not go beyond these evolving standards.73 It is therefore not at liberty to ﬁnd that hunting, eating or experimenting on animals is improper even if it were inclined to do so: every Council of Europe state accepts these practices within certain limits, and nor is there a consensus even among animal activists and scholars on their (im)propriety.74 Although the obligation—both precedential and of prudence—not to stray beyond the understanding of the Convention rights among the states forecloses certain radical steps in using the ECHR to protect animal welfare, the ‘living instrument’ formulation also offers the promise that future developments may be incorporated into the Convention’s protections. Judge Pinto de Albuquerque’s separate opinion in Hermann v. Germany in 2012 gives an indication of the mechanism through which this could take place.75 Animals, Pinto de Albuquerque argued, are protected under the ECHR in two ways. First, they may be property within the deﬁnition of article 1 of Protocol 1.76 More importantly, they may be protected ‘as beings in themselves [. . .] as part of a healthy, balanced and sustainable environment’,77 under the umbrella of the article 8 obligation to ‘avoid acts and activities that could have detrimental consequences for public health and the envi- ronment’.78 Pinto de Albuquerque ﬁnds ‛“clear and uncontested evidence of a continuing international trend” in favour of the protection of animal life and welfare 78Ibid., 33 [references omitted]. 78Ibid., 33 [references omitted]. Protection of Animals Through Human Rights: The Case-Law of the European Court. . 79Ibid., 36, citing ECHR, Goodwin v. UK (n. 50), para. 85. 80Opinion of Pinto de Albuquerque (n. 75), 37 [emphasis and references omitted]. 81Ibid., 37 [references omitted]. 82The ability of animals to appear as “persons” before the courts is discussed below, at note 89. 83Opinion of Pinto de Albuquerque (n. 75), 38. 84See, for example, the recent framework principles prepared by John Knox in his capacity as special rapporteur: Human Rights Council, ‘Report of the Special Rapporteur on the Issue of Human Rights Obligations relating to the Enjoyment of a Safe, Clean, Healthy and Sustainable Environment’, 24 January 2018, UN Document No. A/HRC/37/59. Knox suggests two parallel provisions as his ﬁrst and second framework principles, that ‘[s]tates should ensure a safe, clean, healthy and sustainable environment in order to respect, protect and fulﬁl human rights’, and that ‘[s]tates should respect, protect and fulﬁl human rights in order to ensure a safe, clean, healthy and sustainable environment’ (page 7). 85ECHR K t t G ( 1) 53 79Ibid., 36, citing ECHR, Goodwin v. UK (n. 50), para. 85. 85ECHR, Kyrtatos v. Greece (n. 1), para. 53. 80Opinion of Pinto de Albuquerque (n. 75), 37 [emphasis and references omitted]. 81Ibid 37 [references omitted] 82The ability of animals to appear as “persons” before the courts is discussed below, at note 89. The ability of animals to appear as persons before the courts is discussed below, at note 89. 83Opinion of Pinto de Albuquerque (n. 75), 38. 84See, for example, the recent framework principles prepared by John Knox in his capacity as special rapporteur: Human Rights Council, ‘Report of the Special Rapporteur on the Issue of Human Rights Obligations relating to the Enjoyment of a Safe, Clean, Healthy and Sustainable Environment’, 24 January 2018, UN Document No. A/HRC/37/59. Knox suggests two parallel provisions as his ﬁrst and second framework principles, that ‘[s]tates should ensure a safe, clean, healthy and sustainable environment in order to respect, protect and fulﬁl human rights’, and that ‘[s]tates should respect, protect and fulﬁl human rights in order to ensure a safe, clean, healthy and sustainable environment’ (page 7). 80Opinion of Pinto de Albuquerque (n. 75), 37 [emphasis and references omitted]. 81Ibid., 37 [references omitted]. 4 Obstacles and Opportunities 165 13 [which] is reﬂected in the application of the Convention.’79 He argues that the Court should reject both the ‘commodiﬁcation’ of animals and extensive conceptions of human-like animal personality, instead embracing a ‘qualiﬁed speciesism which builds upon a responsible anthropocentrism.’80 He concludes that recognising the moral differences between humans and animals ‘does not prevent us from acknowl- edging the [. . .] existence of basic comparable interests between humans and other animals and therefore the need to safeguard certain ‘animal rights’, metaphorically speaking, in a similar way to human rights.’81 The mechanism through which this should be achieved is not the grant of legal personality to animals to raise claims before the Court (nor upon human ‘representatives’ to do so),82 but rather through the obligation of states to realise the human right to a healthy environment.83 g g y While there is much here that is attractive, there remain problems with the applica- tion of the approach Pinto de Albuquerque proposes, and ﬂaws in the approach itself. To begin with application, it is increasingly accepted that a healthy environment is an aspect of human rights.84 As yet, however, it is unclear whether the ECHR has the potential adequately to integrate this idea into its provisions. Prima facie, environmental harms are more closely connected to the protection of social and economic rights than the primarily civil and political rights of the ECHR. The disconnect is clear in Kyrtatos v. Greece, in which the Court was asked to decide that the illegal destruction of a wetland habitat next to the applicants’ house was a violation of article 8. The Court chose not to do so, holding that the applicants had not demonstrated that the effect of the environmental degradation on them ‘directly affect[ed] their own rights under article 8’.85 The Court reached that conclusion by six votes to one, with Judge Zagrebelsky the only dissenter. It can be speculated then, that although it would be possible for environmental degradation to have sufﬁciently negative effects to amount to a breach of article 8, such a ﬁnding is likely to be made only where there is a measureable negative effect on individuals’ health or some other equally weighty aspect of their lives. By contrast, and despite that it materially affected their quality of life, ‘the Court 85ECHR, Kyrtatos v. Greece (n. 1), para. 53. T. Sparks 166 T. 88On this idea see Redgwell, ‘Life, The Universe And Everything’ 1996, 71-87, esp. 75-79; Shelton, ‘Environmental Rights’ 2001, 190; Bulto, ‘The Environment and Human Rights’ 2014, 1015-1030; de Lucia, ‘Beyond Anthropocentricism and Egocentrism’, 2017, esp. 184-188. 89 4 Obstacles and Opportunities Sparks [did not] accept that the interference with the conditions of animal life in the swamp constitute[d] an attack on the private or family life of the applicants.’86 If the precedent set in Kyrtatos stands, then, a harm to animal life and the wider environment will have to produce very substantial negative impacts on individuals before it will be possible to assimilate these harms under article 8.87 Yet there are potential problems, too, with the idea of responsible anthropocentricism as a theoretical lens through which to interpret the ECHR in ways conducive to the protection of animal welfare, in that it remains—obviously— anthropocentric.88 Of course, one could hardly expect the ECtHR to move to a position beyond ‘responsible anthropocentricism’ without alteration of the Conven- tion or a substantial leap in its interpretation. Such an interpretive move would, in theory, be possible: indeed, there is nothing in the text of the convention that would prevent it from being extended to apply to (some) animals. Despite its title (Con- vention for the Protection of Human Rights; Convention de sauvegarde des droits de l’homme), the personal scope of the Convention as deﬁned by its ﬁrst article does not refer to ‘humans’ but rather to ‘everyone’ and ‘toute personne’, both terms which seem amenable to a legal rather than scientiﬁc deﬁnition. Nevertheless, it remains to be seen whether the Court is able to go this far,89 and that uncertainty serves to make 87This conclusion is broadly supported by Natalia Kobylarz’s study of the Court’s case-law on wider aspects of environmental protection. While the Court has been able to provide relief under the ECHR in a number of environmental damage scenarios, it remains necessary to show an immediate link to a concrete harm. See Natalia Kobylarz, ‘The European Court of Human Rights: An Underrated Forum for Environmental Litigation’, in European Environmental Law Forum, Sus- tainable Management of Natural Resources – Legal Approaches and Instruments (forthcoming), available at: https://ssrn.com/abstract 3178983, accessed 30/05/2018. ¼ 89Note, for example, the Court’s decision to deny jurisdiction ratione personae over the application in Stibbe v. Austria, a case brought by an animal rights activist on behalf of a chimpanzee known as Matthis Pan. Stibbe sought to be appointed the legal guardian of Matthis Pan, but her application was denied by the Austrian courts on the basis that only humans can have guardians. 87This conclusion is broadly supported by Natalia Kobylarz’s study of the Court’s case-law on wider aspects of environmental protection. While the Court has been able to provide relief under the ECHR in a number of environmental damage scenarios, it remains necessary to show an immediate link to a concrete harm. See Natalia Kobylarz, ‘The European Court of Human Rights: An Underrated Forum for Environmental Litigation’, in European Environmental Law Forum, Sus- tainable Management of Natural Resources – Legal Approaches and Instruments (forthcoming), available at: https://ssrn.com/abstract 3178983, accessed 30/05/2018. ¼ 89Note, for example, the Court’s decision to deny jurisdiction ratione personae over the application in Stibbe v. Austria, a case brought by an animal rights activist on behalf of a chimpanzee known as Matthis Pan. Stibbe sought to be appointed the legal guardian of Matthis Pan, but her application was denied by the Austrian courts on the basis that only humans can have guardians. Her appeal to the ECtHR was declared inadmissible on the basis that ‘[t]he applicant cannot [. . .] claim to have herself been a victim of the violation in accordance with article 34 of the Convention. The complaint is therefore not in accordance with the personal scope of the Convention under article 35 paragraph 3’: Letter from A. Wampach, Deputy Registrar for the First Section, in the matter of ECHR, Stibbe v. Austria, 22 January 2010, Reference No. ECHR-LGer11.0R(CD8); IF/IW/tpe; Application No. 26188/08 [my translation]. Jurisdiction ratione personae, clearly, will be an obstacle to cases of this kind being heard before the ECtHR. This may be contrasted to the now-famous Orangutána Sandra decision before the courts of Argentina, in which it was decided animals may be the subject of rights: ‘Based on a dynamic rather than a static legal interpretation, it is necessary to accord the animal the status of a rights-holder. Non-human subjects (animals) are bearers of rights, and therefore their protection is required within the corresponding jurisdiction’: Camera Federal de Casación Penal, Orangutána Sandra, Judgment of 18 December 2014, LEX No. CCC 68831/2014/ CFC1, para. 2. [I thank Dr Pedro Villarreal for his assistance interpreting the judgment and preparing this translation.] Similar decisions were handed down in 2016 in another Argentinian 86Ibid. 87This conclusion is broadly supported by Natalia Kobylarz’s study of the Court’s case-law on wider aspects of environmental protection. While the Court has been able to provide relief under the ECHR in a number of environmental damage scenarios, it remains necessary to show an immediate link to a concrete harm. See Natalia Kobylarz, ‘The European Court of Human Rights: An Underrated Forum for Environmental Litigation’, in European Environmental Law Forum, Sus- tainable Management of Natural Resources – Legal Approaches and Instruments (forthcoming), available at: https://ssrn.com/abstract 3178983, accessed 30/05/2018. ¼ 88On this idea see Redgwell, ‘Life, The Universe And Everything’ 1996, 71-87, esp. 75-79; Shelton, ‘Environmental Rights’ 2001, 190; Bulto, ‘The Environment and Human Rights’ 2014, 1015-1030; de Lucia, ‘Beyond Anthropocentricism and Egocentrism’, 2017, esp. 184-188. 89Note, for example, the Court’s decision to deny jurisdiction ratione personae over the application in Stibbe v. Austria, a case brought by an animal rights activist on behalf of a chimpanzee known as Matthis Pan. Stibbe sought to be appointed the legal guardian of Matthis Pan, but her application was denied by the Austrian courts on the basis that only humans can have guardians. Her appeal to the ECtHR was declared inadmissible on the basis that ‘[t]he applicant cannot [. . .] claim to have herself been a victim of the violation in accordance with article 34 of the Convention. The complaint is therefore not in accordance with the personal scope of the Convention under article 35 paragraph 3’: Letter from A. Wampach, Deputy Registrar for the First Section, in the matter of ECHR, Stibbe v. Austria, 22 January 2010, Reference No. ECHR-LGer11.0R(CD8); IF/IW/tpe; Application No. 26188/08 [my translation]. Jurisdiction ratione personae, clearly, will be an obstacle to cases of this kind being heard before the ECtHR. This may be contrasted to the now-famous Orangutána Sandra decision before the courts of Argentina, in which it was decided animals may be the subject of rights: ‘Based on a dynamic rather than a static legal interpretation, it is necessary to accord the animal the status of a rights-holder. Non-human subjects (animals) are bearers of rights, and therefore their protection is required within the corresponding jurisdiction’: Camera Federal de Casación Penal, Orangutána Sandra, Judgment of 18 December 2014, LEX No. CCC 68831/2014/ CFC1, para. 2. [I thank Dr Pedro Villarreal for his assistance interpreting the judgment and preparing this translation.] Similar decisions were handed down in 2016 in another Argentinian 4 Obstacles and Opportunities Her appeal to the ECtHR was declared inadmissible on the basis that ‘[t]he applicant cannot [. . .] claim to have herself been a victim of the violation in accordance with article 34 of the Convention. The complaint is therefore not in accordance with the personal scope of the Convention under article 35 paragraph 3’: Letter from A. Wampach, Deputy Registrar for the First Section, in the matter of ECHR, Stibbe v. Austria, 22 January 2010, Reference No. ECHR-LGer11.0R(CD8); IF/IW/tpe; Application No. 26188/08 [my translation]. Jurisdiction ratione personae, clearly, will be an obstacle to cases of this kind being heard before the ECtHR. This may be contrasted to the now-famous Orangutána Sandra decision before the courts of Argentina, in which it was decided animals may be the subject of rights: ‘Based on a dynamic rather than a static legal interpretation, it is necessary to accord the animal the status of a rights-holder. Non-human subjects (animals) are bearers of rights, and therefore their protection is required within the corresponding jurisdiction’: Camera Federal de Casación Penal, Orangutána Sandra, Judgment of 18 December 2014, LEX No. CCC 68831/2014/ CFC1, para. 2. [I thank Dr Pedro Villarreal for his assistance interpreting the judgment and preparing this translation.] Similar decisions were handed down in 2016 in another Argentinian 89Note, for example, the Court’s decision to deny jurisdiction ratione personae over the application in Stibbe v. Austria, a case brought by an animal rights activist on behalf of a chimpanzee known as Matthis Pan. Stibbe sought to be appointed the legal guardian of Matthis Pan, but her application was denied by the Austrian courts on the basis that only humans can have guardians. Her appeal to the ECtHR was declared inadmissible on the basis that ‘[t]he applicant cannot [. . .] claim to have herself been a victim of the violation in accordance with article 34 of the Convention. The complaint is therefore not in accordance with the personal scope of the Convention under article 35 paragraph 3’: Letter from A. Wampach, Deputy Registrar for the First Section, in the matter of ECHR, Stibbe v. Austria, 22 January 2010, Reference No. ECHR-LGer11.0R(CD8); IF/IW/tpe; Application No. 26188/08 [my translation]. Jurisdiction ratione personae, clearly, will be an obstacle to cases of this kind being heard before the ECtHR. 90And there are many who argue that it should not. See, for example, Elder, ‘Legal Rights for Nature – The Wrong Answer to the Right Question’ 1984, 285-295; Livingston, ‘Rightness or Rights?’ 1984, 309-321; Machan, ‘Do Animals Have Rights?’ 1991, 163-173; Merrills, ‘Environ- mental Rights’ 2007, 672. 91See, in particular, Peters, ‘Liberté, Égalité, Animalité: Human-Animal Comparisons in Law’ 2016b, 39-44 et seq.; Gearty, ‘Do Human Rights Help or Hinder Environmental Protection’ 2010, 7-22; and further Plass, ‘Exploring Animal Rights as an Imperative for Human Welfare’ 2010, 403-430; Keim/Sosnowski, ‘Human Rights v Animal Rights: Mutually Exclusive or Complemen- tary Causes’ 2012, 78-83. An intriguing (but, in the author’s view, ultimately ill-directed) inversion of this debate is Shikubu, ‘Work like a Dog’ 2014, 44-65. case (Tercer Juzgado de Garantías de Mendoza, Chimpanzee ‘Cecilia’, Judgment of 3 November 2016, No. P-72.254/15), and by the Colombian Supreme Court in 2017, granting habeas corpus in favour of a spectacled bear: Corte Suprema de Justicia de Colombia, Judgment of 26 July 2017, AHC4806–2017, Radicación no. l7001–22–13–000–2017–00468–02. In the common law world such cases have to date been raised only in the USA, and as yet without great success. In the most recent development (at time of writing), application to appeal to the New York Court of Appeals was denied on 5 April 2018 in joined cases submitted on behalf of two chimpanzees, in which a writ of habeas corpus was denied at ﬁrst instance: State of New York Court of Appeals, In re the Nonhuman Rights Project, inc., on behalf of Tommv v. Patrick C. Lavery and In re the Nonhuman Rights Project, inc., on behalf of Kiko v. Carmen Presti et al., Judgment of 8 May 2018, unreported, Motion No. 2018-268. 4 Obstacles and Opportunities This may be contrasted to the now-famous Orangutána Sandra decision before the courts of Argentina, in which it was decided animals may be the subject of rights: ‘Based on a dynamic rather than a static legal interpretation, it is necessary to accord the animal the status of a rights-holder. Non-human subjects (animals) are bearers of rights, and therefore their protection is required within the corresponding jurisdiction’: Camera Federal de Casación Penal, Orangutána Sandra, Judgment of 18 December 2014, LEX No. CCC 68831/2014/ CFC1, para. 2. [I thank Dr Pedro Villarreal for his assistance interpreting the judgment and preparing this translation.] Similar decisions were handed down in 2016 in another Argentinian 167 13 Protection of Animals Through Human Rights: The Case-Law of the European Court. . . 1 the anthropocentrism problem and its study more urgent. If anthropocentricism is a barrier to the formulation of meaningful principles to undergird animal welfare (let alone animal rights), then one must necessarily conclude that the human rights framework cannot contribute to the development of global animal law.90 That question has been discussed elsewhere (and is taken up in several of the other contributions to this volume), and is too large and complex adequately to be discussed here.91 However, in the present author’s opinion, this proposition is not correct. On the contrary, human rights law can meaningfully contribute to the development of global animal law. Though it may be that global animal law will eventually need to separate itself from human rights law if it is to realise its potential, in its early stages of development there are numerous opportunities for synergistic interactions with frameworks such as the ECHR. 93Ibid., 7. [References omitted]. A similar argument is made by Knox, ‘Climate Ethics and Human Rights’ 2014, 22-34; but compare the problematisation of this aspect of human rights discourse in Blouin Genest/Paquerot, ‘Environmental Human Rights as a Battleﬁeld’ 2016, 132-154. 92Gearty, ‘Human Rights and Environmental Protection’ 2010. 94Peters, ‘Liberté, Égalité, Animalité’ 2016a, 26. Motion No. 2018-268. 95Gearty, ‘Human Rights and Environmental Protection’ 2010, 15-18. 96Ibid., 21; for a similar argument grounded in the concept of dignity see Kotzmann/Seery, ‘Dignity in International Human Rights Law’ 2017, 1-41. 97Gearty, ‘Human Rights and Environmental Protection’ 2010, 22. The signiﬁcance of empathy is also persuasively emphasised by Peters, who notes not only the transformative power of empathy on discourses and societies (39-42), but also the potential for deﬁnitions to structure empathic reactions. She begins by recalling the hideous nineteenth and twentieth century practice of displaying people of non-European origin as zoo exhibits, and notes that ‘[t]he “primitives” were relegated to the animal side of an imagined boundary’: Peters, ‘Liberté, Égalité, Animalité’ 2016a, 25-26; see also Peters, ‘Introduction: Animal Law – A Paradigm Change’ 2015, 17-18. For an examination of empathy as a basis for distinctively human rights see Robinson, ‘Biological Foundations of Human Rights’ 2013, 54-81. 4 Obstacles and Opportunities This is the argument forcefully and convincingly made by Connor Gearty in the wider context of environmental protection.92 Gearty begins by acknowledging that environmental concerns (and, for our purposes, animal welfare and rights) do not sit easily alongside the human rights framework’s proud anthropocentricism: The subject of human rights is, as it declares for all to see in the way that it describes itself, a ﬁeld that is concerned not only with humans but also with the rights that ﬂow from being human, rather than from being anything else[.]93 Human rights law exempliﬁes and makes explicit a sin Anne Peters identiﬁes more generally, that ‘the law as it stands mirrors and reiﬁes a human-animal divide’.94 Yet 91See, in particular, Peters, ‘Liberté, Égalité, Animalité: Human-Animal Comparisons in Law’ 2016b, 39-44 et seq.; Gearty, ‘Do Human Rights Help or Hinder Environmental Protection’ 2010, 7-22; and further Plass, ‘Exploring Animal Rights as an Imperative for Human Welfare’ 2010, 403-430; Keim/Sosnowski, ‘Human Rights v Animal Rights: Mutually Exclusive or Complemen- tary Causes’ 2012, 78-83. An intriguing (but, in the author’s view, ultimately ill-directed) inversion of this debate is Shikubu, ‘Work like a Dog’ 2014, 44-65. 93Ibid., 7. [References omitted]. A similar argument is made by Knox, ‘Climate Ethics and Human Rights’ 2014, 22-34; but compare the problematisation of this aspect of human rights discourse in Blouin Genest/Paquerot, ‘Environmental Human Rights as a Battleﬁeld’ 2016, 132-154. T. Sparks T. Sparks 168 Gearty argues that human rights has the potential to support environmental protection both through the protection of environmental activism (‘protecting the messenger’),95 and by offering a vocabulary of empowerment that activists can use. It speaks meaningfully across the whole spectrum of a community, from the weak across to the powerful, deploying the convictions of the latter—rooted in the battles of the past—to force recognition of the need for similar struggles today. [. . .] This chameleonism is often a source of frustration for sure, but it is what gives the idea of human rights the power that it undeniably enjoys in the world today.96 Though human rights are intrinsically anthropocentric, the human rights project is a legally-embedded socio-linguistic mobilisation of empathy for the other.97 Using the language of human rights carries with it the historical experience of the manifold struggles for justice that have been fought under its banner. 4 Obstacles and Opportunities Embedded in the frame- work are the memories of many claims once bitterly contested as radical oppositions to an entrenched power-structure which have succeeded in breaking into the mainstream consciousness, have overturned centuries of social practice, or have been codiﬁed as a minimum standard of positive morality in international declarations and conventions. The language, experience, and historical legitimacy-claim of human rights can be powerful tools in the campaign for animal (and wider environmental) rights, notwith- standing the inevitable friction between zoo- and anthropos-centrism. 5 Final Thoughts Although that friction is more pronounced (and the radical discourse more constrained) within human rights viewed as a legal framework rather than a socio- political project, nevertheless many of the same arguments hold true. There are barriers to the direct treatment of animal concerns by human rights fora as a result of personal and material limitations on their scope of jurisdiction, but the case-law of the ECtHR demonstrates that there remain opportunities to bring animal concerns under the umbrella of human welfare. This does, it is true, raise moral questions, in particular the “speaking for the other’ problem’, as Catharine MacKinnon has 169 Protection of Animals Through Human Rights: The Case-Law of the European Court. . . 13 pointed out.98 Animal law remains human law, and it aspires towards a human interpretation of what ‘animal welfare’ looks like. Yet though the interpretative divide is deeper, Peters is clearly correct to ask where the differences lie between speaking for animals and speaking for humans who lack legal capacity (Peters’ example is children).99 Arguably in the case of animals the situation is more problematic: where we raise children’s concerns before Courts we do so for the beneﬁt of the children involved, while animal rights at present ﬂow from human rights only as a corollary of human concerns. The former is a case of speaking for, with all the moral difﬁculties that ﬂow from that; the latter is an example of instrumentalisation. Yet there is also a zone of conﬂuence,100 in which human and animal wellbeing and rights coincide insofar as it can be demonstrated that protecting the one beneﬁts the other.101 Peters uses the phrase ‘liberté, égalité, animalité’ as ‘a reminder that humans need legal protection not least on account of their animal nature, their physical vulnerability and their “nakedness”, which they share with all other animals.’102 It is indeed a salutary reminder that the human/ animal divide is bridged in many respects, including the ‘vital interests’ of both groups.103 Articulating those conﬂuences within the language of the ECHR and other human rights frameworks has the potential to catalyse the development of animal welfare as a sub-genre of the international human rights story, as well as to provide norms, ideas and impetuses which will cross into other jurisdictions and disciplines, and scholars should now take up this task. 04Anne Peters, ‘Global Animal Law: What it is and why we need it’, Transnational Environmental aw 5(1) (2016b), 9-23, 20. 98MacKinnon, ‘Of Mice and Men: A Feminist Fragment on Animal Rights’ 2004, 270. 99Peters, ‘Liberté, Égalité, Animalité’ 2016a, 48. 100This idea is similar to Bulto’s substantive regime complementarity: Bulto, ‘Environment’ 2014, 1025-1028. References Blouin Genest, G., & Paquerot, S. (2016). Environmental human rights as a Battleﬁeld: A grammar of political confrontation. Journal of Human Rights and the Environment, 7, 132–154. Bulto, T. S. (2014). The environment and human rights. In A. Mihr & M. Gibney (Eds.), The SAGE handbook of human rights (Vol. II, pp. 1015–1030). London: Sage Publishing. Çoban, A. R. (2004). Protection of property rights within the European Convention on Human Rights. Aldershot: Ashgate. Cochrane, A. (2012). Animal rights without liberation: Applied ethics and human obligations. New York: Columbia University Press. de Lucia, V. (2017). Beyond anthropocentricism and egocentrism: A biopolitical reading of environmental law. Journal of Human Rights and the Environment, 8(2), 181–202. Elder, P. S. (1984). Legal rights for nature – the wrong answer to the right question. Osgoode Hall Law Journal, 22(2), 285–295. Fenwick, H., Phillipson, G., & Williams, A. (2017). Texts, cases and materials on public law and human rights (4th ed.). Abingdon: Routledge. Gearty, C. (2010). Do human rights help or hinder environmental protection. Journal of Human Rights and the Environment, 1, 7–22. Harrop, S. (2011). Climate change, conservation and the place for wild animal welfare in interna tional law. Journal of Environmental Law, 23(3), 441–462. Keim, S., & Sosnowski, J. (2012). Human rights v animal rights: Mutually exclusive or comple mentary causes. Australian Animal Protection Law Journal, 8, 78–83. Knox, J. (2014). Climate ethics and human rights. Journal of Human Rights and the Environment, 5 (Special Issue), 22–34. ¼ Kobylarz, N. (forthcoming). The European Court of Human Rights: An underrated forum for environmental litigation. In European Environmental Law Forum, Sustainable Management of Natural Resources – Legal Approaches and Instruments. Available at: https://ssrn.com/ abstract 3178983 Kotzmann, J., & Seery, C. (2017). Dignity in international human rights law: Potential applicability in relation to international recognition of animal rights. Michigan State International Law Review, 26(1), 1–41. Kymlicka, W., & Donaldson, S. (2014). Animal rights, multiculturalism, and the left. Journal o Social Philosophy, 45(1), 116–135. Livingston, J. (1984). Rightness or rights? Osgoode Hall Law Journal, 22(2), 309–32 MacKinnon, C. (2004). Of mice and men: A feminist fragment on animal rights. In C. Sunstein & M. Nussbaum (Eds.), Animal rights: Current debates and new directions (pp. 263–276). Oxford: OUP. Merrills, J. (2007). Environmental rights. In D. Bodansky, J. Brunnée, & E. Hey (Eds.), The Oxfor handbook of international environmental law (pp. 663–680). Oxford: OUP. Peters, A. (2015). 5 Final Thoughts It is in these interactions that global animal law is growing and will continue to grow,104 and this brief 98MacKinnon, ‘Of Mice and Men: A Feminist Fragment on Animal Rights’ 2004, 270. 99Peters, ‘Liberté, Égalité, Animalité’ 2016a, 48. 100This idea is similar to Bulto’s substantive regime complementarity: Bulto, ‘Environment’ 2014, 1025-1028. 101An example of such an approach in practice can be seen in the Court’s decision on admissibility in Friend and Others v UK (n. 1). In that case, a challenge to the UK ban on hunting wild mammals with dogs, the Court ﬁrst ruled that the Convention articles claimed by the applicants were not engaged, before noting (in particular in relation to article 11) that ‘the measures served the legitimate aim of (. . .) “the protection of . . . morals”, in the sense that they were designed to eliminate the hunting and killing of animals for sport in a manner which the legislature judged to cause suffering and to be morally and ethically objectionable’ (at 18). The Court thus found that had the convention rights been engaged, the limitation would nevertheless have fallen within the State’s margin of appreciation. Though at best indicative, as no full examination was undertaken, the admissibility decision shows one way in which the interests of animals can condition human rights—in this case as a limitation, elsewhere through a zone of conﬂuence approach. 102Peters, ‘Liberté, Égalité, Animalité’ 2016a, 53; citing Saskia Stucki, ‘Sind die Menschenrechte in Zukunft noch Menschen-Rechte?’, Völkerrechtsblog, 13 May 2014, available at: http:// voelkerrechtsblog.com/category/sind-die-menschenrechte-in-zukunft-noch-menschen-rechte/. 103This idea I take from Mark Rowlands, Animals Like Us (London: Verso 2002), 125-136 et seq. Rowlands uses the term to refer to the interest all animals have in remaining alive, as well as the basic goods that enable them to do so. He argues that the non-vital interests of any (human or non-human) animal should not outweigh the vital interests of any other. 104Anne Peters, ‘Global Animal Law: What it is and why we need it’, Transnational Environmental Law 5(1) (2016b), 9-23, 20. 170 T. Sparks examination of the ECtHR suggests that human rights law has a meaningful contri- bution to make to that process. examination of the ECtHR suggests that human rights law has a meaningful contri- bution to make to that process. References Introduction: Animal law – a paradigm change. In A. Peters, S. Stucki, & L. Boscardin (Eds.), Animal law: Reform or revolution (pp. 15–32). Zürich: Schulthess. É Peters, A. (2016a). Liberté, Égalité, Animalité: Human-Animal Comparisons in Law. Transna- tional Environmental Law, 5, 25–53. Peters, A. (2016b). Global animal law: What it is and why we need it. Transnational Environmental Law, 5(1), 9–23. 13 Protection of Animals Through Human Rights: The Case-Law of the European Court. . . 17 171 Plass, S. A. (2010). Exploring animal rights as an imperative for human welfare. West Virginia Law Review, 112, 403–430. Redgwell, C. (1996). Life, the universe and everything: A critique of anthropocentric rights. In A. Boyle & M. Anderson (Eds.), Human rights approaches to environmental protection (pp. 71–87). Oxford: Clarendon Press. Robinson, C. (2013). Biological foundations of human rights. In D. Shelton (Ed.), The Oxford handbook of international human rights law (pp. 54–81). Oxford: OUP. Rowlands, M. (2002). Animals like us. London: Verso. Rowlands, M. (2002). Animals like us. London: Verso. Shelton, D. (2001). Environmental Rights. In P. Alston (Ed.), Peoples’ rights (pp. 185–258). Oxford: OUP. Shikubu, M. (2014). Work like a dog: Expanding animal cruelty statutes to gain human rights for migrant farmworkers in the US. National Lawyers Guild Review, 71, 44–65. Stucki, S. (2014, May 13). Sind die Menschenrechte in Zukunft noch Menschen-Rechte?. Völkerrechtsblog. Available at http://voelkerrechtsblog.com/category/sind-die- menschenrechte-in-zukunft-noch-menschen-rechte/ Taylor, N. (1999). Whiter rights? Animal rights and the rise of New Welfarism. Animal Issues, 3(1), 27–41. Tom Sparks is a senior research fellow at the Max Planck Institute for Comparative Public Law and International Law in Heidelberg, where he works in the research group of Professor Anne Peters. His research interests focus on public international law, international environmental law, the humanisation of law, and legal theory. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), 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. g The images or other third party material in this chapter are included in the chapter’s Creative Commons licence, unless indicated otherwise in a credit line to the material. References If material is not included in the chapter’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.
https://openalex.org/W4206689914	https://www.business-inform.net/export_pdf/business-inform-2021-11_0-pages-235_240.pdf	Ukrainian	null	Development of Private Pension Provision in the Conditions of Reforming the Pension System of Ukraine	Bìznes ìnform/Bìznes ìnform	2,021	cc-by-sa	4,149	JEL: H55; G23 DOI: https://doi.org/10.32983/2222-4459-2021-11-235-240 РОЗВИТОК НЕДЕРЖАВНОГО ПЕНСІЙНОГО ЗАБЕЗПЕЧЕННЯ В УМОВАХ РЕФОРМУВАННЯ ПЕНСІЙНОЇ СИСТЕМИ УКРАЇНИ 2021 РУДИК В. К. УДК 331.25(477) JEL: H55; G23 Рудик В. К. Розвиток недержавного пенсійного забезпечення в умовах реформування пенсійної системи України Ефективне функціонування системи недержавного пенсійного забезпечення, яка формує третій рівень вітчизняної пенсійної системи, має важ ливе значення для підвищення доходів громадян пенсійного віку. Метою статті є визначення основних шляхів подальшого розвитку системи недержавного пенсійного забезпечення в умовах реформування національної пенсійної системи, формування сприятливих умов для залучення все більшої кількості населення до участі в добровільних накопичувальних пенсійних програмах. У статті дана оцінка стану системи недержавного пенсійного забезпечення в Україні, проаналізовано її основні показники за останні три роки. Результати дослідження показали, що на сьогодніш ній день добровільні накопичувальні пенсійні програми, які складають основу системи недержавного пенсійного забезпечення, не користуються популярністю серед населення. Визначено основні виклики та чинники, які заважають її розвиткові в нашій країні. Запропоновано основні напрям ки розвитку системи недержавного пенсійного забезпечення на найближчу перспективу. Особливу увагу звернуто на доцільність запровадження неоподатковуваних добровільних автоматичних індивідуальних ощадних пенсійних рахунків у банках. Серед пріоритетів покращення функціону вання третього рівня важливе місце належить пошуку шляхів подальшого розвитку недержавних пенсійних фондів (НПФ), серед яких виділяють підвищення їх надійності. Для оцінки надійності НПФ як соціально-економічної системи необхідно аналізувати такі параметри, як: наявність довіри громадян до фонду; здатність фонду до виплати грошових коштів; фінансовий стан фонду. Доведено, що вдосконалення законодавчої бази третього рівня вітчизняної пенсійної системи повинно забезпечувати захист майнових прав та інтересів учасників недержавного пенсій ного забезпечення, а також впорядкування оподаткування учасників накопичувальної системи пенсійного забезпечення. Надзвичайно важливим напрямком подальшого розвитку третього рівня національної пенсійної системи вважається зменшення й оптимізація витрат на адміністру вання індивідуальних пенсійних рахунків учасників накопичувальних пенсійних програм. ЕКОНОМІКА ПРАЦІ ТА СОЦІАЛЬНА ПОЛІТИКА Ключові слова: система недержавного пенсійного забезпечення, накопичувальні пенсійні програми, індивідуальні пенсійні заощадження, пенсійні активи, недержавні пенсійні фонди. Рудик Володимир Касянович – доктор економічних наук, професор, завідувач кафедри фінансів, банківської справи, страхування та електронних платіжних систем, Подільський державний аграрно-технічний університет (вул. Шевченка, 13, Кам'янець-Подільський, 32300, Україна) E-mail: Rudyk_vk63@gmail.com ORCID ht // id /0000 0001 9011 4543 Researcher ID: https://publons.com/researcher/2939367/volodymyr-rudyk/ S A th ID htt // / thid/d t il i? th Id 57210895061 Researcher ID: https://publons.com/researcher/2939367/volodymyr-rudyk/ Scopus Author ID: https://www.scopus.com/authid/detail.uri?authorId=57210895061 Researcher ID: https://publons.com/researcher/2939367/volodymyr-rudyk/ Scopus Author ID: https://www.scopus.com/authid/detail.uri?authorId=57210895061 t p //p / / / y y y / Scopus Author ID: https://www.scopus.com/authid/detail.uri?authorId=57210895061 t p //p / / / y y y / Scopus Author ID: https://www.scopus.com/authid/detail.uri?authorId=57210895061 УДК 331.25(477) JEL: H55; G23 DOI: https://doi.org/10.32983/2222-4459-2021-11-235-240 УДК 331.25(477) JEL: H55; G23 DOI: https://doi.org/10.32983/2222-4459-2021-11-235-240 UDC 331.25(477) JEL: H55; G23 Researcher ID: https://publons.com/researcher/2939367/volodymyr-rudyk/ Scopus Author ID: https://www.scopus.com/authid/detail.uri?authorId=57210895061 235 www.business-inform.net М М ісія пенсійної системи для сучасного сус пільства полягає в забезпеченні високого рівня життя пенсіонерів через викорис тання фінансових ресурсів трьох її основних рівнів. Сучасні реалії у сфері пенсійного забезпечення по казують, що солідарна пенсійна система та недер жавне пенсійне забезпечення не можуть забезпечити повною мірою достатнє фінансування пенсіонерів навіть на нинішньому рівні, який набагато нижчий за міжнародні соціальні стандарти. У зв’язку з цим у достатньо великих розмірах відраховуються кошти з державного бюджету, які покривають дефіцит бю джету Пенсійного фонду України. тку, починаючи з 2004 р., не задовольняють потреби пенсійної сфери та самих українських пенсіонерів у забезпеченні додатковим фінансовими ресурсами. Хоча його практичне використання в багатьох кра їнах свідчить про достатньо високий фінансовий потенціал системи недержавного пенсійного забез печення. Для успішного застосування добровільних накопичувальних пенсійних програм, підвищення довіри населення до них важливим завданням для фахівців, експертів, науковців виступають: розробка системи захисту пенсійних активів їх учасників від прояву різноманітних фінансових ризиків; зниження оплати за надання послуг по адмініструванню даних активів; пошук ефективних фінансових інструментів для їх інвестування. Як показує зарубіжний досвід, система недер жавного пенсійного забезпечення має досить великі фінансові резерви, які можна використати для підви щення доходів громадян пенсійного віку. Ефективне застосування добровільних накопичувальних пенсій них програм, розвиток фондового ринку, довіра насе лення до них, законодавче регулювання відносин на третьому рівні сприяли розвиткові цієї ланки пенсій ної системи в багатьох країнах. Метою дослідження є визначення основних шляхів подальшого розвитку системи недержавного пенсійного забезпечення в умовах реформування на ціональної пенсійної системи, формування сприят ливих умов для залучення все більшої кількості на селення до участі в добровільних накопичувальних пенсійних програмах. Ураховуючи, що система загальнообов’язкового накопичувального пенсійного страхування в Україні на сьогодні ще не запроваджена, науковці, експерти, фахівці у сфері пенсійного забезпечення приділяють розвиткові системи недержавного пенсійного забез печення багато уваги. Основне завдання полягає в тому, щоб розробити такий механізм функціонуван ня добровільних накопичувальних програм, який би зацікавив населення та підвищив довіру до них. О с г н О сновною метою запровадження недержавно го пенсійного забезпечення було отриман ня учасниками накопичувальних пенсійних програм додаткових до загальнообов’язкового дер жавного пенсійного страхування пенсійних виплат разом із забезпеченням дохідності пенсійних активів вище рівня інфляції та залучення довгострокових ін вестиційних ресурсів, необхідних для модернізації економіки. UDC 331.25(477) JEL: H55; G23 Вона формує третій рівень вітчизняної пенсійної системи, який, за переконаннями вітчиз няних фінансистів, базується на засадах добровіль ної участі громадян, роботодавців та їх об’єднань у формуванні пенсійних накопичень і який забезпе чує застрахованій особі після виходу на пенсію отри мання, крім пенсії за загальнообов’язковим держав ним пенсійним страхуванням, додаткової пенсії шля хом добровільних пенсійних внесків до недержавних пенсійних фондів, які стають основою цієї системи, в тому числі й ті, що створені за корпоративними та професійними ознаками [12; с 362]. ЕКОНОМІКА ПРАЦІ ТА СОЦІАЛЬНА ПОЛІТИКА ЕКОНОМІКА ЕКОНОМІКА ПРАЦІ ТА СОЦІАЛЬ ру Дослідженню питань розвитку системи недер жавного пенсійного забезпечення приділяли ува гу у своїх наукових працях і публікаціях В. Грушко, Ю. Скулиш, С. Лаптєв, В. Фатхутдінов, А. Француз, І. Румик, О. Пилипенко [9], О. Піщуліна, О. Коваль, Т. Бурляй [11], співробітники Науково-дослідного інституту демографії та соціальних досліджень імені М. В. Птухи НАН України [8]. Значний вклад у висвіт лення питань щодо розвитку недержавного пенсій ного забезпечення в Україні, виявлення причин недо статньої його популярності серед населення, пошуку шляхів покращення функціонування в національній пенсійній системі зробили фахівці й експерти Проєк ту USAID «Трансформація фінансового сектору» [6]. Аналіз наукових праць по даній проблематиці показав, що система недержавного пенсійного забез печення в Україні ще не достатньо розвинута; визна чено чинники, що впливають на її розвиток в нашій державі; підкреслено її важливість для підвищення пенсійних доходів громадян. Тому напрацювання шляхів подальшого розвитку недержавного пенсій ного забезпечення являє науковий інтерес і має важ ливе практичне значення для успішної реалізації пен сійної реформи в Україні. Оцінка стану третього рівня вітчизняної пен Дослідженню питань розвитку системи недер жавного пенсійного забезпечення приділяли ува гу у своїх наукових працях і публікаціях В. Грушко, Ю. Скулиш, С. Лаптєв, В. Фатхутдінов, А. Француз, І. Румик, О. Пилипенко [9], О. Піщуліна, О. Коваль, Т. Бурляй [11], співробітники Науково-дослідного інституту демографії та соціальних досліджень імені М. В. Птухи НАН України [8]. Значний вклад у висвіт лення питань щодо розвитку недержавного пенсій ного забезпечення в Україні, виявлення причин недо статньої його популярності серед населення, пошуку шляхів покращення функціонування в національній пенсійній системі зробили фахівці й експерти Проєк ту USAID «Трансформація фінансового сектору» [6]. Система недержавного пенсійного забезпечен ня базується на використанні принципів добровіль ного накопичувального пенсійного страхування та, відповідно до діючого вітчизняного пенсійного зако нодавства, застосовує накопичувальні пенсійні про грами [4]. UDC 331.25(477) JEL: H55; G23 Rudyk V. K. Development of Private Pension Provision in the Conditions of Reforming the Pension System of Ukraine The efficient functioning of the non-state pension system, which forms the third level of the domestic pension system, is essential for increasing the incomes of citizens of retirement age. The article is aimed at defining the main ways of further development of the non-state pension system in the context of reforming the national pension system, creating favorable conditions for attracting an increasing number of the population to participate in voluntary accumulation pension programs. The article provides an assessment of the status of the non-state pension system in Ukraine, analyzes its main indicators for the last three years. The results of the study showed that today voluntary accumulation pension programs, which form the basis of the system of non-state pension provision, are not popular among the population. The main challenges and factors that hinder its development in our country are determined. The main directions of development of the system of non-state pension provision for the near future are proposed. Particular attention is paid to the expediency of introducing the tax-free voluntary automatic individual savings pension accounts in banks. Among the priorities for improving the functioning of the third level, an important place is to find ways to further develop the non-state pension funds (NSPFs), among which an increase in their reliability is to be highlighted. To assess the reliability of NSPFs as a socio-economic system, it is necessary to analyze such parameters as: presence of public trust in the fund; the fund’s ability to cash payments; financial condi tion of the fund. It is proved that the improvement of the legislative framework of the third level of the domestic pension system should ensure the protection of property rights and interests of participants in the non-state pension provision, as well as ordering the taxation of participants in the accumulation pension system. An extremely important direction for the further development of the third level of the national pension system is the reduction and optimization of the costs of administering individual pension accounts of participants in the accumulation pension programs.i ЕКОНОМІКА ii ywords: system of the non-state pension provision, accumulation pension programs, individual pension savings, pension assets, non-state pension bl.: 2. Bibl.: 13. UDC 331.25(477) JEL: H55; G23 Одержані результати показали, що третій рівень на сьогоднішній день недостатньо розвину тий, не має значного впливу на збільшення пенсійних доходів українських пенсіонерів і не користується до вірою в населення. Про це свідчать дані табл. 1. Аналіз наукових праць по даній проблематиці показав, що система недержавного пенсійного забез печення в Україні ще не достатньо розвинута; визна чено чинники, що впливають на її розвиток в нашій державі; підкреслено її важливість для підвищення пенсійних доходів громадян. Тому напрацювання шляхів подальшого розвитку недержавного пенсій ного забезпечення являє науковий інтерес і має важ ливе практичне значення для успішної реалізації пен сійної реформи в Україні. ЕКОНОМІКА Протягом трьох останніх років хоча і спостері гається динаміка до зростання вартості пенсійних ак тивів і пенсійних внесків у системі недержавного пен сійного забезпечення, але темпи зростання незначні. Оцінка стану третього рівня вітчизняної пен сійної системи показує, що тенденції його розви БІЗНЕСІНФОРМ № 11 ’2021 236 www.business-inform.net Основні показники системи недержавного пенсійного забезпечення Показник 2018 р. 2019 р. 2020 р. Темпи приросту, % 2019 р. до 2018 р. 2020 р. до 2019 р. Кількість укладених пенсійних контрактів, тис. шт. 68,8 77,1 87,8 12,1 13,9 Загальна кількість учасників НПФ, тис. осіб 855,3 874,6 883,0 2,3 1,0 Загальна вартість активів НПФ, млн грн. 2745,2 3143,3 3563,7 14,5 13,4 Пенсійні внески, всього, млн грн 2000,5 2160,8 2377,9 8,0 10,0 у тому числі: – від фізичних осіб 172,1 222,7 300,7 29,4 35,0 – від фізичних осіб – підприємців 0,2 0,2 0,3 0,0 50,0 – від юридичних осіб 1827,7 1937,3 2075,5 6,0 7,1 Пенсійні виплати, млн грн 809,9 947,4 1 107,4 17,0 16,9 Кількість учасників, що отримали/отримують пенсійні виплати, тис. осіб 81,3 83,9 87,0 3,2 3,7 Сума інвестиційного доходу, млн грн 1767,7 2200,0 2635,7 24,5 19,8 Прибуток від інвестування активів недержавного пенсійного фонду, млн грн 1440,5 1810,9 2179,5 25,7 20,4 Сума витрат, що відшкодовуються за рахунок пенсійних активів, млн грн 327,2 389,1 456,1 18,9 17,2 Співвідношення пенсійних виплат і пенсійних активів, % 29,5 30,1 31,1 х х Співвідношення витрат, що відшкодовуються, та пенсійних активів, % 11,9 12,4 12,8 х х Співвідношення суми інвестиційного доходу та пенсійних активів, % 64,4 70,0 73,9 х х Джерело: складено за даними [7; 10]. ЕКОНОМІКА ПРАЦІ ТА СОЦІАЛЬНА ПОЛІТИКА КОНОМІКА ЕКОНОМІКА ПРАЦІ ТА СОЦІАЛЬНА ПОЛІТИК Джерело: складено за даними [7; 10]. (у 2019 р. – 7,5 тис. грн, у 2018 р. – 7,1 тис. грн). UDC 331.25(477) JEL: H55; G23 Серед ній розмір пенсійної виплати на визначений строк на одного учасника НПФ, який отримав/отримує пен сійну виплату, становив 106,5 тис. грн (у 2019 р. – 82,7 тис. грн, у 2018 р. – 63,4 тис. грн). Звертає на себе увагу той факт, що загальна кількість учасників недержавних пенсійних фондів за дослі джуваний період зростала щорічно всього на 1–2%. В В раховуючи, що третій рівень вітчизняної пен сійної системи практично почав використову ватися з 2004 р., важливо розглянути пенсійні виплати із системи недержавного пенсійного забез печення, їх величину та як вони впливають на пенсій ні доходи українських пенсіонерів. Пенсійні виплати (одноразові та на визначений строк) у 2020 р. стано вили 1107,4 млн грн, що на 16,9% більше порівняно з аналогічним періодом 2019 р. (табл. 2). При цьому одноразові виплати зросли на 9,3%, пенсійні виплати на визначений строк – на 29,6%. Вітчизняні науковці й експерти з даної пробле матики, враховуючи такий стан системи недержав ного пенсійного забезпечення, відмічають основні виклики та чинники, що заважають її розвиткові в нашій країні. До них можна віднести такі:  часті та глибокі економічні кризи в Україні протягом останніх 25 років;  низький розмір наявних (реальних) доходів працівників; ЕКОНОМІКА  слабкість банківського та небанківського фі нансових секторів, а також недостатній рівень розвитку ринку капіталу (у тому числі брак надійних інвестиційних інструментів); Сукупно недержавними пенсійними фондами у 2020 р. було здійснено пенсійних виплат (одноразо вих і на визначений строк) 87,0 тис. учасникам, тобто 9,9% від загальної кількості учасників, які отримали/ отримують пенсійні виплати.  винятково високий рівень оплати послуг, наданих учасникам адміністраторами НПФ, КУА та зберігачами за рахунок їхніх пенсій них коштів (у середньому 4,93% пенсійних АВУ); Середній розмір одноразової пенсійної випла ти на одного учасника НПФ, який отримав/отримує пенсійну виплату у 2020 р., становив 7,9 тис. грн 237 БІЗНЕСІНФОРМ № 11 ’2021 www.business-inform.net Таблиця 2 Динаміка пенсійних виплат Показник Од. виміру Роки Приріст 2020 р. до 2019 р., % 2018 р. 2019 р. 2020 р. Одноразові пенсійні виплати млн грн 545,5 594,5 649,9 9,3 Пенсійні виплати на визначений строк млн грн 264,4 352,9 457,5 29,6 Усього млн грн 809,9 947,4 1107,4 16,9 Джерело: систематизовано за даними [10]. Таблиця 2 Динаміка пенсійних виплат розроблення надійних фінансових інструментів для інвестування пенсійних активів потрібен час.  недовіра до фінансових установ і пенсійної системи серед населення;  недовіра до фінансових установ і пенсійної системи серед населення;  слабкість нагляду та регулювання у фінансо вому секторі, включаючи недержавне пенсій не забезпечення [5].  слабкість нагляду та регулювання у фінансо вому секторі, включаючи недержавне пенсій не забезпечення [5]. С еред пріоритетів покращення функціонування третього рівня важливе місце належить пошу ку шляхів подальшого розвитку недержавних пенсійних фондів. На нашу думку, важливим у вирі шенні цієї проблеми є підвищення надійності НПФ. Для оцінки надійності НПФ як соціально-економіч ної системи необхідно аналізувати такі параметри, як: наявність довіри громадян до фонду; здатність фонду до виплати грошових коштів; фінансовий стан фонду. Кожен із цих параметрів повинен формувати свою систему показників. Досвід функціонування НПФ в Україні свідчить про те, що для їх становлен ня та повноцінного розвитку ще потрібно докладати дуже багато зусиль. Це пов’язано з великою кількіс тю об’єктивних чинників, серед яких дуже важливим є значне старіння населення, а прогнози поліпшення такого становища в найближчі роки невтішні. С Для подальшого розвитку системи недержавно го пенсійного забезпечення важливим є визначення основних її пріоритетів на перспективу. Вони пови нні дати поштовх розвитку добровільних накопичу вальних пенсійних програм і сприяти ефективному функціонуванню третього рівня вітчизняної пенсій ної системи. ЕКОНОМІКА ПРАЦІ ТА СОЦІАЛЬНА ПОЛІТИКА ВИСНОВКИ податкового та пенсійного законодавства в частині оподаткування учасників накопичувальної пенсійної системи. Вони повинні бути направленими на запо бігання неправильному оподаткуванню обов’язкових і добровільних пенсійних накопичень громадян. Крім того, розробляючи нові законодавчі документи й удо сконалюючи вже діючі, потрібно адаптувати їх до за конодавства ЄС. Результати досліджень показують, що на відміну від національних пенсійних систем багатьох європей ських країн, в яких система недержавного пенсійного забезпечення розвинута на досить високому рівні, в Україні третій рівень не користується значною дові рою населення та не має значного впливу на зростання доходів українських пенсіонерів. У зв’язку з цим про понуються основні напрямки подальшого розвитку системи недержавного пенсійного забезпечення, се ред яких – розробка механізму стимулів, що будуть за охочувати людей заощаджувати на пенсію. Для цього необхідно сформувати систему індивідуальних пенсій них заощаджень, яка б могла використовувати неопо датковувані, добровільні, автоматичні, індивідуальні ощадні пенсійні рахунки, відкриті в установах банків. Важливим для поліпшення законодавчої бази системи недержавного пенсійного забезпечення вва жається внесення змін до чинних законодавчих доку ментів, які будуть посилювати вимоги та відповідаль ність учасників третього рівня, а також ураховувати положення Євродиректив, що стосуються пенсійної сфери, тобто імплементувати їх вимоги [3, с. 10–21]. Застосовуючи міжнародний досвід щодо викорис тання й інвестування пенсійних активів накопичу вальних пенсійних програм, потрібно розвивати нор мативно-правову базу, що стосується використання правила «розумного інвестора» [1]. Ураховуючи, що недержавні пенсійні фонди і на далі будуть відігравати основну роль в адмініструван ні й управлінні пенсійними активами накопичуваль них пенсійних програм, запропоновано шляхи поліп шення їх надійності. Визначено основні чинники, що впливають на надійність системи недержавних пен сійних фондів, а саме: на інвестування коштів пенсій них активів. Удосконалення законодавчої бази дасть змогу поліпшити правові взаємовідносини в системі недержавного пенсійного забезпечення, забезпечити захист майнових прав та інтересів учасників добро вільних накопичувальних пенсійних програм.  Н Н Н адзвичайно важливим напрямком подаль шого розвитку третього рівня національної пенсійної системи вважається зменшення й оптимізація витрат на адміністрування індивідуаль них пенсійних рахунків учасників накопичувальних пенсійних програм. Недержавні пенсійні фонди й інші фінансові посередники: адміністратори НПФ, компанії з управління активами (КУА) і зберігачі (банки) здійснюють адміністрування й інвестиційну діяльність щодо пенсійних активів і утримують пла ту за надання послуг. За інших однакових умов, чим вища вартість адміністрування, управління активами (інвестування пенсійних коштів), тим меншою є сума активів для отримання інвестиційного доходу та, в кінцевому підсумку, для здійснення пенсійних виплат. ЕКОНОМІКА ПРАЦІ ТА СОЦІАЛЬНА ПОЛІТИКА КОНОМІКА ЕКОНОМІКА ПРАЦІ ТА СОЦІАЛЬНА ПОЛІТИК А ЕКОНОМІКА ПРАЦІ ТА СОЦІАЛЬНА ПОЛІТИКА В р д с В раховуючи, що основною метою системи не державного пенсійного забезпечення є до сягнення гідного рівня життя громадян після виходу на пенсію шляхом доповнення солідарної системи загальнообов’язкового державного пенсій ного страхування, важливим напрямом її розвитку вважається розробка механізму стимулів, які будуть мотивувати людей заощаджувати на пенсію. Для цьо го необхідно з боку уряду країни розробити комплекс заходів, які б сприяли формуванню системи індиві дуальних пенсійних заощаджень. На думку експертів у сфері пенсійного забезпечення, у короткостроковій перспективі з боку державних фінансових інститутів доцільно було б розглянути можливість запроваджен ня неоподатковуваних, добровільних автоматичних індивідуальних ощадних пенсійних рахунків у банках [5; с. 43]. Адже для запровадження системи індиві дуальних пенсійних заощаджень потрібні відповідні передумови. По-перше, населенню треба добре розу міти недержавні фінансові установи й інструменти та довіряти їм. По-друге, мають бути надійні фінансові інструменти та фінансові ринки для стимулювання внутрішніх інвестицій і створення робочих місць. По- третє, зважаючи на високі адміністративні вимоги накопичувальної системи, приватний сектор повинен мати значний адміністративний потенціал. Якщо фінансовий стан і здатність фонду виплачу вати кошти можна оцінити за кількісними характерис тиками, то параметр довіри/недовіри досить складно оцінити кількісно, отже, оцінювати його потрібно з ви користанням якісних характеристик [2, с. 292]. Важливим показником для збільшення довіри населення до НПФ є наявність інформаційної політи ки, раціональна побудова якої забезпечить збільшен ня кола вкладників та учасників НПФ. Інформаційна політика НПФ спрямована на досягнення реалізації прав вкладників, учасників, є істотною для прийнят тя ними рішення про формування недержавної пенсії або про переведення накопичувальної частини пенсії в НПФ (у майбутньому), а також захист конфіденцій ної інформації про фонд, розголошення якої може за вдати шкоди фонду і його вкладникам та учасникам. ЕКОНОМІКА Для успішного розвитку системи недержавного пенсійного забезпечення необхідно вдосконалювати її нормативно-правову базу. Правове регулювання третього рівня вітчизняної пенсійної системи пови нно забезпечувати захист майнових прав та інтересів учасників недержавного пенсійного забезпечення, а також упорядкування оподаткування учасників накопичувальної системи пенсійного забезпечення. Законодавчі документи мають узгоджувати норми Проведені дослідження показують, що таких передумов у нашій країні ще не сформовано, тому використання індивідуальних ощадних пенсійних рахунків є корисним і дало б можливість громадя нам у такий спосіб заощадити пенсійні кошти. Це є важливим, оскільки для розвитку ринків капіталу та БІЗНЕСІНФОРМ № 11 ’2021 238 БІЗНЕСІНФОРМ № 11 ’2021 www.business-inform.net www.business-inform.net ЛІТЕРАТУРА 1. Поліщук Є. Альтернативні підходи до управління активами небанківських фінансових установ та можливості їх використання в Україні. Ринок цінних паперів України. 2015. № 11–12. С. 77–81. 2. Бабушкін А. І., Свєтлова Г. Р. Формування соціально- економічного інституту недержавного пенсійного забезпечення населення України. Вісник інженерної академії України. 2008. № 3–4. С. 291–295. у у у У зв’язку з цим, для підвищення ефективнос ті функціонування добровільних накопичувальних пенсійних програм, потрібно зменшити витрати на адміністрування послуг, які надаються фінансовими посередниками, підвищити рівень розкриття інфор мації, запровадити регулювання ціноутворюючих паперів і прийняти структурні рішення для всього фінансового ринку. Оптимізація оплати витрат за об слуговування пенсійних активів буде сприяти підви щенню довіри громадян до третього рівня, дозволить зберегти учасникам накопичувальних пенсійних про грам пенсійні накопичення в більших розмірах. При розгляді цього питання заслуговують на увагу висно вки відомого американського інвестора та підприєм ця Джона Богла (J. C. Bogle): «У сфері інвестицій час не загоює рани. Він роз’ятрює їх. Для доходів час – ваш друг, а для витрат – ворог»; «чудова магія склад ного відсотка руйнується свавіллям сукупних витрат на інвестування» [13, с. 41]. 3. Директива 2003/41/ЄС Європейського Парламенту та Ради від 3 червня 2003 р. про діяльність установ трудового пенсійного забезпечення та нагляд над ними. Офіційний вісник ЄС, L 235/10. 23.09.2003. С. 10–21. URL: https://eur-lex.europa.eu/legal-content/ EN/TXT/PDF/?uri=CELEX:32003L0041&from=LV 4. Закон України «Про недержавне пенсійне забезпе чення» від 09.07.2003 № 1057-IV. URL: https://zakon. rada.gov.ua/laws/show/1057-15#Text 5. Недержавне пенсійне забезпечення в Україні: оцін ка та рекомендації. Проект USAID «Трансформація фінансового сектору». Київ, липень 2019 р. 58 с. URL: http://www.fst-ua.info/wp-content/uploads/2019/07/ Voluntary_Private_Pensions_in_Ukraine-Assessment_ jul2019_ua.pdf ЕКОНОМІКА 6. Основні питання реформування пенсійної систе ми в Україні. Проект USAID «Трансформація фінан сового сектору». Київ, серпень 2018 р. 65 c. URL: http://www.fst-ua.info/wp-content/uploads/2019/02/ Key-Issues-in-Pension-System-Reform-in-Ukraine_ Aug2018_UA.pdf 6. Основні питання реформування пенсійної систе ми в Україні. Проект USAID «Трансформація фінан сового сектору». Київ, серпень 2018 р. 65 c. URL: http://www.fst-ua.info/wp-content/uploads/2019/02/ Key-Issues-in-Pension-System-Reform-in-Ukraine_ Aug2018_UA.pdf БІЗНЕСІНФОРМ № 11 ’2021 www.business-inform.net 239 БІЗНЕСІНФОРМ № 11 ’2021 www.business-inform.net 7. Офіційний сайт Пенсійного фонду України. URL: http://www.pfu.gov.ua/ lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX: 32003L0041&from=LV lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX: 32003L0041&from=LV Hrushko, V. I. et al. Pensiina systema [Pension System]. Kyiv, 2019. 8. Пенсійна реформа в України: напрями реалізації : колективна монографія / за ред. Е. М. Лібанової. Київ : Ін-т демографії та соціальних досліджень імені М. В. Птухи НАН України, 2010. 270 с. 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D.	2,020	cc-by	21,912	University of Groningen Improved model-independent determination of the strong-phase difference between D0 and ¯D0 → K0 S,LK+K− decays BESIII Collaboration; Ablikim, M.; Achasov, M.N.; Adlarson, P.; Ahmed, S.; Albrecht, M.; Aliberti, R.; Amoroso, A.; Kalantar-Nayestanaki, Nasser; Kappert, Rosa Published in: Physical Review D University of Groningen Improved model-independent determination of the strong-phase difference between D0 and ¯D0 → K0 S,LK+K− decays BESIII Collaboration; Ablikim, M.; Achasov, M.N.; Adlarson, P.; Ahmed, S.; Albrecht, M.; Aliberti, R.; Amoroso, A.; Kalantar-Nayestanaki, Nasser; Kappert, Rosa Published in: Physical Review D University of Groningen Improved model-independent determination of the strong-phase difference between D0 and ¯D0 → K0 S,LK+K− decays BESIII Collaboration; Ablikim, M.; Achasov, M.N.; Adlarson, P.; Ahmed, S.; Albrecht, M.; Aliberti, R.; Amoroso, A.; Kalantar-Nayestanaki, Nasser; Kappert, Rosa Published in: Physical Review D DOI: 10.1103/PhysRevD.102.052008 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2020 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): BESIII Collaboration, Ablikim, M., Achasov, M. N., Adlarson, P., Ahmed, S., Albrecht, M., Aliberti, R., Amoroso, A., Kalantar-Nayestanaki, N., Kappert, R., Kavatsyuk, M., Messchendorp, J., & Rodin, V. (2020). Improved model-independent determination of the strong-phase difference between D0 and ¯D0 → K0 S,LK+K− decays. Physical Review D, 102(5), Article 052008. https://doi.org/10.1103/PhysRevD.102.052008 DOI: 10.1103/PhysRevD.102.052008 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2020 Publication date: 2020 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): BESIII Collaboration, Ablikim, M., Achasov, M. N., Adlarson, P., Ahmed, S., Albrecht, M., Aliberti, R., Amoroso, A., Kalantar-Nayestanaki, N., Kappert, R., Kavatsyuk, M., Messchendorp, J., & Rodin, V. (2020). Improved model-independent determination of the strong-phase difference between D0 and ¯D0 → K0 S,LK+K− decays. Physical Review D, 102(5), Article 052008. https://doi.org/10.1103/PhysRevD.102.052008 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). 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Physical Review D, 102(5), Article 052008. https://doi.org/10.1103/PhysRevD.102.052008 University of Groningen Improved model-independent determination of the strong-phase difference between D0 and ¯D0 → K0 S,LK+K− decays BESIII Collaboration; Ablikim, M.; Achasov, M.N.; Adlarson, P.; Ahmed, S.; Albrecht, M.; Aliberti, R.; Amoroso, A.; Kalantar-Nayestanaki, Nasser; Kappert, Rosa Published in: Physical Review D Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 Download date: 24-10-2024 PHYSICAL REVIEW D 102, 052008 (2020) Improved model-independent determination of the strong-phase difference between D0 and ¯D0 →K0 S;LK + K −decays g g g g y B. Liu,42,g B. J. Liu,1 C. X. Liu,1 D. Liu,60,48 D. Y. Liu,42,g F. H. Liu,44 Fang Liu,1 Feng Liu,6 H. B. Liu,13 H. M. Liu,1,52 1 17 60 48 1 52 1 34 6 60 48 52 60 48 Huanhuan Liu,1 Huihui Liu,17 J. B. Liu,60,48 J. Y. Liu,1,52 K. Liu,1 K. Y. Liu,34 Ke Liu,6 L. Liu,60,48 Q. Liu,52 S. B. Liu,60,48 Shuai Liu,46 T. Liu,1,52 X. Liu,32 Y. B. Liu,37 Z. A. Liu,1,48,52 Z. Q. Liu,41 Y. F. Long,38,k X. C. Lou,1,48,52 F. X. Lu,16 H. J. Lu,18 J. D. Lu,1,52 J. G. Lu,1,48 X. L. Lu,1 Y. Lu,1 Y. P. Lu,1,48 C. L. Luo,35 M. X. Luo,67 P. W. Luo,49 T. Luo,9,h X. L. Luo,1,48 S. Lusso,63c X. R. Lyu,52 F. C. Ma,34 H. L. Ma,1 L. L. Ma,41 M. M. Ma,1,52 Q. M. Ma,1 R. Q. Ma,1,52 Huanhuan Liu, Huihui Liu, J. B. Liu, J. Y. Liu, K. Liu, K. Y. Liu, Ke Liu, L. Liu, Q. Liu, S. B. Liu, Shuai Liu,46 T. Liu,1,52 X. Liu,32 Y. B. Liu,37 Z. A. Liu,1,48,52 Z. Q. Liu,41 Y. F. Long,38,k X. C. Lou,1,48,52 F. X. Lu,16 H. J. Lu,18 J. D. Lu,1,52 J. G. Lu,1,48 X. L. Lu,1 Y. Lu,1 Y. P. Lu,1,48 C. L. Luo,35 M. X. Luo,67 P. W. Luo,49 T. Luo,9,h X L L 1 48 S L 63c X R L 52 F C M 34 H L M 1 L L M 41 M M M 1 52 Q M M 1 R Q M 1 52 Q , g , , , , g, p, G. Mezzadri,24a T. J. Min,36 R. E. Mitchell,22 X. H. Mo,1,48,52 Y. J. Mo,6 N. Yu. Muchnoi,10,c H. Muramatsu,56 S. Nakhoul,11,f Y. Nefedov,29 F. Nerling,11,f I. B. Nikolaev,10,c Z. Ning,1,48 S. Nisar,8,i S. L. Olsen,52 Q. Ouyang,1,48,52 S. Pacetti,23b,23c X. Pan,9,h Y. Pan,55 A. Pathak,1 P. Patteri,23a M. Pelizaeus,4 H. P. Peng,60,48 K. Peters,11,f J. Pettersson,64 35 1 52 4 56 60 48 60 48 50 36 9 2 2470-0010=2020=102(5)=052008(27) 052008-1 PHYS. REV. D 102, 052008 (2020) Budker Institute of Nuclear Physics SB RAS (BINP), Novosibirsk 630090, Russia 11GSI Helmholtzcentre for Heavy Ion Research GmbH, D-64291 Darmstadt, Germany 12Guangxi Normal University, Guilin 541004, People’s Republic of China 13Guangxi University, Nanning 530004, People’s Republic of China 14Hangzhou Normal University, Hangzhou 310036, People’s Republic of China 15Helmholtz Institute Mainz, Johann-Joachim-Becher-Weg 45, D-55099 Mainz, Germany 16Henan Normal University, Xinxiang 453007, People’s Republic of China 17Henan University of Science and Technology, Luoyang 471003, People’s Republic of China 18Huangshan College, Huangshan 245000, People’s Republic of China 19Hunan Normal University, Changsha 410081, People’s Republic of China 20Hunan University, Changsha 410082, People’s Republic of China 21Indian Institute of Technology Madras, Chennai 600036, India 22Indiana University, Bloomington, Indiana 47405, USA 23aINFN Laboratori Nazionali di Frascati, I-00044, Frascati, Italy 23bINFN Sezione di Perugia, I-06100, Perugia, Italy 23cUniversity of Perugia, I-06100, Perugia, Italy 24aINFN Sezione di Ferrara, I-44122, Ferrara, Italy 24bUniversity of Ferrara, I-44122, Ferrara, Italy 25Institute of Modern Physics, Lanzhou 730000, People’s Republic of China 26Institute of Physics and Technology, Peace Ave. 54B, Ulaanbaatar 13330, Mongolia 27Jilin University, Changchun 130012, People’s Republic of China 28Johannes Gutenberg University of Mainz, Johann-Joachim-Becher-Weg 45, D-55099 Mainz, Germany 29Joint Institute for Nuclear Research, 141980 Dubna, Moscow region, Russia 30Justus-Liebig-Universitaet Giessen, II. Physikalisches Institut, Heinrich-Buff-Ring 16, D-35392 Giessen, Germany 31KVI-CART, University of Groningen, NL-9747 AA Groningen, Netherlands 32Lanzhou University, Lanzhou 730000, People’s Republic of China 33Liaoning Normal University, Dalian 116029, People’s Republic of China 34Liaoning University, Shenyang 110036, People’s Republic of China 35Nanjing Normal University, Nanjing 210023, People’s Republic of China 36Nanjing University, Nanjing 210093, People’s Republic of China 37Nankai University, Tianjin 300071, People’s Republic of China 38Peking University, Beijing 100871, People’s Republic of China 39Qufu Normal University, Qufu 273165, People’s Republic of China 40Shandong Normal University, Jinan 250014, People’s Republic of China 41Shandong University, Jinan 250100, People’s Republic of China 42Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China 43Shanxi Normal University, Linfen 041004, People’s Republic of China 44Shanxi University, Taiyuan 030006, People’s Republic of China D-35392 Giessen, Germany , y 31KVI-CART, University of Groningen, NL-9747 AA Groningen, Netherlands 32Lanzhou University, Lanzhou 730000, People’s Republic of China 33Liaoning Normal University, Dalian 116029, People’s Republic of China 34Liaoning University, Shenyang 110036, People’s Republic of China 35Nanjing Normal University, Nanjing 210023, People’s Republic of China 36Nanjing University, Nanjing 210093, People’s Republic of China 37Nankai University, Tianjin 300071, People’s Republic of China 38Peking University, Beijing 100871, People’s Republic of China 39Qufu Normal University, Qufu 273165, People’s Republic of China 40Shandong Normal University, Jinan 250014, People’s Republic of China 41Shandong University, Jinan 250100, People’s Republic of China 42Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China 43Shanxi Normal University, Linfen 041004, People’s Republic of China 44Shanxi University, Taiyuan 030006, People’s Republic of China 052008-1 Published by the American Physical Society (BESIII Collaboration) 1Institute of High Energy Physics, Beijing 100049, People’s Republic of China 2Beihang University, Beijing 100191, People’s Republic of China 3Beijing Institute of Petrochemical Technology, Beijing 102617, People’s Republic of China 4Bochum Ruhr-University, D-44780 Bochum, Germany 5Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA 6Central China Normal University, Wuhan 430079, People’s Republic of China 7China Center of Advanced Science and Technology, Beijing 100190, People’s Republic of China 8COMSATS University Islamabad, Lahore Campus, Defence Road, Off Raiwind Road, 54000 Lahore, Pakistan 9Fudan University, Shanghai 200443, People’s Republic of China 10G.I. Budker Institute of Nuclear Physics SB RAS (BINP), Novosibirsk 630090, Russia 11GSI Helmholtzcentre for Heavy Ion Research GmbH, D-64291 Darmstadt, Germany 12Guangxi Normal University, Guilin 541004, People’s Republic of China 13Guangxi University, Nanning 530004, People’s Republic of China 14Hangzhou Normal University, Hangzhou 310036, People’s Republic of China 15Helmholtz Institute Mainz, Johann-Joachim-Becher-Weg 45, D-55099 Mainz, Germany 16Henan Normal University, Xinxiang 453007, People’s Republic of China 17Henan University of Science and Technology, Luoyang 471003, People’s Republic of China 18Huangshan College, Huangshan 245000, People’s Republic of China 19Hunan Normal University, Changsha 410081, People’s Republic of China 20Hunan University, Changsha 410082, People’s Republic of China 21Indian Institute of Technology Madras, Chennai 600036, India 22Indiana University, Bloomington, Indiana 47405, USA 23aINFN Laboratori Nazionali di Frascati, I-00044, Frascati, Italy 23bINFN Sezione di Perugia, I-06100, Perugia, Italy 23cUniversity of Perugia, I-06100, Perugia, Italy 24aINFN Sezione di Ferrara, I-44122, Ferrara, Italy 24bUniversity of Ferrara, I-44122, Ferrara, Italy 25Institute of Modern Physics, Lanzhou 730000, People’s Republic of China 26Institute of Physics and Technology, Peace Ave. 54B, Ulaanbaatar 13330, Mongolia 27Jilin University, Changchun 130012, People’s Republic of China 28Johannes Gutenberg University of Mainz, Johann-Joachim-Becher-Weg 45, D-55099 Mainz, Germany 29Joint Institute for Nuclear Research, 141980 Dubna, Moscow region, Russia 30Justus-Liebig-Universitaet Giessen, II. (BESIII Collaboration) Physikalisches Institut, Heinrich-Buff-Ring 16, D-35392 Giessen, Germany 31KVI-CART, University of Groningen, NL-9747 AA Groningen, Netherlands 32Lanzhou University, Lanzhou 730000, People’s Republic of China 33Liaoning Normal University, Dalian 116029, People’s Republic of China 34Liaoning University, Shenyang 110036, People’s Republic of China 35Nanjing Normal University, Nanjing 210023, People’s Republic of China 36Nanjing University, Nanjing 210093, People’s Republic of China 37Nankai University, Tianjin 300071, People’s Republic of China 38Peking University, Beijing 100871, People’s Republic of China 39Qufu Normal University, Qufu 273165, People’s Republic of China 40Shandong Normal University, Jinan 250014, People’s Republic of China 41Shandong University, Jinan 250100, People’s Republic of China 42Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China 43Shanxi Normal University, Linfen 041004, People’s Republic of China 44Shanxi University, Taiyuan 030006, People’s Republic of China Institute of High Energy Physics, Beijing 100049, People’s Republic of China 2 PHYS. 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Zou1 (BESIII Collaboration) 1Institute of High Energy Physics, Beijing 100049, People’s Republic of China 2Beihang University, Beijing 100191, People’s Republic of China 3Beijing Institute of Petrochemical Technology, Beijing 102617, People’s Republic of China 4Bochum Ruhr-University, D-44780 Bochum, Germany 5Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA 6Central China Normal University, Wuhan 430079, People’s Republic of China 7China Center of Advanced Science and Technology, Beijing 100190, People’s Republic of China 8COMSATS University Islamabad, Lahore Campus, Defence Road, Off Raiwind Road, 54000 Lahore, Pakistan 9Fudan University, Shanghai 200443, People’s Republic of China 10G.I. 052008-2 PHYS. REV. D 102, 052008 (2020) Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. g , p p iAlso at Harvard University, Department of Physics, Cambridge, Massachusetts 02138, USA. jCurrently at: Institute of Physics and Technology Peace Ave 54B Ulaanbaatar 13330 Mongo cAlso at the Novosibirsk State University, Novosibirsk, 630090, Russia. d g p p iAlso at Harvard University, Department of Physics, Cambridge, Massachusetts 02138, USA. jCurrently at: Institute of Physics and Technology, Peace Ave.54B, Ulaanbaatar 13330, Mongolia. k y y gy g kAlso at State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871, P lSchool of Physics and Electronics, Hunan University, Changsha 410082, China. DOI: 10.1103/PhysRevD.102.052008 dAlso at the NRC “Kurchatov Institute”, PNPI, 188300, Gatchina, Russia. IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … 45Sichuan University, Chengdu 610064, People’s Republic of China 46Soochow University, Suzhou 215006, People’s Republic of China 47Southeast University, Nanjing 211100, People’s Republic of China 48State Key Laboratory of Particle Detection and Electronics, Beijing 100049, Hefei 230026, People’s Republic of China 49Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China 50Tsinghua University, Beijing 100084, People’s Republic of China 51aAnkara University, 06100 Tandogan, Ankara, Turkey 51bIstanbul Bilgi University, 34060 Eyup, Istanbul, Turkey 51cUludag University, 16059 Bursa, Turkey 51dNear East University, Nicosia, North Cyprus, Mersin 10, Turkey 52University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China 53University of Hawaii, Honolulu, Hawaii 96822, USA 54University of Jinan, Jinan 250022, People’s Republic of China 55University of Manchester, Oxford Road, Manchester, M13 9PL, United Kingdom 56University of Minnesota, Minneapolis, Minnesota 55455, USA 57University of Muenster, Wilhelm-Klemm-Str. 9, 48149 Muenster, Germany 58University of Oxford, Keble Rd, Oxford OX13RH, United Kingdom 59University of Science and Technology Liaoning, Anshan 114051, People’s Republic of China 60University of Science and Technology of China, Hefei 230026, People’s Republic of China 61University of South China, Hengyang 421001, People’s Republic of China 62University of the Punjab, Lahore-54590, Pakistan 63aUniversity of Turin, I-10125, Turin, Italy 63bUniversity of Eastern Piedmont, I-15121, Alessandria, Italy 63cINFN, I-10125, Turin, Italy 64Uppsala University, Box 516, SE-75120 Uppsala, Sweden 65Wuhan University, Wuhan 430072, People’s Republic of China 66Xinyang Normal University, Xinyang 464000, People’s Republic of China 67Zhejiang University, Hangzhou 310027, People’s Republic of China 68Zhengzhou University, Zhengzhou 450001, People’s Republic of China University of the Punjab, Lahore-54590, Pakistan 63aUniversity of Turin, I-10125, Turin, Italy y f y 63bUniversity of Eastern Piedmont, I-15121, Alessandria, Italy 66Xinyang Normal University, Xinyang 464000, People’s Republic of China 67 67Zhejiang University, Hangzhou 310027, People’s Republic of China 68 68Zhengzhou University, Zhengzhou 450001, People’s Republic of China We present a measurement of the strong-phase difference between D0 and ¯D0 →K0 S;LKþK−decays, performed through a study of quantum-entangled pairs of charm mesons. The measurement exploits a data sample equivalent to an integrated luminosity of 2.93 fb−1, collected by the BESIII detector in eþe− collisions corresponding to the mass of the ψð3770Þ resonance. The strong-phase difference is an essential input to the determination of the Cabibbo-Kobayashi-Maskawa (CKM) angle γ=ϕ3 through the decay B−→DK−, where D can be either a D0 or a ¯D0 decaying to K0 S;LKþK−. This is the most precise measurement to date of the strong-phase difference in these decays. aAlso at Bogazici University, 34342 Istanbul, Turkey. y gy; y , g , p p hAlso at Key Laboratory of Nuclear Physics and Ion-beam Application (MOE) and Institute of Modern Physics, Fudan University, Shanghai 200443, People’s Republic of China. i aAlso at Bogazici University, 34342 Istanbul, Turkey. bAlso at the Moscow Institute of Physics and Technology, Moscow 141700, Russia. cAlso at the Novosibirsk State University, Novosibirsk, 630090, Russia. dAlso at the NRC “Kurchatov Institute”, PNPI, 188300, Gatchina, Russia. eAlso at Istanbul Arel University, 34295 Istanbul, Turkey. fAlso at Goethe University Frankfurt, 60323 Frankfurt am Main, Germany. gAlso at Key Laboratory for Particle Physics, Astrophysics and Cosmology, Ministry of Education; Shanghai Key L Particle Physics and Cosmology; Institute of Nuclear and Particle Physics, Shanghai 200240, People’s Republic of Ch hAlso at Key Laboratory of Nuclear Physics and Ion-beam Application (MOE) and Institute of Modern Physics, Fuda Shanghai 200443, People’s Republic of China. iAlso at Harvard University, Department of Physics, Cambridge, Massachusetts 02138, USA. jCurrently at: Institute of Physics and Technology, Peace Ave.54B, Ulaanbaatar 13330, Mongolia. kAlso at State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871, People’s Repub lSchool of Physics and Electronics, Hunan University, Changsha 410082, China. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International lice distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and by SCOAP3. eAlso at Istanbul Arel University, 34295 Istanbul, Turkey. f I. INTRODUCTION luminosity of 818 pb−1 that was collected at a center-of- mass energy corresponding to the mass of the ψð3770Þ resonance [14]. In this paper, we present an improved measurement of the strong-phase parameters for D → K0 S;LKþK−decays, using a ψð3770Þ data sample corre- sponding to an integrated luminosity of 2.93 fb−1 recorded by the BESIII detector. This measurement can be used as an input to the model-independent measurement of γ using the BPGGSZ method. Moreover, these strong-phase parame- ters serve as an essential input to the model-independent determination of charm-mixing parameters and in probing CP violation with D →K0 S;LKþK−decays [15]. In the Standard Model (SM), the charged-weak inter- action in the quark sector is described by the Cabibbo- Kobayashi-Maskawa matrix (CKM) [1]. One of the pri- mary goals of flavor physics experiments is to determine the angles α, β and γ (or ϕ2; ϕ1 and ϕ3) of the b −d CKM unitary triangle precisely. Currently, the most precise measurements of γ are extracted using tree-level B−→ DK−decays [2]. Here and elsewhere in this paper D refers to either a D0 or a ¯D0 meson decaying into the same final state and charge conjugation is implicit, unless stated otherwise. The sensitivity to γ arises from the interference of two amplitudes: b →c¯us that results in the B−→D0K− decay, and b →u¯cs that leads to the B−→¯D0K−decay. The latter amplitude is both CKM- and color-suppressed relative to the former. The value of γ measured with such tree-level transitions is insensitive to loop-level contribu- tions [3]. Therefore, tests for new physics that are made by comparing unitarity triangle parameters measured using tree and loop processes can be improved by more precise determinations of γ [4,5]. ; The D →K0 SKþK−decay proceeds via various inter- mediate resonances, which leads to a significant strong- phase variation over the phase space. We define the kinematic variables m2 ¼ ðPK0 S þ PKÞ2, which serve as the basis of the D →K0 SKþK−Dalitz plot. Here, Piði ¼ K0 S; Kþ; K−Þ is the four-momentum of the D decay product. The amplitude for B−→DðK0 SKþK−ÞK−at ðm2 þ; m2−Þ can be written as Different methods of determining γ are classified based upon the decay products of the D decay: CP eigenstates (GLW method) [2], flavor-eigenstates (ADS method) [6], and self-conjugate multibody states (BPGGSZ method) [7–9]. IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … DOI: 10.1103/PhysRevD.102.052008 g y, , y bAlso at the Moscow Institute of Physics and Technology, Moscow 141700, Russia. y gy cAlso at the Novosibirsk State University, Novosibirsk, 630090, Russia. d y dAlso at the NRC “Kurchatov Institute”, PNPI, 188300, Gatchina, Russia. kAlso at State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871, People’s Republic of China. lSchool of Physics and Electronics, Hunan University, Changsha 410082, China. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. 052008-3 PHYS. REV. D 102, 052008 (2020) M. ABLIKIM et al. I. INTRODUCTION The most widely used D decays for the BPGGSZ method are D →K0 Shþh−, where h ¼ π, K. Measurements of γ using these final states have been performed by the Belle, BABAR and LHCb Collabo- rations [9–11]. Recently the first constraints on γ using the BPGGSZ method with a four-body D decay were reported [12]. BPGGSZ analyses require an understanding of the interference effects between D0 and ¯D0 decays, especially concerning the strong-phase difference between the D0 and ¯D0 decay amplitudes. fB−ðm2 þ; m2−Þ ∝fDðm2 þ; m2−Þ þ rBeiðδB−γÞf ¯Dðm2 þ; m2−Þ; ð1Þ ð1Þ ð1Þ where rB is the ratio of the magnitude of the suppressed to the favored B-decay amplitude, δB is the CP-conserving strong-phase difference between favored and suppressed B-decay amplitudes, γ is the weak-phase difference between the B decay amplitudes, and fDðm2 þ; m2−Þ ðf ¯Dðm2 þ; m2−ÞÞ is the amplitude of the D0 →K0 SKþK−ð ¯D0 →K0 SKþK−Þ decay. We neglect CP violation in D decays as in Ref. [8], and can thus use the relation f ¯Dðm2 þ; m2−Þ ¼ fDðm2−; m2 þÞ so that Eq. (1) can be written as A precise measurement of the strong-phase difference in D →K0 S;Lπþπ−decays was reported by the BESIII Collaboration recently [13]. The first measurements of the strong-phase difference between D0 and ¯D0 decaying to the K0 S;LKþK− final state were reported by the CLEO Collaboration, using a data set equivalent to an integrated fB−ðm2 þ; m2−Þ ∝fDðm2 þ; m2−Þ þ rBeiðδB−γÞfDðm2−; m2 þÞ: fB−ðm2 þ; m2−Þ ∝fDðm2 þ; m2−Þ þ rBeiðδB−γÞfDðm2−; m2 þÞ: ð2Þ fB−ðm2 þ; m2−Þ ∝fDðm2 þ; m2−Þ þ rBeiðδB−γÞfDðm2−; m2 þÞ: ð2Þ Therefore, the decay rate of a B−meson is m2 þÞj2 þ 2rBjfDðm2 þ; m2−ÞjjfDðm2−; m2 þÞj cos ðΔδD þ δB −γÞ; ð3Þ ΓB−ðm2 þ; m2−Þ ∝jfDðm2 þ; m2−Þj2 þ r2 BjfDðm2−; m2 þÞj2 þ 2rBjfDðm2 þ; m2−ÞjjfDðm2−; m2 þÞj cos ðΔδD þ δB −γÞ; ð3Þ ð3Þ D →D0ðK0 SKþK−Þπ decay, which is fit to an ampli- tude model describing the decay of D0 →K0 SKþK−[16] to determine fDðm2 þ; m2−Þ. The amplitude model is then used in an unbinned likelihood fit to the B-meson data sample to determine γ, δB, and rB. However, this method results in a model-dependent systematic uncertainty on the measured value of γ which is difficult to quantify [17]. I. INTRODUCTION These model- dependent uncertainties have been estimated to lie between D →D0ðK0 SKþK−Þπ decay, which is fit to an ampli- tude model describing the decay of D0 →K0 SKþK−[16] to determine fDðm2 þ; m2−Þ. The amplitude model is then used in an unbinned likelihood fit to the B-meson data sample to determine γ, δB, and rB. However, this method results in a model-dependent systematic uncertainty on the measured value of γ which is difficult to quantify [17]. These model- dependent uncertainties have been estimated to lie between where ΔδD ≡δDðm2 þ;m2−Þ −δDðm2−;m2 þÞ, and δDðm2 þ; m2−Þ is the strong phase of fDðm2 þ; m2−Þ. Hence, knowledge of ΔδD is essential for the determination of γ in B−→DK− decays. In the literature, both model-dependent and model- independent BPGGSZ methods are used. In the model- dependent approach, the D0 amplitude is obtained using a flavor-tagged D0 meson sample selected from the 052008-4 PHYS. REV. D 102, 052008 (2020) IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … PHYS. RE Ki ¼ aD Z i jfDðm2 þ; m2−Þj2dm2 þdm2−¼ aDFi; ð4Þ 3° to 9° [18,19], which limits the precision on γ that future measurements performed with much larger B-meson data samples [20,21] can obtain. where aD is a normalization factor equal to the total number of D0 →K0 SKþK−decays in the flavor-tagged charm sample, Fi is the fraction of D0 →K0 SKþK−decays in the ith bin, and the integral is over the ðm2 þ; m2−Þ region defined by the ith bin. Here and elsewhere the values of Kð−Þi are corrected for efficiency and also for the presence of any doubly Cabibbo-suppressed (DCS) component (see Sec. VII A). We assume fDðm2 þ; m2−Þ has been normalized such that p An alternative method of measuring γ is in a model- independent manner that relies on defining a number of bins in the D0 →K0 SKþK−Dalitz plot [8]. This approach determines γ from the measured rate in each bin of the Dalitz plot, rather than fitting the Dalitz plot distribution to an amplitude model. The method requires information about ΔδDðm2 þ; m2−Þ in each bin, which is accessible at the ψð3770Þ resonance by exploiting the quantum coherence of the D0 ¯D0 pair produced in ψð3770Þ decays. The advantage of this method is that the hard-to-quantify systematic uncertainty related to the model assumption is replaced by the uncertainty on the binned strong-phase parameters of the D decay mode. I. INTRODUCTION These strong-phase parameter uncertainties are statistically dominated, and thus well understood. The major disadvantage of the model-independent method is the inevitable loss of information that arises from binning, which reduces the statistical sensitivity of the γ measurement by approximately 20% compared to the model-dependent method [14]. Z jfDðm2 þ; m2−Þj2dm2 þdm2−¼ 1; ð5Þ ð5Þ where the integral is over the whole Dalitz plot. For each bin the interference between D0 and ¯D0 decays can be parametrized by two variables ci and si, which are the amplitude-weighted averages of cos ΔδD and sin ΔδD, defined as: ci ≡ 1 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ FiF−i p Z i jfDðm2 þ; m2−ÞjjfDðm2−; m2 þÞj × cos½ΔδDðm2 þ; m2−Þdm2 þdm2−; ð6Þ The remainder of this paper is structured as follows. In Sec. II we define the formalism used to measure the strong-phase parameters with ψð3770Þ data. We explain the Dalitz-plot binning in Sec. III. In Sec. IV we outline the features of the BESIII detector and the simulation techniques used in the analysis. We describe the event- selection criteria and the procedure for estimating the data yields in Secs. V and VI, respectively. In Sec. VII we explain the procedure for estimating the bin yields, including the various corrections applied. We describe the extraction of strong-phase parameters and the calcu- lation of systematic uncertainties in Secs. VIII and IX, respectively. We present a discussion on the impact of these results on γ in Sec. X. In Sec. XI we give the conclusion and outlook. ð6Þ and si ≡ 1 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ FiF−i p Z i jfDðm2 þ; m2−ÞjjfDðm2−; m2 þÞj × sin½ΔδDðm2 þ; m2−Þdm2 þdm2−: ð7Þ ð7Þ From Eqs. (6) and (7) it is evident that ci ¼ c−i; si ¼ −s−i and c2 i þ s2 i ≤1. The condition c2 i þ s2 i ¼ 1 is satisfied only if fD is constant throughout the bin. Thus the yield of B decays in the ith bin, Ni, is obtained by integrating Eq. (3), which results in N∓ i ∝Ki þ r2 BK∓i þ 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ KiK−i p ðxB∓· ci þ yB∓· siÞ; ð8Þ ð8Þ II. FORMALISM The model-independent method [8] for a three-body D decay is implemented as follows. The entire Dalitz plot is divided into 2N bins, with N bins symmetrically placed on either side of the m2 þ ¼ m2−line. We follow the convention in which bins with m2 þ ≥m2−are labelled with i and bins with m2 þ < m2−are labelled with −i. Thus, the 2N bins are assigned labels from −N to N excluding zero. The interchange of the Dalitz plot variables m2 þ ↔m2−corre- sponds to the interchange of positions of the bins i ↔−i. In order to extract the strong-phase difference parameters, we need to determine the yield in each bin for flavor-, CP- and mixed-CP tagged D →K0 SKþK−decays. The number of flavor-tagged D0 →K0 SKþK−decays Ki in the ith bin of the Dalitz plot is defined as where xB∓≡rB cosðδB ∓γÞ, yB∓≡rB sinðδB ∓γÞ and r2 B ¼ x2 B∓þ y2 B∓. A maximum likelihood fit to binned B−→DK−decay yields, using Eq. (8) as a probability density function with externally measured values of ci and si as inputs, then allows γ to be determined along with rB and δB. We now describe how ψð3770Þ data are used to determine the values of ci and si. The D0 ¯D0 pair from the decay of the ψð3770Þ (or if directly produced from the virtual photon in an eþe−annihilation) is in a C-odd eigenstate, as long as there are no additional particles in the final state. This quantum correlation between the mesons leads to the total D0 ¯D0 decay rate being sensitive to the strong-phase difference between the D0 and ¯D0 052008-5 PHYS. REV. D 102, 052008 (2020) M. ABLIKIM et al. amplitudes. For example, the decay of one D to a CP-even eigenstate fixes the other D to the CP-odd admixture of ðD0 −¯D0Þ= ﬃﬃﬃ 2 p . Hence, if the other D decays to KS;LKþK−, the total rate will be sensitive to the interference between the D0 and ¯D0 amplitudes and the strong-phase parameters. Generally, this interference affects the decays of one D in combination with the other. If only one D meson is reconstructed, leaving the companion D meson to decay to any final state, the decay rate is largely insensitive to the effects of quantum correlations; we refer to the reconstructed samples of such events as single-tag (ST) decays. II. FORMALISM If both D mesons are required to be in specific final states, the rates can be significantly enhanced or suppressed in the quantum- correlated events compared to the expected rate if the decays are uncorrelated; we refer to the reconstructed samples of such events as double-tag (DT) decays. Hereafter, all the D decay final states, except the signal mode K0 S;LKþK−, are referred to as “tags.” where þð−Þ indicates a CP-even (CP-odd) state. Therefore, the expected number of events hM i i in the ith bin of a sample that has been tagged with a decay that has a CP-even fraction Fþ is hM i i ¼ ϵDT;i S 2Sf ðKi −2cið2Fþ −1Þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ KiK−i p þ K−iÞ; f ð10Þ ð10Þ where S ðSfÞ are the efficiency-corrected single-tag yields of the CP-eigenstate (flavor) modes used in the analysis and ϵDT;i is the DT efficiency in the ith bin. The value of Fþ is equal to 1 (0) for a pure CP-even (CP-odd) tag mode. We refer to modes with intermediate values of Fþ as quasi-CP tags. The values of ci alone can be extracted using Eq. (10). The relation si ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −c2 i p is a good approximation only for N > 200 [22], which is not feasible with the available data sample. However, analysing D →K0 SKþK−decays tagged by D →K0 Shþh−(h ¼ π, K) decays gives access to both ci and si. The amplitude of the D0 ¯D0 pair produced by the ψð3770Þ decaying to K0 SKþK−and K0 Shþh−is where S ðSfÞ are the efficiency-corrected single-tag yields of the CP-eigenstate (flavor) modes used in the analysis and ϵDT;i is the DT efficiency in the ith bin. The value of Fþ is equal to 1 (0) for a pure CP-even (CP-odd) tag mode. We refer to modes with intermediate values of Fþ as quasi-CP tags. The values of ci alone can be extracted using Eq. (10). The relation si ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −c2 i p is a good approximation only for N > 200 [22], which is not feasible with the available data sample. However, analysing D →K0 SKþK−decays tagged by D →K0 Shþh−(h ¼ π, K) decays gives access to both ci and si. II. FORMALISM The amplitude of the D0 ¯D0 pair produced by the ψð3770Þ decaying to K0 SKþK−and K0 Shþh−is The D →K0 SKþK−decay amplitude from a CP eigen- state is fðm2 þ; m2−Þ ¼ 1ﬃﬃﬃ 2 p ½fDðm2 þ; m2−Þ fDðm2−; m2 þÞ; ð9Þ ð11Þ we expect a significant fraction of the D mesons to decay to the K0 LKþK−final state as well. Although so far γ has only been determined using D →K0 Shþh−decays, the decay D →K0 LKþK−has a close connection with D →K0 SKþK− that can be exploited to improve the precision with which ci and si are determined. In the absence of CP violation, CPjK0 Si ¼ jK0 Si and CPjK0 Li ¼ −jK0 Li. Hence K0 LKþK− has opposite CP to K0 SKþK−. We define the decay amplitude for D0 →K0 LKþK− [ ¯D0 →K0 LKþK−] as f0 Dðm2 þ; m2−Þ [f0 ¯Dðm2 þ; m2−Þ] such that where ð ¯m2 þ; ¯m2−Þ are the Dalitz plot coordinates corres- ponding to the phase space of the K0 Shþh−decay. The expected event rate in which one D decays in the region of phase space defined by the ith bin of the D →K0 SKþK− Dalitz plot and the other D in the region of phase space defined by the jth bin of the D →K0 Shþh−Dalitz plot can be written as hMiji ¼ ϵDT;ij ND0 ¯D0 2S2 f ðKiK−j þ K−iKj −2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ KiK−jK−iKj p ðcicj þ sisjÞÞ; ð12Þ hMiji ¼ ϵDT;ij ND0 ¯D0 2S2 f ðKiK−j þ K−iKj ð12Þ f0 ¯Dðm2 þ; m2−Þ ¼ −f0 Dðm2−; m2 þÞ: ð13Þ ð13Þ where ND0 ¯D0 is the number of D0 ¯D0 pairs in the ψð3770Þ data sample and ϵDT;ij is the DT efficiency in the ith and jth pair of bins. The two-fold ambiguity in the sign of si can be resolved using weak amplitude-model assumptions. Note that Eq. (10) is symmetric under the interchange of i ↔−i and Eq. (12) is symmetric under the interchange of pair, ði;jÞ↔ð−i;−jÞ and ði; −jÞ ↔ð−i; jÞ. No such sym- metry exists for the values of Ki because fDðm2 þ; m2−Þ ≠ fDðm2−; m2 þÞ. III. BINNING OF THE D0 →K0 SK + K − DALITZ PLOT All the relations given in Sec. II are independent of the shape of the Dalitz plot bins. The original proposal [8] was to divide the Dalitz plot into rectilinear bins. The reduction in sensitivity of such an approach compared to an unbinned analysis is about 30% even with 20 bins [22]. The sensitivity of the model-independent method as a function of the bin shape is discussed in Ref. [22]; this paper concludes that binning schemes that minimize the varia- tions of ΔδD within each Dalitz plot bin give significantly improved statistical sensitivity compared to the rectilinear binning. An amplitude model can be used to guide the definition of bin boundaries in order to minimize the ΔδD variation. The number of bins that can be used in the analysis is restricted by the available statistics in either the ψð3770Þ or B-decay data samples. Since the amplitude model is used only to define the bin shapes, the model neither leads to any bias nor introduces any model- dependent uncertainties on the measurement of γ. However, a model that poorly describes the phase variation of the amplitude over the Dalitz plot may lead to a lower than expected statistical sensitivity to γ. 2πði −3=2Þ=N ≤ΔδDðmþ; m−Þ < 2πði −1=2Þ=N ; ð16Þ ð16Þ as shown in Fig. 1 for N ¼ 2, 3 and 4. Here i ¼ 1; 2; …; N are the bin numbers. The bins in the region m2 þ > m2−are defined symmetrically. The class of binning defined by Eq. (16) is referred to as the “equal-ΔδD” binning scheme. A smaller number of bins is the best choice to measure ci and si precisely, but this will potentially reduce the sensitivity to γ. On the other hand, a larger number of bins provides increased sensitivity to γ, because it is a better approximation to the unbinned method. Keeping this trade- off in mind, we perform the analysis with N ¼ 2, 3 and 4 bins. Binning the Dalitz plot with N > 4 is not yet feasible with the size of the sample of ψð3770Þ data collected by BESIII; the fit to determine ci and si described in Sec. VIII fails with N > 4. FIG. 1. Equal-ΔδD binning of D0 →K0 SKþK−phase-space based on the BABAR model [16] for N ¼ 2 (left), N ¼ 3 (middle) and N ¼ 4 (right) bins. IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … PHYS. REV. D 102, 052008 (2020) hM0 iji ¼ ϵ0 DT;ij ND0 ¯D0 2SfS0 f KiK0 −j þ K−iK0 j þ 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ KiK0 −jK−iK0 j q ðc0 icj þ s0 isjÞ : ð15Þ In the current analysis we employ an amplitude model for D0 →K0 SKþK−decays developed by the BABAR Collaboration [16] to define the bin shapes. Our choice of model and bin definitions is consistent with the previous measurement [14]. The amplitude model is constructed in the isobar formalism, where the amplitude at a phase-space point is defined as a coherent sum of two-body amplitudes and a nonresonant amplitude. There are eight intermediate resonances used in the model. The a0ð980Þ0 and a0ð980Þ∓ resonances are modeled by the Flatt´e parametrization [23], while all other resonances are parameterized by Breit- Wigner line shapes. The model-based lookup table (LUT) containing the moduli and phases of the D0 →K0 SKþK− amplitudes at different phase points (m2−; M2 KþK−) was supplied by the authors of Ref. [16]. The granularity of the (m2−; M2 KþK−) grid in the LUT is 0.00179 GeV2=c4× 0.00536 GeV2=c4. Based on the LUT, the values of ΔδD at a position (m2 þ; m2−) in the phase-space are calculated. Half of the Dalitz plot, m2 þ < m2−, is divided into equally spaced regions (bins) of ΔδD satisfying the condition ð15Þ The symmetries between the exchange of coordinates in the cases of Mi and Mij are also present for M0 i and M0 ij. In general ðci; siÞ ≠ðc0 i; s0 iÞ because f0 Dðm2 þ; m2−Þ ≠ fDðm2 þ; m2−Þ. In order to improve the precision of the extracted values of ci and si constraints are imposed on the difference Δci ¼ c0 i −ci and Δsi ¼ s0 i −si; these con- straints are explained in Sec. VIII. II. FORMALISM Therefore, the number of events in the ith bin of a CP- or quasi-CP tagged D →K0 LKþK−sample is hM0 i i ¼ ϵ0 DT;i S∓ 2S0 f ðK0 i þ 2c0 ið2Fþ −1Þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ K0 iK0 −i p þ K0 −iÞ; ð14Þ hM0 i i ¼ ϵ0 DT;i S∓ 2S0 f ðK0 i þ 2c0 ið2Fþ −1Þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ K0 iK0 −i p þ K0 −iÞ; ð14Þ ð14Þ where K0 i is defined in analogous fashion to Ki [see Eq. (4)]. Furthermore, the expected event rate in the ith bin of the D →K0 LKþK−Dalitz plot and the jth bin of the D →K0 Shþh−Dalitz plot can be written as Ignoring the very low level of CP violation in the neutral kaon system, the K0 state is an equal admixture of K0 S and K0 L states. Therefore, in the decays of correlated D0 ¯D0 pairs 052008-6 V. EVENT SELECTION We analyze an eþe−collision data sample produced by the Beijing Electron Positron Collider II (BEPCII), which corresponds to an integrated luminosity of 2.93 fb−1 [25], collected by the BESIII detector at a center-of-mass energy of ﬃﬃﬃs p ¼ 3.773 GeV. The BESIII experiment is a general purpose solenoidal detector with a geometrical acceptance of 93% of the 4π solid angle. It has a He-gas-based multilayer drift chamber (MDC) for measuring the momen- tum and specific ionization loss (dE=dx) of the charged particles, a plastic-scintillator-based time-of-flight (TOF) measurement system for the identification of charged particles, and an electromagnetic calorimeter (EMC) con- sisting of CsI(Tl) crystal, which is used to measure the energy of the neutral showers and identify electrons. The detector is encapsulated in a magnetic field of 1 T provided by a superconducting solenoid. A resistive-plate-chamber- based muon counter is interleaved between the flux-return yoke of the magnet. The MDC has a transverse-momentum resolution of 0.5% at 1 GeV=c. The time resolution of the TOF is about 80 ps in the barrel region and 110 ps in the endcap region, enabling a 2σ K=π separation up to a momentum of 1 GeV=c. The energy resolution of the EMC for 1 GeV photons is about 2.5% in the barrel region and 5% in the endcap regions. More details about the BESIII detector can be found in Ref. [26]. In this section we initially describe the requirements for selecting the reconstructed particles that are combined to form the final states of interest. Then we present the selection criteria of fully reconstructed tag modes and partially reconstructed tag modes in Secs. VA and V B, respectively. p y Table I summarizes the set of tag modes used to reconstruct D0 final states. The decay channels are split into five categories: signal, flavored, mixed CP, CP-odd and CP-even. A highlight of this analysis is that the quasi- CP mode D →πþπ−π0, which has a large branching fraction, is used for the first time for the strong-phase measurements in the D →K0 S;LKþK−analysis. The Fþ value of πþπ−π0 is measured in Ref. [34] and the mode is found to be overwhelmingly CP-even. Hence in this analysis we treat πþπ−π0 as a CP-even tag taking into account its Fþ value. In the analysis, daughter particles are reconstructed as: K0 S →πþπ−, η →γγ, π0 →γγ, ω →πþπ−π0, η0 →πþπ−η. M. ABLIKIM et al. M. ABLIKIM et al. [29] using branching fractions taken from the Particle Data Group [30], and the remaining unknown decays from the charmonium states with LUNDCHARM [31]. The final state radiation (FSR) from charged final-state particles is incor- porated with the PHOTOS package [32]. The simulation of quantum-correlations in the process ψð3770Þ →D0 ¯D0 is done outside the EvtGen framework, using an algorithm developed by the CLEO Collaboration [33]. The effective integrated luminosity of the generated D0 ¯D0 sample is about ten times that of the data. For the efficiency determination we use signal MC samples. Signal MC samples consist of D0 →Stag; ¯D0 →X decays for the reconstruction of STs and D0 →K0 S;LKþK−; ¯D0 →Stag decays for the reconstruction of DTs, where Stag is a tag final state and X is any inclusive final state. Each signal MC sample corresponds to a specific ST or DT decay mode studied in this paper and contains 2 × 105 events. In order to ascertain the quality of the binning, a figure- of-merit based on the ratio of statistical sensitivity of the binned to the unbinned approach, known as the binning quality factor, Q, is defined in Ref. [22]. The predicted values of Q for this model are determined to be 0.771, 0.803 and 0.822 for N ¼ 2, 3 and 4 bins, respectively [14]. The measured values were 0.94þ0.16 −0.06, 0.87þ0.14 −0.06 and 0.94þ0.21 −0.06 for N ¼ 2, 3 and 4 bins respectively [24]. Since these values are close to one it implies that the loss of sensitivity due to the current bin definitions is small. An optimal binning scheme, which accounts for the distribu- tion of the B-meson data sample across the Dalitz plot, as well as the ΔδD variation, is found to give negligible improvement to the projected sensitivity compared to the “equal-ΔδD” binning [14]; hence, it is not pursued further. III. BINNING OF THE D0 →K0 SK + K − DALITZ PLOT The color scale represents the absolute value of the bin number and the black curve represents the kinematic boundary of the Dalitz plot. FIG. 1. Equal-ΔδD binning of D0 →K0 SKþK−phase-space based on the BABAR model [16] for N ¼ 2 (left), N ¼ 3 (middle) and N ¼ 4 (right) bins. The color scale represents the absolute value of the bin number and the black curve represents the kinematic boundary of the Dalitz plot. 052008-7 PHYS. REV. D 102, 052008 (2020) A. Selection of fully reconstructed tags Fully reconstructed tags are decay modes that do not contain an undetected particle in the final state. Before describing the kinematic variables used to select fully reconstructed tags, we introduce two additional vetoes that remove specific backgrounds to certain tag modes. The first veto is to suppress backgrounds arising from cosmic rays and lepton-pair events in the ST reconstruction of the two- body decay channels KþK−, πþπ−and Kπ∓. Here, we reject events in which the two charged tracks that recon- struct the ST candidate are consistent with being an eþe−or μþμ−pair. In addition, to suppress cosmic muons, we reject events where the time-of-flight difference between the two tracks is greater than 5 ns. Further, an event that has neither an EMC shower with an energy greater than 50 MeV nor an additional charged track in the MDC is rejected. The second veto is to remove the CP-odd K0 Sπ0, K0 S →πþπ− background to the predominantly CP-even πþπ−π0 tag mode; here we reject events that satisfy the condition jMπþπ−−mK0 Sj < 0.018 GeV=c2, where mK0 S refers to the nominal mass of the K0 S meson given in Ref. [30]. g The particle identification (PID) is performed by com- bining the dE=dx information from the MDC with the time-of-flight of the charged particle. The likelihoods for the kaon hypothesis LK and pion hypothesis Lπ are calculated. Tracks satisfying the condition LK > Lπ are identified as kaons and vice versa for pions. For electrons the PID is performed by defining a likelihood based on information about dE=dx in the MDC, time-of-flight and deposited energy and shape of the electromagnetic shower from the EMC. The track is identified as an electron if Le=ðLe þ LK þ LπÞ > 0.8 and Le > 0.001, where Le is the likelihood of the electron hypothesis. A K0 S candidate is formed by considering a pair of intersecting oppositely charged tracks. These tracks are not subject to any track quality requirement or PID. The closest approach of these tracks to the interaction point is required to be less than 20 cm along the beam direction with no requirement in the transverse direction. A secondary vertex fit is performed to form the K0 S vertex, and candidates with χ2 < 100 are selected. The updated four momenta after the secondary vertex fit are used later in this analysis. V. EVENT SELECTION In this section we will describe the selection criteria implemented to reconstruct these final states. For the charged tracks the polar angle θ is required to be within the MDC acceptance, which is j cos θj < 0.93. The distance of closest approach of a primary track from the Simulated samples produced with the GEANT4-based [27] Monte Carlo (MC) package, which includes the geometric description of the BESIII detector and the detector response, are used to determine the detection efficiency and to estimate the backgrounds. The simulation includes the beam-energy spread and initial-state radiation (ISR) in the eþe−annihilations modelled with the generator KKMC [28]. The inclusive MC samples consist of the production of D ¯D pairs, the non-D ¯D decays of the ψð3770Þ, the ISR production of the J=ψ and ψð3686Þ states, and the continuum processes incorporated in KKMC [28]. The known decay modes are modelled with EvtGen TABLE I. D0 decays used in this analysis. Type Tag modes Signal K0 SKþK−, K0 LKþK− Flavored K−πþ, K−πþπ0, K−eþνe Mixed CP K0 Sπþπ−, K0 Lπþπ− CP-odd K0 Sπ0, K0 Sη, K0 Sη0, K0 Sω CP-even KþK−, πþπ−, πþπ−π0, K0 Sπ0π0, K0 Lπ0, K0 Lη, K0 Lη0, K0 Lω TABLE I. D0 decays used in this analysis. TABLE I. D0 decays used in this analysis. 052008-8 IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … PHYS. REV. D 102, 052008 (2020) interaction region is required to be less than 10 cm in beam direction and less than 1 cm in the plane perpendicular to the beam direction to remove tracks not originating from eþe−collisions. For neutral showers the energy deposited in the EMC is required to be larger than 0.025 GeV in the barrel region ðj cos θj < 0.8Þ and larger than 0.050 GeV in the endcap region ð0.86 < j cos θj < 0.92Þ, which reduces the effect of electronic noise and deposits resulting from beam-related backgrounds. Moreover, the angle between the position of the shower and any extrapolated charged track in the EMC must be greater than 10° to reduce the number of showers related to charged tracks. Furthermore, we require the time of the shower to be less than 700 ns after the event start-time to further suppress fake photons associated with electronic noise and beam backgrounds. momenta are used later in the analysis. V. EVENT SELECTION For ω candidates the invariant mass of the πþπ−π0 combination is required to be within the range (0.760, 0.805) GeV/c2 and for η0 candi- dates the invariant mass of the πþπ−η combination is required to be within the range ð0.938; 0.978Þ GeV=c2. All the invariant mass intervals described correspond to approximately 3 times the standard deviation about the mean of the reconstructed distribution. A. Selection of fully reconstructed tags The mass of a K0 S candidate is required to be within the range (0.487, 0.511) GeV/c2. In order to suppress combinatorial back- grounds from two pions that are not from a true K0 S, the flight significance, L=σL, is required to be greater than two, where L is the flight length and σL is the uncertainty in L from the secondary vertex fit. For all fully reconstructed tag modes, the selected final- state particles are combined to reconstruct the D decay. Since the D ¯D pair production occurs at the ψð3770Þ resonance, there are no additional particles in the final state, so the energy of each D meson is equal to ﬃﬃﬃs p =2. Thus, with a well measured beam energy Ebeam (¼ ﬃﬃﬃs p =2) we define two quantities to reconstruct the D candidates: the energy difference, ΔE ≡ X i Ei −Ebeam; ð17Þ ð17Þ y Both π0 and η candidates are reconstructed from a pair of photons, where at least one of the photons must be reconstructed in the barrel region; this requirement reduces combinatorial backgrounds that arise from the large num- ber of showers in the endcap region that are related to beam backgrounds. The invariant mass of the two photon candidates must be in the range ð0.110; 0.155Þ GeV=c2 or ð0.480; 0.580Þ GeV=c2 for π0 and η candidates, respec- tively. In order to improve the momentum resolution, a kinematic fit of the two photons is performed with their invariant mass constrained to the nominal mass of π0 or η meson taken from the PDG [30]. Only π0 and η candidates with χ2 < 20 are selected. The improved values of the and the beam-constrained mass, MBC ≡1 c2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ E2 beam −j X ipicj2 q : ð18Þ ð18Þ Here Ei and pi are the energies and momenta of the D decay products in the center-of-mass frame. Properly reconstructed candidates will peak at zero in the ΔE distribution and at the nominal mass of the D0 meson [30] in the MBC distribution. For all the reconstructed final states mode-dependent criteria are applied to the ΔE distribution to reduce the level of combinatorial back- grounds. The ΔE distribution is fit with a combination of a 052008-9 PHYS. REV. D 102, 052008 (2020) M. ABLIKIM et al. double-Gaussian function and a polynomial to describe the signal and background, respectively. A. Selection of fully reconstructed tags The value of ΔE is required to be within the range 3σ [ð−4σ; 3σÞ] from the mean of the signal distribution for modes without [with] a π0 in the final state. Here σ is the total width of the ΔE signal shape. If multiple ST candidates are reconstructed in an event, the candidate with the minimum value of jΔEj is selected. If multiple DT candidates are selected, the candidate with a value ¯M ≡½MðD0Þ þ Mð ¯D0Þ=2 closest to the nominal D mass is selected. which peaks at m2 K0 for signal, where mK0 is the mass of the neutral kaon given in Ref. [30]. The presence of a neutrino is inferred using the variable Umiss ≡Emiss −jpmissjc; ð20Þ ð20Þ which peaks at zero for signal. Again we take advantage of resonant production and the knowledge of beam energy to determine Emiss and pmiss. Figure 2 shows example dis- tributions of M2 miss and Umiss. Reconstruction using the missing-mass technique inevitably results in a higher level of background than the full-reconstruction method. To reduce the background further, we do not consider events that have more charged tracks or neutral particles than required in the final state. The angle α, between the pmiss and the nearest unassigned shower is calculated. All the events with cos α > 0.98 are retained. For the events with cos α < 0.98 mode-dependent criteria are applied on the energy of the unassigned shower. Even though we reject events with additional neutral particles in the final state, there is significant background in the modes with neutral particles in the final states, arising from the final states having additional neutral particles that are not recon- structed. For example, in the case of D →K0 Lπ0 decays there are backgrounds from K0 Lπ0π0 where one π0 meson is not reconstructed, so the event passes all our selection criteria. These backgrounds can be further reduced by applying criteria on the momentum spectrum of recon- structed π0 or η candidates wherever applicable. The values of these criteria are selected based on optimization studies that use the inclusive MC samples. This optimization maximizes the figure-of-merit defined as S= ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ S þ B p , where SðBÞ are the number of signal (background) events in the A. ST yields ST yields of fully reconstructed tag modes are deter- mined from maximum likelihood fits to the MBC distribu- tion. Our probability density function (PDF) is a sum of the signal shape derived from the signal MC sample convolved with a Gaussian function to account for any difference in resolution between data and MC simulation, and an ARGUS function [35] to model the background. The threshold of the ARGUS function is fixed at MBC ¼ 1.8865 GeV=c2, which corresponds to the kin- ematic limit of D0 production at the ψð3770Þ. The peaking The ST yield, SST, of a partially reconstructed tag is calculated using the relation SST ¼ 2ND0 ¯D0BST; ð21Þ ð21Þ where ND0 ¯D0 is the number of D0 ¯D0 pairs in the BESIII data sample [36] and BST is the branching fraction of the tag mode, which is taken from Ref. [30] where available. The branching fractions of all D →K0 LX modes except TABLE II. Single-tag (ST) and D0 →K0 S;LKþK−double-tag (DT) yields and efficiencies. The DT yields are the observed number of events in the signal region prior to background and efficiency corrections. The ST yields are background subtracted because they are the result of fits to the MBC distributions. TABLE II. Single-tag (ST) and D0 →K0 S;LKþK−double-tag (DT) yields and efficiencies. The DT yields are the observed number of events in the signal region prior to background and efficiency corrections. The ST yields are background subtracted because they are the result of fits to the MBC distributions. VI. ESTIMATION OF ST AND DT YIELDS In Secs. VI A and VI B we will describe the methods of estimating ST and DT yields, respectively. Note that DT yields are only required bin-by-bin, not integrated over the Dalitz plot as given in Table II. B. Selection of partially reconstructed tags Partially reconstructed tags collectively refer to the tag modes where there is one particle in the final state, either a K0 L meson or a neutrino, which is not reconstructed. Modes with more than one missing particle in the final state are not considered in this analysis. Due to the presence of a missing particle in the final state, these tag modes can be recon- structed only as DTs so that four-momentum conservation can be exploited in the reconstruction. Selections of partially reconstructed tag modes proceed as follows. The opposite-side D candidate is reconstructed as a ST candidate using the criteria given in the Sec. VA. All the particles except the missing particle in the final state are reconstructed from the unused tracks and showers that satisfy the selection criteria already described. The pres- ence of an unreconstructed K0 L is inferred from the missing- mass distribution, calculated from the missing energy, Emiss, and the missing momentum, pmiss, in the center- of-mass frame as M2 miss ≡E2 miss c4 −jpmissj2 c2 ; ð19Þ ð19Þ ð Þ FIG. 2. (a) M2 miss distribution for ¯D0 →K0 LKþK−candidates reconstructed against the flavor-tags D0 →K−πþ and D0 →K−πþπ0. The points with error bars are the data, the red histogram denotes the peaking background due to ¯D0 →K0 SKþK−events from the inclusive MC sample, the blue-shaded histogram shows the combinatorial backgrounds from inclusive MC samples, and the magenta vertical lines indicates the signal region. (b) Umiss distribution for events in which ¯D0 →K0 SKþK−candidates are reconstructed against the D0 →K−eþνe tag. The black points with error bars are data, the blue-shaded histogram shows the backgrounds estimated from the inclusive MC sample and the magenta vertical line shows the signal region. FIG. 2. (a) M2 miss distribution for ¯D0 →K0 LKþK−candidates reconstructed against the flavor-tags D0 →K−πþ and D0 →K−πþπ0. The points with error bars are the data, the red histogram denotes the peaking background due to ¯D0 →K0 SKþK−events from the inclusive MC sample, the blue-shaded histogram shows the combinatorial backgrounds from inclusive MC samples, and the magenta vertical lines indicates the signal region. (b) Umiss distribution for events in which ¯D0 →K0 SKþK−candidates are reconstructed against the D0 →K−eþνe tag. The black points with error bars are data, the blue-shaded histogram shows the backgrounds estimated from the inclusive MC sample and the magenta vertical line shows the signal region. 052008-10 052008-10 052008-10 PHYS. B. Selection of partially reconstructed tags REV. D 102, 052008 (2020) IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … background is modeled by the shapes and yields obtained from the inclusive MC sample; this assumption is consid- ered as a source of systematic uncertainty. The flavor-tag modes D0 →K−πþ and D0 →K−πþπ0 have a peaking background of approximately 0.2% from DCS decays. The dominant peaking background to the decays D →K0 Sπ0 and D →K0 Sπ0π0 is from D →πþπ−π0 (0.5%) and D → πþπ−π0π0 (7%) decays, respectively. The MBC distribution is fit over the range ð1.83; 1.88Þ GeV=c2. The STyields are obtained by integrating the MBC distribution in the range ð1.86; 1.87Þ GeV=c2. In order to eliminate the small effect of D0 ¯D0 mixing, the measured ST yields of CP modes are multiplied by a correction factor of 1=ð1 −ηyDÞ, where η is the CP eigenvalue of the mode and yD is the charm- mixing parameter taken from Ref. [30]. signal region retained by the selection; the signal region for the optimization is the interval 0.2 < M2 miss < 0.3 GeV2=c4. The values of the shower energy and π0 (η) momentum criteria are varied, and the value that maximizes the figure-of-merit is chosen. A. ST yields ST DT Mode NST ϵST(%) N K0 SKþK− DT N K0 LKþK− DT ϵ K0 SKþK− DT (%) ϵ K0 LKþK− DT (%) Flavor-tags K−πþ 524307 742 63.31 0.06 323 743 12.43 0.07 15.85 0.08 K−πþπ0 995683 1117 31.70 0.03 596 1769 15.86 0.05 17.94 0.06 K−eþνe 752387 12795 263 13.23 0.04 CP-even tags KþK− 53481 247 61.02 0.11 42 112 12.07 0.07 15.52 0.08 πþπ− 19339 163 64.52 0.11 10 31 12.16 0.07 15.70 0.08 K0 Sπ0π0 19882 233 14.86 0.08 7 45 12.49 0.04 13.79 0.04 πþπ−π0 199981 618 37.65 0.11 51 254 16.79 0.06 19.54 0.07 K0 Lπ0 209445 14796 90 18.88 0.06 K0 LηðγγÞ 40009 2543 19 16.60 0.06 K0 Lω 207376 11498 44 13.42 0.04 K0 Lη0ðπþπ−ηÞ 33683 1909 7 13.23 0.04 CP-odd tags K0 Sπ0 65072 281 36.92 0.11 39 89 16.75 0.06 9.33 0.07 K0 SηðγγÞ 9524 134 32.94 0.11 9 10 16.05 0.05 9.05 0.06 K0 Sω 19262 157 12.14 0.07 16 27 12.20 0.03 3.42 0.04 K0 Sη0ðπþπ−ηÞ 3301 62 12.46 0.07 2 5 12.20 0.03 3.46 0.04 Mixed CP tags K0 Sπþπ− 78 265 16.35 0.05 8.32 0.06 K0 Lπþπ− 282 19.56 0.07 K0 SKþK− 12949 119 18.35 0.09 4 19 12.99 0.04 3.40 0.04 052008-11 PHYS. REV. D 102, 052008 (2020) M. ABLIKIM et al. expected to be less than 10% [37], which is considered as a systematic uncertainty; the difference will barely affect our final results, as the ST yields are used only for yield normalization, as given in Eqs. (10) and (14). The STyields calculated using Eq. (21) have larger uncertainties com- pared to the fully reconstructed tags, largely due to the uncertainty of the assumed values of BST. The ST MBC fits D →K0 Lπ0 are not available in Ref. [30], hence we assume the branching fractions of these modes to be the same as for the corresponding D →K0 SX modes. A. ST yields We note that this reasoning is not strictly valid, as the interference between Cabibbo-favored (CF) (D0 →¯K0X) and DCS transitions (D0 →K0X) can lead to a difference in the decay rates for D →K0 LX and D →K0 SX. However, this difference is (D0 →K0X) can lead to a difference in the decay rates for D →K0 LX and D →K0 SX. However, this difference is pared to the fully reconstructed tags, largely due to the uncertainty of the assumed values of BST. The ST MBC fits FIG. 3. Fits to the MBC distributions of ST decay modes. The points with error bars are data, the red curve is the total fit result and the blue dashed curve is the background component. Beneath each distribution the pull (σp) between the data and the fit is shown. The significant pulls observed in the flavor-tag modes D0 →K−πþ and D0 →K−πþπ0 are a consequence of the large sample size but studies of MC samples indicate that there is no significant bias on the ST yield introduced as a result. FIG. 3. Fits to the MBC distributions of ST decay modes. The points with error bars are data, the red curve is the total fit result and the blue dashed curve is the background component. Beneath each distribution the pull (σp) between the data and the fit is shown. The significant pulls observed in the flavor-tag modes D0 →K−πþ and D0 →K−πþπ0 are a consequence of the large sample size but studies of MC samples indicate that there is no significant bias on the ST yield introduced as a result. 052008-12 052008-12 IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … PHYS. REV. D 102, 052008 (2020) ) 2 c (GeV/ 0 D BC M 1.84 1.86 1.88 ) 2 c (GeV/ 0 D BC M 1.84 1.86 1.88 S S A B C D D FIG. 4. Distribution of events across ðMD0 BC; M ¯D0 BCÞ plane for K0 SKþK−reconstructed against flavor-tags. The signal region is denoted as S, while A, B, C and D are the various sideband regions. ) 2 c (GeV/ 0 D BC M 1.84 1.86 1.88 ) 2 c (GeV/ 0 D BC M 1.84 1.86 1.88 S S A B C D D In the case of partially reconstructed tag modes we follow a similar sideband-estimation method as in Ref. [24]. A. ST yields Here three regions are defined on the M2 miss or Umiss distributions: low sideband (L), signal region (S) and high sideband (H). The total yield is estimated as In the case of partially reconstructed tag modes we follow a similar sideband-estimation method as in Ref. [24]. [ ] Here three regions are defined on the M2 miss or Umiss distributions: low sideband (L), signal region (S) and high sideband (H). The total yield is estimated as YS ¼ ðNS −NP SÞ −δðNL −NP LÞ −γðNH −NP HÞ 1 −δα −γβ ; ð23Þ ð23Þ where NS, NL and NH are the uncorrected yields in the signal and sideband regions, NP i refers to the peaking background in the ith region, δ and γ refer to the ratio of combinatorial backgrounds in the signal region to that in the L and H sideband regions, respectively, and α and β refer to the ratio of signal in region S to that in the regions L and H, respectively. The values of α, β, δ and γ are derived from MC samples. Here the definitions of sidebands are mode dependent. We follow the same optimization procedure described in Sec. V B to define the signal regions. The peaking backgrounds are estimated from MC samples as described in Sec. VII B. FIG. 4. Distribution of events across ðMD0 BC; M ¯D0 BCÞ plane for K0 SKþK−reconstructed against flavor-tags. The signal region is denoted as S, while A, B, C and D are the various sideband regions. VII. D →K0 S;LK + K −DALITZ PLOTS In this section we discuss the Dalitz plot distributions of events when D →K0 S;LKþK−candidates are tagged with pure CP eigenstates and mixed CP states; we highlight the important differences. are shown in Fig. 3 and the yields are given in Table II. The effect on the final measurement due to the uncertainty in the measured values of the ST yields is treated as a systematic uncertainty. The ST yield uncertainty includes systematic uncertainties related to the fit procedure. In order to improve the resolution on the Dalitz plot variables ðm2 þ; m2−Þ, a kinematic fit is performed for D → K0 S;Lhþh−candidates. For D →K0 Shþh−tags, the two pions from the K0 S candidate obtained after the secondary vertex fit are combined with the hþ and h−into a common fit to the nominal mass of the D0 meson taken from Ref. [30]. In the case of a D →K0 Lhþh−candidate, a missing particle is created using the position of an EMC shower associated with the K0 L candidate. The mass of this object is set to the nominal mass of the K0 L meson taken from Ref. [30]; it is combined with hþh−tracks and fit to the nominal mass of the D0 meson. A 35% to 40% (30% to 35%) improvement in the m2 resolution across the Dalitz plot is achieved for D →K0 SKþK−(D →K0 LKþK−) can- didates after the kinematic fit. The resolutions are quanti- fied using the signal MC samples. Events that fail the kinematic fit are rejected. The improved values of ðm2 þ; m2−Þ are used to define the position of the event within the Dalitz plot and assign its bin index. B. DT yield This is a consequence of the quantum-correlation in data. Since each D meson is of opposite CP eigenvalue, the K0 SKþK− candidates reconstructed against CP-odd tags should decay through CP-even intermediate states. Hence it cannot decay through the D →K0 Sϕ state. The dominant CP-even intermediate state is the D →K0 Sað980Þ0 decay. The distribution of events in the Dalitz plot is observed to be flatter than in the case of K0 SKþK−tagged against a CP- even state. Since K0 S and K0 L have opposite CP eigenvalues, the entire scenario is reversed in the case of D →K0 LKþK− decays as shown in Fig. 6. The Dalitz plot distribution of D →K0 LKþK−candidates against CP-even modes resem- bles that of D →K0 SKþK−candidates against CP-odd modes and vice versa. separately for signal and tag sides in Fig. 7. The Dalitz plot of D →K0 Sπþπ−tags is consistent with that presented in Ref. [13]. In Fig. 7(d), the enhancement above M2 πþπ−∼ 1.3 GeV=c2 corresponds to D →Kð892Þπ∓decays, whereas the peak around M2 πþπ−∼0.6 GeV=c2 corresponds to D →K0 Sρ0 decays. The D →Kð892Þπ∓decays can be seen as two bands that are parallel to the vertical and horizontal axes of the Dalitz plane. The decay D →K0 Sρ0 lies close and parallel to the diagonal boundary. Since the D →K0 LKþK−decays reconstructed against D →K0 Sπþπ− decays are not in a CP eigenstate, the Dalitz plot distri- bution is a combination of both the CP-even and CP-odd tagged K0 LKþK−Dalitz plots. The Dalitz plot structure of D →K0 SKþK−reconstructed against D →K0 S;Lπþπ−has similar features to those shown in Fig. 7. B. DT yield For fully reconstructed DT modes we follow a sideband- estimation method developed by the CLEO Collaboration [38] to determine the DT yield. The sidebands are defined on the two-dimensional ðMD0 BC; M ¯D0 BCÞ plane as shown in Fig. 4. Here, the MD0 BC (M ¯D0 BC) refers to the MBC distribution of signal (tag) side. In Fig. 4, S refers to the signal region, sideband A (B) contains events which are from misrecon- structed tag (signal) decays, sideband C consists of con- tinuum events and sideband D consists of events that are purely combinatoric. The amount of combinatorial (non- peaking) backgrounds in the signal region is estimated from the events in the sideband regions. Thus the total DT yield, NDT, of K0 SKþK−is estimated as NDT, of K0 SKþK−is estimated as NDT ¼ NS −NP − aS aD ND þ X i¼A;B;C aS ai Ni −aS ai ND ; ð22Þ The Dalitz plot distribution of the D →K0 SKþK−can- didates reconstructed against CP-even tag modes and their corresponding M2 KþK− projections are given in Fig. 5. The presence of a significant peak around M2 KþK−∼ 1.04 GeV2=c4 is due to the decay D0 →K0 Sϕ; ϕ →KþK−. These events are distributed along the diagonal boundary of the Dalitz plot. As D0 →K0 Sϕ constitutes a large fraction of the total D0 →K0 SKþK−decay width [30], a higher ð22Þ where ai is the area of the corresponding region i ¼ A, B, C, D or S, Ni refers to the yields in the sideband region, NS is the yield in the signal region before background correc- tion (uncorrected yield) and NP is the peaking-background yield estimated from the MC simulation (see Sec. VII B). 052008-13 PHYS. REV. D 102, 052008 (2020) M. ABLIKIM et al. FIG. 5. (a) Dalitz plot and (b) M2 KþK−distributions for D →K0 SKþK−reconstructed against CP-even final states. (c) Dalitz plot and (d) M2 KþK−distributions for D →K0 SKþK−reconstructed against CP-odd final states. FIG. 5. (a) Dalitz plot and (b) M2 KþK−distributions for D →K0 SKþK−reconstructed against CP-even final states. (c) Dalitz plot and (d) M2 KþK−distributions for D →K0 SKþK−reconstructed against CP-odd final states. population of events is seen in the region enclosing the ϕ resonance. A similar peak is absent in the M2 KþK−distri- bution of D →K0 SKþK−candidates reconstructed against CP-odd tag modes shown in Figs. 5(c) and (d). A. Dalitz plot binning, bin yield estimation and corrections The Dalitz plot distribution of D →K0 LKþK−candidates against the self-conjugate mode D →K0 Sπþπ−is given In this section we describe our method of binning the Dalitz plots and calculating the bin yields and efficiencies. 052008-14 INDEPENDENT DETERMINATION OF THE … PHYS. REV. D 102, 052008 (2020 ROVED MODEL-INDEPENDENT DETERMINATION OF THE … PHYS. REV. D IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … PHYS. REV. D 102, 052008 (2020) FIG. 6. (a) Dalitz plot and (b) M2 KþK−distributions for D →K0 LKþK−reconstructed against CP-even final states. (c) Dalitz plot and (d) M2 KþK−distributions for D →K0 LKþK−reconstructed against CP-odd final states. FIG. 6. (a) Dalitz plot and (b) M2 KþK−distributions for D →K0 LKþK−reconstructed against CP-even final states. (c) Dalitz plot and (d) M2 KþK−distributions for D →K0 LKþK−reconstructed against CP-odd final states. The procedures for correcting the bin migration and DCS correction for flavor-tag yields are also explained. reconstructed against CP tags, along with their efficiencies, are given in Table II. The binning prescription followed in our analysis is described in Sec. II. The entire D0 →K0 S;LKþK−Dalitz plot is divided into N ¼ 2; N ¼ 3 and N ¼ 4 equal-ΔδD bins. In the case of D →K0 S;Lπþπ−tag modes, the entire Dalitz plot is divided into N ¼ 8 equal-ΔδD bins identical to those defined in Ref. [13]. The uncorrected bin yields are obtained by counting the number of events in each bin. The bin yield M i [see Eq. (10)] of D →K0 SKþK−recon- structed against CP tags and the flavor-tag yield, Ki [see Eq. (4)] are calculated separately for each mode. The events in the ith bin of the D →K0 SKþK−Dalitz plot and the jth bin of the D →K0 Shþh−Dalitz plot are counted to obtain Mij [see Eq. (12)]. A similar procedure is followed to obtain the yields K0 i, M0 i and M0 ij [see Eqs. (14) and (15)] for the D →K0 LKþK−decay. The flavor-tag yield for the D0 →K0 S;Lπþπ−mode is taken from Ref. [13]. The yields of D →K0 SKþK−decays reconstructed against CP tags are quite low. The inclusion of the D →πþπ−π0 tag mode results in an approximately 50% increase in the CP-even tag yield. The uncorrected yields of D →K0 SKþK−decays Due to the finite ðm2 þ; m2−Þ resolution, events migrate between bins. A. Dalitz plot binning, bin yield estimation and corrections Often these migrations are asymmetric between the bins because of the differing event densities in each bin. We correct for this using an unfolding method based on correction factors derived from the signal MC samples. For D →K0 S;LKþK−decays reconstructed against CP and flavor tags, we define a 2N × 2N migration matrix U as U as Ui;j ≡ mji PN k¼−N ;k≠0 mjk ; ð24Þ ð24Þ where mji are the events generated in the jth bin and reconstructed in the ith bin. The vector of migration- corrected data yields N and the vector of reconstructed yields in the signal region NS are related by N ¼ U−1NS: ð25Þ ð25Þ In the case of D →K0 S;LKþK−reconstructed against the D →K0 S;Lhþh−tags, the correlation between the bins on 052008-15 PHYS. REV. D 102, 052008 (2020) M. ABLIKIM et al. FIG. 7. (a) Dalitz plot and (b) M2 KþK−distributions for D →K0 LKþK−reconstructed against ¯D →K0 Sπþπ−final states. (c) Dalitz plot and (d) M2 πþπ−distributions for D →K0 Sπþπ−decay in the same events. FIG. 7. (a) Dalitz plot and (b) M2 KþK−distributions for D →K0 LKþK−reconstructed against ¯D →K0 Sπþπ−final states. (c) Dalitz plot and (d) M2 πþπ−distributions for D →K0 Sπþπ−decay in the same events. the signal and tag sides needs to be taken into account. Hence the total migration matrix is a tensor (Kronecker) product of signal- and tag-migration matrices. For a given number of signal bins N , the dimension of the migration matrix for K0 S;LKþK−against K0 S;LKþK−is 4N 2 × 4N 2 and for K0 S;LKþK−against K0 S;Lπþπ−it is 32N × 32N . The uncertainties in the matrix elements due to the finite size of the signal MC sample are considered as a source of systematic uncertainty. An example of the migration matrix for D →K0 SKþK−candidates reconstructed against the D →KþK−tag mode is given in Table III. Typically the rate of migration out of bin 1, which contains the narrow ϕ resonance, is about 3% for D →K0 SKþK−decays and about 5% for D →K0 LKþK−decays. The rate of migration into bin 1 is significantly smaller due to the broader structures that occupy the remainder of the Dalitz plot away from the ϕ resonance. Throughout this unfolding procedure we assume signal and background migrate in identical fashion, because the background is dominated by peaking components. A. Dalitz plot binning, bin yield estimation and corrections Since these decays are used fF i ¼ R i jfðm2 þ; m2−Þj2dm2 þdm2− R iðjfðm2 þ; m2−Þj2 þ ðrF DÞ2jfðm2−; m2 þÞj2 −2rF DRFR½eiδF Dfðm2 þ; m2−Þfðm2−; m2 þÞÞdm2 þm2− ; ð26Þ fF0 i ¼ R i jf0ðm2 þ; m2−Þj2dm2 þdm2− R iðjf0ðm2 þ; m2−Þj2 þ ðrF DÞ2jf0ðm2−; m2 þÞj2 þ 2rF DRFR½eiδF Df0ðm2 þ; m2−Þf0ðm2−; m2 þÞÞdm2 þdm2− ; ð27Þ fF i ¼ R i jfðm2 þ; m2−Þj2dm2 þdm2− R iðjfðm2 þ; m2−Þj2 þ ðrF DÞ2jfðm2−; m2 þÞj2 −2rF DRFR½eiδF Dfðm2 þ; m2−Þfðm2−; m2 þÞÞdm2 þm2− ; ð26Þ fF0 i ¼ R i jf0ðm2 þ; m2−Þj2dm2 þdm2− R iðjf0ðm2 þ; m2−Þj2 þ ðrF DÞ2jf0ðm2−; m2 þÞj2 þ 2rF DRFR½eiδF Df0ðm2 þ; m2−Þf0ðm2−; m2 þÞÞdm2 þdm2− ; ð27Þ R i jfðm2 þ; m2−Þj2dm2 þdm2− m2−; m2 þÞj2 −2rF DRFR½eiδF Dfðm2 þ; m2−Þfðm2−; m2 þÞÞdm2 þm2− ; ð26Þ ð26Þ ð27Þ where rF D is the ratio of the moduli of the DCS to CF amplitudes, for example jAðD0 →Kþπ−Þ=AðD0 → K−πþÞj for Kπ∓, and δD F is the strong-phase difference between the DCS and CF amplitudes. The coherence factor, RF for flavor-mode F, accounts for the dilution in inter- ference effects that arises when integrating over the phase space of multibody decays [40]. The values of the param- eters used to determine the correction factors are listed in Table IV. The fraction of events in each bin Fð0Þ i , defined in Eq. (4), is given in Table V. The D0 →K0 LKþK−ampli- tude model is developed by modifying the intermediate resonances of D0 →K0 SKþK−as presented in Ref. [24]. Good agreement with the predicted values [14] is observed for the results given in Table V. The uncertainties in the final result due to the correction factors are small and are treated as a systematic uncertainty. The DCS correction is not required for the D0 →K−eþνe flavor-tag. B. Bin-by-bin background estimation In this section we explain the method used to estimate the peaking background. The amount of combinatorial background in each bin is estimated from the sideband- estimation methods described in Sec. VI B. The peaking backgrounds are identified from the inclu- sive MC samples using the tool described in Ref. [43]. The backgrounds to fully reconstructed tags are found to be negligible. However, all the D →K0 LX modes contain backgrounds from D →K0 SX modes, where the π0 mesons from K0 S →π0π0 decays are not reconstructed, so that the K0 S is treated as a missing particle. The D →K0 LX and D → K0 SX decays are of opposite CP, hence the distribution of background events across the Dalitz plot is not the same as that for signal events. The level of these backgrounds varies between 2 to 4% depending on the tag mode. The bin-by- bin background estimation using the inclusive MC sample is not reliable for two reasons. First, there can be a difference between the branching fraction in data and that assumed in the MC simulation. Second, the MC samples are not tuned to reflect the distributions of events across the Dalitz plot. Both these issues will result in an incorrect estimation of the bin-by-bin background. Hence, we use a combination of data and background MC samples to estimate the backgrounds. TABLE IV. Values of the parameters used to calculate the DCS correction factors. F rF D (%) δF D (°) RF Kπ 5.86 0.02 [41] 194.7þ8.4 −17.6 [41] 1 Kππ0 4.47 0.12 [42] 198þ14 −15 [42] 0.81 0.06 [42] TABLE IV. Values of the parameters used to calculate the DCS correction factors. TABLE IV. Values of the parameters used to calculate the DCS correction factors. F rF D (%) δF D (°) RF Kπ 5.86 0.02 [41] 194.7þ8.4 −17.6 [41] 1 Kππ0 4.47 0.12 [42] 198þ14 −15 [42] 0.81 0.06 [42] TABLE V. Values of Fð−Þi and F0 ð−Þi (%) measured from the flavor-tagged D0 →K0 SKþK−and D0 →K0 LKþK−data for the different number of bins N . A. Dalitz plot binning, bin yield estimation and corrections The bin efficiency for each tag mode is evaluated from the signal MC sample. The signal MC yield in each bin is corrected for migration before calculating the efficiency. The bin efficiency is defined as the ratio of events reconstructed in each bin to the number of events gen- erated. The bins are combined appropriately taking into account their symmetry (see Sec. II) when estimating the efficiencies. The total DT efficiencies are given in Table II. In the case of D →K0 SKþK−, the efficiencies vary between ð12.43 0.07Þ% for K0 SKþK−vs K−πþ tags to ð2.20 0.03Þ% for K0 SKþK−vs K0 Sη0 tags, whereas for D → K0 LKþK−the efficiency varies between (15.85 0.08Þ% for K0 LKþK−vs K−πþ tags and ð3.40 0.04Þ% for K0 SKþK−vs K0 LKþK−tags. The uncertainty on the TABLE III. Migration matrix for K0 SKþK−vs KþK−events when the D →K0 SKþK−Dalitz plot is divided into N ¼ 3 bins. i Ui;1 Ui;2 Ui;3 Ui;−1 Ui;−2 Ui;−3 1 0.968 0.020 0.001 0.011 0.000 0.000 2 0.036 0.967 0.001 0.000 0.001 0.003 3 0.007 0.001 0.992 0.000 0.000 0.000 −1 0.010 0.000 0.000 0.972 0.018 0.000 −2 0.000 0.000 0.000 0.032 0.967 0.001 −3 0.000 0.000 0.000 0.006 0.006 0.988 TABLE III. Migration matrix for K0 SKþK−vs KþK−events when the D →K0 SKþK−Dalitz plot is divided into N ¼ 3 bins. 052008-16 PHYS. REV. D 102, 052008 (2020) IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … efficiency is related to the size of the MC sample. The bin efficiencies are used to calculate the expected yield for each tag mode as given in Eqs. (10), (12), (14) and (15). to determine Kð0Þ i , the presence of this DCS contamination may bias the values [39]. In order to correct for this effect, the yield in each bin is multiplied by a correction factor estimated using the decay model reported in Ref. [16]. The correction factors fF i for D0 →K0 SKþK−against F and fF0 i for D0 →K0 LKþK−against F are given by Both pseudoflavor DTyields with F ∈ðK−πþ; K−πþπ0Þ have contamination from DCS decays whose contribution is enhanced compared to ST yields due to the quantum correlation between the D0 ¯D0. B. Bin-by-bin background estimation i Fi (%) F−i (%) F0 i (%) F0 −i (%) N ¼ 2 1 24.4 1.7 30.4 1.9 23.5 1.2 27.7 1.3 2 19.6 1.6 25.6 1.9 23.1 1.3 25.6 1.3 N ¼ 3 1 21.9 1.5 27.7 1.8 21.1 1.1 25.1 1.2 2 21.3 1.7 24.7 1.8 22.6 1.3 25.1 1.4 3 1.3 0.4 3.1 0.5 2.8 0.3 3.3 0.4 N ¼ 4 1 21.1 1.5 27.0 1.8 19.5 1.0 23.2 1.7 2 6.5 0.9 3.6 0.6 7.2 0.7 4.1 0.5 3 16.3 1.5 22.4 1.8 19.5 1.2 23.0 1.3 4 0.5 0.2 2.6 0.5 0.9 0.2 2.6 0.3 TABLE V. Values of Fð−Þi and F0 ð−Þi (%) measured from the flavor-tagged D0 →K0 SKþK−and D0 →K0 LKþK−data for the different number of bins N . i Fi (%) F−i (%) F0 i (%) F0 −i (%) N ¼ 2 1 24.4 1.7 30.4 1.9 23.5 1.2 27.7 1.3 2 19.6 1.6 25.6 1.9 23.1 1.3 25.6 1.3 N ¼ 3 1 21.9 1.5 27.7 1.8 21.1 1.1 25.1 1.2 2 21.3 1.7 24.7 1.8 22.6 1.3 25.1 1.4 3 1.3 0.4 3.1 0.5 2.8 0.3 3.3 0.4 N ¼ 4 1 21.1 1.5 27.0 1.8 19.5 1.0 23.2 1.7 2 6.5 0.9 3.6 0.6 7.2 0.7 4.1 0.5 3 16.3 1.5 22.4 1.8 19.5 1.2 23.0 1.3 4 0.5 0.2 2.6 0.5 0.9 0.2 2.6 0.3 Our method of peaking background estimation is as follows. We generate dedicated background MC samples corresponding to each type of background decay. The background retention efficiencies for these backgrounds are calculated for each bin. The expected yields are calculated using the values of ci and si obtained from the previous 052008-17 M. ABLIKIM et al. PHYS. REV. D 102, 052008 (2020) measurement [14] through the relations given in Eqs. (10), (12), (14) and (15). The yields are multiplied by the retention efficiencies to obtain the expected background yields in data. VIII. EXTRACTION OF ci AND si The uncorrected yields in related bins are combined according to the symmetry relations described in Sec. II. This procedure reduces the number of degrees of freedom in the fit. The quantities KiðK0 iÞ and ci, si (c0 i; s0 i) for D → K0 Sπþπ−ðD →K0 Lπþπ−Þ are taken from Ref. [13]. The values of cð0Þ i and sð0Þ i are obtained by minimizing the negative log likelihood expression −2 ln L ¼ −2 X i ln PðN i ; hN i iÞK0 SKþK−;CP −2 X i ln PðN0 i ; hN0 i iÞK0 LKþK−;CP −2 X i;j ln PðNij; hNijiÞK0 SKþK−;K0 SKþK−−2 X i;j ln PðN0 ij; hN0 ijiÞK0 SKþK−;K0 LKþK− −2 X i;j ln PðNij; hNijiÞK0 SKþK−;K0 Sπþπ−−2 X i;j ln PðN0 ij; hN0 ijiÞK0 SKþK−;K0 Lπþπ− −2 X i;j ln PðN0 ij; hN0 ijiÞK0 LKþK−;K0 Sπþπ−þ χ2: ð2 ð28Þ between the extracted values of ciðsiÞ and c0 iðs0 iÞ to lie within the uncertainties of the predicted differences ΔciðΔsiÞ. The χ2 is defined as between the extracted values of ciðsiÞ and c0 iðs0 iÞ to lie within the uncertainties of the predicted differences ΔciðΔsiÞ. The χ2 is defined as Here, hNi is the expected migration-corrected yield in a particular bin whose measured yield is N. PðN; hNiÞ is the Poisson probability of observing a yield N given the mean hNi: χ2 ¼ X i c0 i −ci −Δci σΔci 2 þ X i s0 i −si −Δsi σΔsi 2 ; ð30Þ ð30Þ PðN; hNiÞ ¼ hNiNe−hNi N! : ð29Þ ð29Þ where Δci ¼c0 i;BABAR −ci;BABARðΔsi ¼s0 i;BABAR −si;BABARÞ is the predicted difference from the BABAR model [16] and σΔciðσΔsiÞ is the uncertainty on ΔciðΔsiÞ. The values of ΔciðΔsiÞ and σΔciðσΔsiÞ are given in Table VI. If hMi represents the expected signal yield and hBi repre- sents the expected background then hNi ¼ hMi þ hBi. It is to be noted that in Eq. (28) the comparison is between the uncorrected yield and the sum of expected signal and expected background in each bin. This method eliminates the possibility of unphysical negative bin yields arising from the subtraction of backgrounds from bins having low yields. The χ2 term in Eq. (28) constrains the difference Large values of Δci and Δsi are observed in bins i ¼ 3 and i ¼ 4 in the N ¼ 3 and N ¼ 4 binnings, respectively. B. Bin-by-bin background estimation Though most of the combinatorial back- ground beneath the D →K0 LX signal decays are from non- resonant D →KþK−π0π0 decays, these decays only con- tribute approximately 2% of the background in the signal region. The contributions from continuum backgrounds and low-lying charmonium resonance decays are found to be negligible for all DT modes, which is expected given the tight kinematic criteria that can be imposed close to open- charm threshold. The expected background yields are not subtracted from signal yield but added to the expected signal yield in the fit as explained in Sec. VIII. VIII. EXTRACTION OF ci AND si These bins correspond to the lobes on the Dalitz plot that encompass the neutral resonance a0ð1450Þ0. The model has TABLE VI. Model-predicted values of Δci and Δsi along with the uncertainties σΔci and σΔsi for equal-ΔδD binnings N ¼ 2, 3 and 4. The values are those reported in Ref. [24]. N i ci c0 i Δci si s0 i Δsi 2 1 0.742 0.768 0.026 0.017 0.275 0.286 0.011 0.023 2 −0.679 −0.680 −0.001 0.036 0.318 0.397 0.079 0.021 3 1 0.801 0.827 0.026 0.014 0.268 0.269 0.001 0.023 2 −0.657 −0.593 0.064 0.019 0.411 0.435 0.024 0.010 3 −0.043 −0.680 −0.637 0.311 −0.661 0.126 0.787 0.161 4 1 0.845 0.864 0.019 0.011 0.239 0.242 0.003 0.021 2 −0.028 0.095 0.123 0.029 0.531 0.512 −0.019 0.022 3 −0.804 0.779 0.025 0.019 0.332 0.382 0.050 0.015 4 0.232 −0.718 −0.950 0.355 0.738 0.262 1.000 0.254 TABLE VI. Model-predicted values of Δci and Δsi along with the uncertainties σΔci and σΔsi for e binnings N ¼ 2, 3 and 4. The values are those reported in Ref. [24]. INDEPENDENT DETERMINATION OF THE … PHYS. REV. D 102, 052008 (2020 PHYS. REV. D 102, 052008 (2020) MPROVED MODEL-INDEPENDENT DETERMINATION OF THE … PHYS. REV. IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … TABLE VII. Fit results for cð0Þ i and sð0Þ i for different N . The first uncertainty is statistical and the second uncertainty is systematic. VIII. EXTRACTION OF ci AND si N i ci si c0 i s0 i 2 1 0.704 0.034 0.003 −0.038 0.144 0.039 0.730 0.035 0.003 −0.028 0.144 0.039 2 −0.760 0.040 0.007 0.590 0.198 0.085 −0.785 0.034 0.006 0.669 0.198 0.086 3 1 0.724 0.035 0.003 −0.037 0.174 0.049 0.751 0.036 0.003 −0.037 0.174 0.049 2 −0.576 0.050 0.009 0.616 0.146 0.047 −0.512 0.050 0.009 0.640 0.146 0.047 3 −0.174 0.173 0.040 −0.669 0.370 0.119 −0.382 0.145 0.040 0.045 0.384 0.116 4 1 0.783 0.034 0.003 −0.242 0.173 0.051 0.802 0.034 0.003 −0.239 0.174 0.051 2 −0.053 0.106 0.017 0.306 0.294 0.125 0.070 0.106 0.017 0.286 0.294 0.124 3 −0.654 0.057 0.011 0.659 0.210 0.059 −0.630 0.056 0.011 0.709 0.210 0.059 4 0.090 0.208 0.041 −0.713 0.387 0.195 −0.290 0.201 0.036 0.122 0.422 0.206 very small amplitudes in this region, hence a small differ- ence between the D →K0 SKþK−and D →K0 LKþK− models is proportionally more significant. Consequently, the uncertainties are also large. Improvement in the precision due to the χ2 term is more significant for si than ci. The minimization of Eq. (28) is performed using the MINUIT [44] package. The value of parameters obtained from the fit are given in Table VII. procedure as smearing. This process is repeated 1000 times and the distribution of the resulting cð0Þ i and sð0Þ i values is fit with a Gaussian distribution. The width of the Gaussian distribution is then reported as the systematic uncertainty. If there is a set of correlated input quantities assumed in our fit, we use a procedure based on the Cholesky decom- position of the covariance matrix to smear the value taking the correlation into account. The procedure involves gen- erating a vector of correlated variables X ¼ μ þ AZ, where μ is a vector of the reported values of the input parameter, Z is a vector with random values that follow a normal distribution, and A is the decomposed matrix. VIII. EXTRACTION OF ci AND si The fit is repeated with the new value of X. The systematic covari- ance matrix is calculated using the distributions of the cð0Þ i and sð0Þ i values as well as the correlations between them. The fitting procedure is validated using pseudodata samples generated by a standalone simulation. The bin yields and backgrounds are generated according to the relations given in Sec. II. The effects of bin migration are also considered in the simulation. The bin yields are fluctuated assuming a Poisson distribution. The procedure is repeated 500 times to obtain a pull distributions for cð0Þ i and sð0Þ i . The means and widths of the pulls in each bin are consistent with zero and one, respectively, indicating no bias and proper estimation of the uncertainties. The systematic uncertainties related to the ST yields are evaluated as follows. First, a combined systematic uncer- tainty in the yields due to various assumptions made in the fit is estimated. For example, the endpoints of the ARGUS function are fixed in our fits. We rerun the fits by changing the endpoints by 0.5 MeV=c2, which is the approximate uncertainty related to the beam-energy spread in the end- point. The difference between the new value of the yield and the nominal value is taken as a systematic uncertainty in the ST yield. Other assumptions include the estimation of peaking backgrounds from MC simulations and the assumed IX. SYSTEMATIC UNCERTAINTIES The evaluation of systematic uncertainties arising due to various inputs to the cð0Þ i and sð0Þ i fit are described in this section. In general, our method of evaluating systematic uncertainties uses a procedure that varies the nominal input values within their uncertainties randomly; we refer to this TABLE VIII. Summary of the contributions to the systematic uncertainty for the N ¼ 3 equal-ΔδD binning. Systematic c1 c2 c3 s1 s2 s3 c0 1 c0 2 c0 3 s0 1 s0 2 s0 3 ST yield 0.001 0.005 0.007 0.001 0.002 0.001 0.001 0.005 0.002 0.001 0.002 0.001 Kð0Þ i statistics 0.001 0.006 0.033 0.006 0.001 0.010 0.001 0.006 0.033 0.006 0.001 0.010 K0πþπ−ðcð0Þ i ; sð0Þ i Þ 0.002 0.003 0.010 0.045 0.044 0.085 0.002 0.003 0.004 0.045 0.044 0.083 K0πþπ−ðKð0Þ i Þ 0.001 0.001 0.003 0.009 0.015 0.018 0.001 0.001 0.001 0.009 0.015 0.018 ND ¯D 0.000 0.000 0.000 0.001 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.001 MC statistics 0.001 0.003 0.013 0.000 0.001 0.000 0.001 0.003 0.020 0.000 0.001 0.000 Background 0.001 0.002 0.012 0.015 0.009 0.082 0.001 0.002 0.008 0.015 0.009 0.079 DCS correction 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Statistical 0.035 0.050 0.173 0.174 0.146 0.370 0.036 0.050 0.145 0.174 0.146 0.384 Total systematic 0.003 0.009 0.040 0.049 0.047 0.119 0.003 0.009 0.040 0.049 0.047 0.116 Total 0.035 0.058 0.178 0.181 0.154 0.389 0.036 0.051 0.150 0.181 0.154 0.401 052008-19 PHYS. REV. D 102, 052008 (2020) fractions in partially reconstructed modes. The uncertainties in the yields from the fits are added in i h h i i i b i h within the total uncertainty and the distribution of resulting cð0Þ i and sð0Þ i values is obtained as laid out abo Measured values of c½0 i and s½0 i for N ¼ 2 (a) [(b)], N ¼ 3 (c) [(d)], and N ¼ 4 (e) [(f)] equal-ΔδD bins are given by the bl h error bars. Also shown for comparison are the measurements reported by the CLEO Collaboration [14] (pink points w and the predictions of the model reported by the BABAR Collaboration [16] (blue stars). The black circle corresponds to hysical region c2 i þ s2 i ¼ 1. KIM et al. PHYS. REV. D 102, 052008 (202 M. ABLIKIM et al. FIG. 8. IX. SYSTEMATIC UNCERTAINTIES ð Þ ð Þ The systematic uncertainties due to the assumed values of the DCS correction factors to the flavor-tag yields fFð0Þ i are estimated by smearing the correction factors within their uncertainties assuming a Gaussian distribution. The fFð0Þ i uncertainties are small, hence the associated system- atic uncertainties on the values of cð0Þ i and sð0Þ i are also small. An example of the individual contributions to the sys- tematic uncertainties for the N ¼ 3 equal-ΔδD binning is shown in Table VIII; corresponding tables for the N ¼ 2 and 4 binning schemes are given in Appendix XIII. The systematic uncertainties are significantly smaller than stat- istical uncertainties for all binning schemes. Appendix XIV contains the statistical and systematic correlation matrices for the N ¼ 2, 3 and 4 binning schemes. The final results for ci; si; c0 i and s0 i are shown in Fig. 8. In our fit the values of cð0Þ i and sð0Þ i of D →K0 S;Lπþπ−are fixed to the values reported in Ref. [13]. The covariance matrix used to smear the values is also taken from Ref. [13]. This uncertainty is the dominant systematic uncertainty. The value of the total number of D0 ¯D0 pairs is fixed in our fits. This information is used to normalize the bin yields of D →K0 S;LKþK−tagged by D →K0 S;Lhþh−decays. The related systematic uncertainty is evaluated by smearing the value of ND0 ¯D0 within its uncertainty assuming a Gaussian distribution. Since the value of ND0 ¯D0 pairs is measured precisely [36], the systematic uncertainty due to this input is negligible. i i An example of the individual contributions to the sys- tematic uncertainties for the N ¼ 3 equal-ΔδD binning is shown in Table VIII; corresponding tables for the N ¼ 2 and 4 binning schemes are given in Appendix XIII. The systematic uncertainties are significantly smaller than stat- istical uncertainties for all binning schemes. Appendix XIV contains the statistical and systematic correlation matrices for the N ¼ 2, 3 and 4 binning schemes. The final results for ci; si; c0 i and s0 i are shown in Fig. 8. Signal MC samples are widely used in this analysis for various purposes such as constructing migration matrices and determining the selection efficiencies. IX. SYSTEMATIC UNCERTAINTIES Measured values of c½0 i and s½0 i for N ¼ 2 (a) [(b)], N ¼ 3 (c) [(d)], and N ¼ 4 (e) [(f)] equal-ΔδD bins are given by the black points with error bars. Also shown for comparison are the measurements reported by the CLEO Collaboration [14] (pink points with error bars) and the predictions of the model reported by the BABAR Collaboration [16] (blue stars). The black circle corresponds to the allowed physical region c2 i þ s2 i ¼ 1. branching fractions in partially reconstructed modes. The statistical uncertainties in the yields from the fits are added in quadrature with the systematic uncertainties to obtain the total uncertainty related to the ST fit. The STyield is smeared within the total uncertainty and the distribution of the resulting cð0Þ i and sð0Þ i values is obtained as laid out above. The systematic uncertainties due to the ST yield are found to be small. The systematic uncertainty due to the factors used 052008-20 PHYS. REV. D 102, 052008 (2020) IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … to the fully reconstructed tags, hence the systematic uncertainties due to the background parametrization for partially reconstructed tags are larger than those for the fully reconstructed tags. The effect of bin-by-bin peaking background from D →K0 Shþh−decays in the D → K0 Lhþh−signal sample is estimated accounting for quan- tum correlations by using the ci and si values reported in Ref. [14]. Since the backgrounds are only identified but not estimated from MC simulations, there is no systematic uncertainty arising due to any difference in branching fractions between data and MC simulations. to correct for the effect of charm mixing is found to be negligible. Furthermore, uncertainties related to the absolute efficiency do not affect the results due to the use of yield ratios and normalization constants fit to data. The systematic uncertainty due to the finite statistics of the flavor-tag yields Ki are evaluated by smearing the input values assuming a Gaussian distribution around the nominal value with width equal to the uncertainty. The systematic uncertainty due to the flavor-tag yield of the D →K0 Sπþπ−decay is estimated using the covariance matrix reported in the Ref. [13]. The uncertainties due to flavor-tag yields are small due to the large yields compared to those for CP and D →K0hþh−tags. IX. SYSTEMATIC UNCERTAINTIES The systematic uncertainties due to the finite size of the MC samples are evaluated by smearing the matrix elements within their uncertainties assuming a Gaussian distribution taking corre- lations into account. Any systematic uncertainty due to the incorrect MC modeling is canceled since we use ratios of ST and DT yields in computing the values of hMi. ACKNOWLEDGMENTS The BESIII Collaboration thanks the staff of BEPCII and theIHEPcomputingcenterfortheirstrongsupport.Thiswork is supported in part by National Key Basic Research Program of China under Contract No. 2015CB856700; National Natural Science Foundation of China (NSFC) under Contracts No. 11625523, No. 11635010, No. 11735014, No.11822506,No.11835012,No.11935015,No.11935016, No. 11935018, No. 11961141012; the Chinese Academy of Sciences (CAS) Large-Scale Scientific Facility Program; Joint Large-Scale Scientific Facility Funds of the NSFC and CAS under Contracts No. U1732263, No. U1832207; CAS Key Research Program of Frontier Sciences under Contracts No. QYZDJ-SSW-SLH003, No. QYZDJ-SSW- SLH040; 100 Talents Program of CAS; INPAC and Shanghai Key Laboratory for Particle Physics and Cosmology; ERC under Contract No. 758462; German Research Foundation DFG under Contracts Nos. 443159800, Collaborative Research Center CRC 1044, FOR 2359, FOR 2359, GRK 214; Istituto Nazionale di Fisica Nucleare, Italy; Ministry of Development of Turkey under Contract No. DPT2006K- 120470; National Science and Technology fund; Olle Engkvist Foundation under Contract No. 200-0605; STFC (United Kingdom); The Knut and Alice Wallenberg g y g Biases of up to 0.7° are observed in the central values of γ for all binning schemes. A bias was reported in the previous analysis as well [14]. The bias is smaller with our measure- ment, but non-negligible given the size of the pseudodata sample used. In order to investigate the source of the bias, we rerun the pseudodata experiments ignoring any pairs of ci and si values that lie outside the physical region given by c2 i þ s2 i < 1; the bias is still observed, which is due to the non-Gaussian nature of the truncated ci and si distributions. Therefore, instead of removing the unphysical values the uncertaintiesontheci andsi areartificiallyreducedbyafactor of three; in this case no observable bias is seen in any of the extracted parameters. Hence we conclude that the bias stems from some pairs of ci and si values lying outside the physical region.Therefore,futuremeasurementswithalargerψð3770Þ sample [45] are likely to reduce or remove this bias. X. IMPACT OF ci, si ON MEASUREMENT OF γ This is due to the fact that dividing the data into more bins is a better approximation of the unbinned case. The lack of improve- ment in sensitivity while going from N ¼ 3 to N ¼ 4 is due to the nature of the D0 →K0 SKþK−Dalitz plot. The dominant resonances contributing to D0 →K0 SKþK−are D0 →K0 Sϕ and D0 →K0 Sað980Þ0, which are both located close to the M2 KþK−kinematic limit. In a binning based on equal-ΔδD regions these bins are always enclosed by a similar pair of bins and the new pair of bins always encloses a region with a low density of events, as can be seen in Fig. 1. Good agreement with the previous measurements by the CLEO Collaboration [14] is obtained in all bins. Hence, we have performed a combination of the BESIII and CLEO results, which is reported in Appendix XV. The predictions from the BABAR model [16] lie within one to two standard deviations from our values. We have recently reported an amplitude model and branching fraction for D0 →K0 SKþK− decays [47]; the measured values of ci and si are also in agreement with this model. The estimated uncertainties on γ arising from the uncertainties on the measured values of ci and si are 2.3°, 1.3° and 1.3° for N ¼ 2, N ¼ 3 and N ¼ 4 equal-ΔδD binning, respectively. X. IMPACT OF ci, si ON MEASUREMENT OF γ In order to reduce the uncertainty due to the statistics of the B decay sample to a negligible level, the pseudodata sample size is set to 5 × 106 events. The bin yields are fluctuated according to a Poisson distribution to produce a new bin yield, NI. The expected bin yield, NE, is calculated using ci and si values obtained by smearing the measured values by their uncertainties, assuming a Gaussian distribution and taking the correlations into account. The best fit values of rB, δB and γ are extracted by minimizing χ2 ¼ P iðNI i −NE i Þ2=NE i . The pseudodata simulations are repeated 10000 times and the resulting γ distributions for N ¼ 2, 3 and 4 bins are shown in Fig. 9. detector, we report a measurement of the strong-phase difference parameters for D →K0 S;LKþK−decays with the best precision to date. The results presented here are an important input to model-independent measurements of the CKM angle γ using the BPGGSZ method. These values are also essential for the model-independent determination of charm-mixing parameters and in the search for CP viola- tion in D0 →K0 SKþK−decays [46]. We note that the statistical uncertainties limit the precision of our measure- ments. Therefore, it is desirable to collect larger ψð3770Þ data sets in future [45]. For most values of ci and si, the major source of systematic uncertainty is due to the input strong-phase difference parameters of D →K0 S;Lπþπ−decays [13]. Significant sys- tematic uncertainties on sð0Þ i also arise from the background parametrization of the partially reconstructed D →K0 LX decay modes and the values of Kð0Þ i used. All these uncer- tainties depend on the size of the charm sample available. , g The distributions of all parameters (rB, δB and γ) are found to be asymmetric. Using the root mean square (RMS) of the distribution, we estimate the uncertainties on γ to be 2.3°, 1.3° and 1.3° for the schemes with N ¼ 2, 3 and 4 bins, respectively. We also estimate an asymmetric uncertainty by integrating 16% of the distribution in the lower and upper tails to work out a 68% confidence level; the asymmetric uncertainties on γ are þ2.4° −1.8°, þ1.0° −0.9° and þ0.9° −0.9° for schemes with N ¼ 2, 3 and 4 bins, respectively. Better sensitivity for N ¼ 3 and 4 compared to N ¼ 2 is observed. X. IMPACT OF ci, si ON MEASUREMENT OF γ The values of ci and si are used as an input to the model- independent determination of γ using the B−→DK−; D → K0 SKþK−decay. The uncertainties on the measured values of ci and si introduce a related systematic uncertainty on the measured value of γ, which we here estimate through a simulation study. To estimate the systematic uncertainty from the back- ground parametrization, the estimated amount of back- ground is varied by a Gaussian function within its statistical uncertainty. The level of the background with respect to the partially reconstructed tags is larger than that with respect We simulate the decay rate of B−→DK−; D → K0 SKþK−within the Dalitz plot bins using the relation in Eq. (3). In the simulation, the input values of ci, si, Ti FIG. 9. Distribution of γ obtained from pseudodata experiments for N ¼ 2 (left), N ¼ 3 (middle) and N ¼ 4 (right) binning schemes. FIG. 9. Distribution of γ obtained from pseudodata experiments for N ¼ 2 (left), N ¼ 3 (middle) and N ¼ 4 (right) binning schemes. 052008-21 052008-21 PHYS. REV. D 102, 052008 (2020) M. ABLIKIM et al. and T−i are set to those reported here. The values of rB, δB and γ are taken to be 0.103, 136.9° and 73.5°, respectively (see Ref. [41]). In order to reduce the uncertainty due to the statistics of the B decay sample to a negligible level, the pseudodata sample size is set to 5 × 106 events. The bin yields are fluctuated according to a Poisson distribution to produce a new bin yield, NI. The expected bin yield, NE, is calculated using ci and si values obtained by smearing the measured values by their uncertainties, assuming a Gaussian distribution and taking the correlations into account. The best fit values of rB, δB and γ are extracted by minimizing χ2 ¼ P iðNI i −NE i Þ2=NE i . The pseudodata simulations are repeated 10000 times and the resulting γ distributions for N ¼ 2, 3 and 4 bins are shown in Fig. 9. and T−i are set to those reported here. The values of rB, δB and γ are taken to be 0.103, 136.9° and 73.5°, respectively (see Ref. [41]). XI. SUMMARY Using a sample of ψð3770Þ data corresponding to an integrated luminosity of 2.93 fb−1 collected by the BESIII 052008-22 PHYS. REV. D 102, 052008 (2020) IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … Foundation (Sweden) under Contract No. 2016.0157; The Royal Society, UK under Contracts No. DH140054, No. DH160214; The Swedish Research Council; U.S. Department of Energy under Contracts No. DE-FG02- 05ER41374, No. DE-SC-0012069. schemes are given in Tables IX and X, respectively. These tables complement Table VIII for the N ¼ 3 binning scheme, which is given in Sec. IX. APPENDIX A: ADDITIONAL TABLES OF SYSTEMATIC UNCERTAINTIES We report the statistical (systematic) covariance matrices related to the measurements for the N ¼ 2, 3 and 4 binning schemes in Tables XI (XII), XIII (XIV) and XV (XVI), respectively. The contributions to the systematic uncertainty of the cð0Þ i and sð0Þ i measurements for the N ¼ 2 and N ¼ 4 binning TABLE IX. Summary of the contributions to the systematic uncertainty for the N ¼ 2 equal-ΔδD binning. Systematic c1 c2 s1 s2 c0 1 c0 2 s0 1 s0 2 ST yield 0.002 0.003 0.000 0.000 0.002 0.001 0.000 0.000 Kð0Þ i statistics 0.000 0.003 0.004 0.005 0.001 0.002 0.004 0.004 K0πþπ−ðcð0Þ i ; sð0Þ i Þ 0.001 0.002 0.037 0.075 0.001 0.002 0.037 0.076 K0πþπ−ðKð0Þ i Þ 0.000 0.001 0.007 0.033 0.000 0.000 0.006 0.033 ND ¯D 0.000 0.000 0.001 0.001 0.000 0.000 0.001 0.001 MC statistics 0.001 0.002 0.000 0.000 0.001 0.003 0.000 0.000 Background 0.002 0.003 0.011 0.022 0.002 0.003 0.011 0.022 DCS correction 0.000 0.000 0.000 0.001 0.000 0.000 0.000 0.001 Stat 0.034 0.040 0.144 0.198 0.035 0.034 0.144 0.198 Syst total 0.003 0.007 0.039 0.085 0.003 0.006 0.039 0.086 Total 0.034 0.041 0.149 0.215 0.035 0.035 0.149 0.216 butions to the systematic uncertainty for the N ¼ 2 equal-ΔδD binning. TABLE IX. Summary of the contributions to the systematic uncertainty for the N ¼ 2 equal-ΔδD binning. X. Summary of the contributions to the systematic uncertainty for the N ¼ 2 equal-ΔδD binning. TABLE X. Summary of the contributions to the systematic uncertainty for the N ¼ 4 equal-ΔδD binning. APPENDIX A: ADDITIONAL TABLES OF SYSTEMATIC UNCERTAINTIES Systematic c1 c2 c3 c4 s1 s2 s3 s4 c0 1 c0 2 c0 3 c0 4 s0 1 s0 2 s0 3 s0 4 ST yield 0.001 0.003 0.006 0.004 0.002 0.006 0.002 0.003 0.001 0.003 0.006 0.001 0.002 0.006 0.002 0.000 Kð0Þ i statistics 0.002 0.008 0.007 0.010 0.002 0.009 0.005 0.026 0.002 0.008 0.007 0.010 0.002 0.009 0.005 0.026 K0πþπ−ðci; siÞ 0.002 0.003 0.003 0.015 0.048 0.089 0.053 0.091 0.002 0.003 0.003 0.008 0.048 0.089 0.053 0.087 K0πþπ−ðKð0Þ i Þ 0.001 0.001 0.001 0.003 0.009 0.013 0.017 0.016 0.001 0.001 0.001 0.001 0.009 0.013 0.017 0.016 ND ¯D 0.001 0.000 0.001 0.001 0.001 0.003 0.004 0.000 0.001 0.000 0.001 0.000 0.001 0.003 0.004 0.000 MC statistics 0.000 0.013 0.003 0.015 0.002 0.006 0.002 0.001 0.000 0.012 0.003 0.026 0.001 0.006 0.002 0.001 Background 0.001 0.007 0.003 0.033 0.013 0.086 0.019 0.170 0.001 0.007 0.003 0.018 0.013 0.085 0.019 0.182 DCS correction 0.000 0.000 0.000 0.001 0.001 0.000 0.001 0.000 0.000 0.000 0.000 0.002 0.000 0.000 0.001 0.000 Stat 0.034 0.106 0.057 0.208 0.173 0.294 0.210 0.387 0.034 0.106 0.056 0.201 0.174 0.294 0.210 0.422 Syst total 0.003 0.017 0.011 0.041 0.051 0.125 0.059 0.195 0.003 0.017 0.011 0.036 0.051 0.124 0.059 0.206 Total 0.034 0.107 0.058 0.212 0.180 0.320 0.218 0.433 0.034 0.107 0.057 0.204 0.181 0.319 0.218 0.469 TABLE X. Summary of the contributions to the systematic uncertainty for the N ¼ 4 equal-ΔδD binning. APPENDIX A: ADDITIONAL TABLES OF SYSTEMATIC UNCERTAINTIES Systematic c1 c2 c3 c4 s1 s2 s3 s4 c0 1 c0 2 c0 3 c0 4 s0 1 s0 2 s0 3 s0 4 ST yield 0.001 0.003 0.006 0.004 0.002 0.006 0.002 0.003 0.001 0.003 0.006 0.001 0.002 0.006 0.002 0.000 Kð0Þ i statistics 0.002 0.008 0.007 0.010 0.002 0.009 0.005 0.026 0.002 0.008 0.007 0.010 0.002 0.009 0.005 0.026 K0πþπ−ðci; siÞ 0.002 0.003 0.003 0.015 0.048 0.089 0.053 0.091 0.002 0.003 0.003 0.008 0.048 0.089 0.053 0.087 K0πþπ−ðKð0Þ i Þ 0.001 0.001 0.001 0.003 0.009 0.013 0.017 0.016 0.001 0.001 0.001 0.001 0.009 0.013 0.017 0.016 ND ¯D 0.001 0.000 0.001 0.001 0.001 0.003 0.004 0.000 0.001 0.000 0.001 0.000 0.001 0.003 0.004 0.000 MC statistics 0.000 0.013 0.003 0.015 0.002 0.006 0.002 0.001 0.000 0.012 0.003 0.026 0.001 0.006 0.002 0.001 Background 0.001 0.007 0.003 0.033 0.013 0.086 0.019 0.170 0.001 0.007 0.003 0.018 0.013 0.085 0.019 0.182 DCS correction 0.000 0.000 0.000 0.001 0.001 0.000 0.001 0.000 0.000 0.000 0.000 0.002 0.000 0.000 0.001 0.000 Stat 0.034 0.106 0.057 0.208 0.173 0.294 0.210 0.387 0.034 0.106 0.056 0.201 0.174 0.294 0.210 0.422 Syst total 0.003 0.017 0.011 0.041 0.051 0.125 0.059 0.195 0.003 0.017 0.011 0.036 0.051 0.124 0.059 0.206 Total 0.034 0.107 0.058 0.212 0.180 0.320 0.218 0.433 0.034 0.107 0.057 0.204 0.181 0.319 0.218 0.469 TABLE XI. Statistical correlation matrix (%) for the K0 SKþK− equal-ΔδD N ¼ 2 binning. c2 s1 s2 c0 1 c0 2 s0 1 s0 2 c1 4.8 2.9 −0.2 94.1 3.0 2.9 −0.2 c2 −1.2 −1.5 5.1 63.2 −1.2 −1.5 s1 −0.4 2.8 −0.8 99.4 0.4 TABLE XII. Systematic correlation (%) matrix for K0 SKþK− equal-ΔδD N ¼ 2 binning. c2 s1 s2 c0 1 c0 2 s0 1 s0 2 c1 36.6 −1.3 −0.1 90.0 27.3 −1.3 −0.1 c2 −5.3 −8.4 26.7 58.4 −5.4 −8.4 s1 −21.1 1.6 12.7 99.6 −21.2 TABLE X. Summary of the contributions to the systematic uncertainty for the TABLE XI. Statistical correlation matrix (%) for the K0 SKþK− equal-ΔδD N ¼ 2 binning. c2 s1 s2 c0 1 c0 2 s0 1 s0 2 c1 4.8 2.9 −0.2 94.1 3.0 2.9 −0.2 c2 −1.2 −1.5 5.1 63.2 −1.2 −1.5 s1 −0.4 2.8 −0.8 99.4 0.4 s2 −0.2 −1.6 −0.3 99.5 c0 1 3.2 2.8 −0.2 c0 2 −0.8 1.6 s0 1 0.3 TABLE XII. APPENDIX A: ADDITIONAL TABLES OF SYSTEMATIC UNCERTAINTIES Systematic correlation (%) matrix for K0 SKþK− equal-ΔδD N ¼ 2 binning. c2 s1 s2 c0 1 c0 2 s0 1 s0 2 c1 36.6 −1.3 −0.1 90.0 27.3 −1.3 −0.1 c2 −5.3 −8.4 26.7 58.4 −5.4 −8.4 s1 −21.1 1.6 12.7 99.6 −21.2 s2 0.7 −10.9 −21.0 90.0 c0 1 33.4 −1.6 −0.7 c0 2 12.6 −11.1 s0 1 −21.1 TABLE XI. Statistical correlation matrix (%) for the K0 SKþK− equal-ΔδD N ¼ 2 binning. c2 s1 s2 c0 1 c0 2 s0 1 s0 2 c1 4.8 2.9 −0.2 94.1 3.0 2.9 −0.2 c2 −1.2 −1.5 5.1 63.2 −1.2 −1.5 s1 −0.4 2.8 −0.8 99.4 0.4 s2 −0.2 −1.6 −0.3 99.5 c0 1 3.2 2.8 −0.2 c0 2 −0.8 1.6 s0 1 0.3 TABLE XI. Statistical correlation matrix (%) for the K0 SKþK− equal-ΔδD N ¼ 2 binning. TABLE XII. Systematic correlation (%) matrix for K0 SKþK− equal-ΔδD N ¼ 2 binning. c2 s1 s2 c0 1 c0 2 s0 1 s0 2 c1 36.6 −1.3 −0.1 90.0 27.3 −1.3 −0.1 c2 −5.3 −8.4 26.7 58.4 −5.4 −8.4 s1 −21.1 1.6 12.7 99.6 −21.2 s2 0.7 −10.9 −21.0 90.0 c0 1 33.4 −1.6 −0.7 c0 2 12.6 −11.1 s0 1 −21.1 TABLE XII. Systematic correlation (%) matrix for K0 SKþK− equal-ΔδD N ¼ 2 binning. 052008-23 PHYS. REV. D 102, 052008 (2020) M. ABLIKIM et al. E XIII. Statistical correlation matrix (%) for K0 SKþK−equal-ΔδD N ¼ 3 binning. TABLE XIII. Statistical correlation matrix (%) for K0 SKþK−equal-ΔδD N ¼ 3 binning. c2 c3 s1 s2 s3 c0 1 c0 2 c0 3 s0 1 s0 2 s0 3 c1 1.3 1.8 1.2 0.1 0.0 9.7 1.2 0.4 −1.2 0.1 0.0 c2 0.7 0.1 9.9 1.5 1.3 96.5 0.1 0.1 9.9 1.3 c3 −1.4 −1.3 0.9 1.8 0.7 24.3 −1.4 −1.3 0.8 s1 −5.5 −7.2 1.1 0.1 −0.3 99.7 −5.5 −6.6 s2 11.7 0.1 9.5 −0.4 −5.5 99.9 10.7 s3 0.0 1.4 −0.3 −7.2 11.7 91.3 c0 1 1.3 0.4 −1.1 0.1 0.0 c0 2 0.2 0.1 9.4 1.3 c0 3 −0.3 −0.4 −0.4 s0 1 −5.5 −6.6 s0 2 10.7 TABLE XIV. Systematic correlation matrix (%) for K0 SKþK−equal-ΔδD N ¼ 3 binning. IMPROVED MODEL-INDEPENDENT DETERMINATION OF THE … TABLE XVI. Systematic correlation matrix for K0 SKþK−equal-ΔδD N ¼ 4 binning. c2 c3 c4 s1 s2 s3 s4 c0 1 c0 2 c0 3 c0 4 s0 1 s0 2 s0 3 s0 4 c1 2.8 2.7 −2.7 0.2 −3.8 −1.9 2.0 2.2 2.4 2.7 −1.3 0.2 −3.8 −2.1 1.8 c2 53.2 −23.7 12.6 2.9 −7.7 16.3 −30.8 64.0 49.1 −7.2 −12.6 2.9 −9.0 11.3 c3 −24.8 −28.5 −5.0 −16.7 22.9 −33.3 50.3 34.3 −10.5 −22.7 −5.0 −17.1 20.4 c4 −18.9 13.3 0.1 −65.2 31.6 −18.0 −22.3 58.0 −18.9 13.3 1.1 −35.0 s1 −51.3 3.1 −14.9 2.6 1.0 27.5 −9.5 39.0 −51.3 3.1 −12.1 s2 3.9 3.7 0.9 3.0 −4.8 5.9 −43.0 99.0 3.9 2.4 s3 −13.8 1.0 −8.9 −16.5 −1.5 3.2 3.9 93.0 −8.8 s4 −0.5 2.0 6.1 −19.0 −1.8 3.8 −14.3 39.8 c0 1 −24.2 −29.1 19.4 2.7 −1.1 1.7 −14.6 c0 2 50.4 −14.4 12.8 2.8 −7.4 15.8 c0 3 −13.2 −27.5 −4.9 −16.1 20.9 c0 4 −9.6 6.2 −3.5 −43.8 s0 1 −51.2 3.2 −12.1 s0 2 3.9 2.3 s0 3 −8.4 TABLE XVI. Systematic correlation matrix for K0 SKþK−equal-ΔδD N ¼ 4 binning. APPENDIX A: ADDITIONAL TABLES OF SYSTEMATIC UNCERTAINTIES c2 c3 s1 s2 s3 c0 1 c0 2 c0 3 s0 1 s0 2 s0 3 c1 3.1 −31.0 −1.5 0.9 −7.4 20.0 5.7 6.4 −1.5 0.9 −6.9 c2 71.3 −21.2 2.1 7.4 −2.0 30.1 24.9 21.2 2.1 8.7 c3 −55.4 6.7 −7.0 −13.0 18.8 57.4 −0.9 6.6 5.6 s1 −30.4 −23.8 −0.5 −18.9 −19.3 9.9 −30.4 −23.6 s2 29.2 1.2 1.1 −0.4 −30.4 90.0 31.6 s3 −8.0 5.9 −13.3 23.9 20.0 67.0 c0 1 0.8 5.7 −0.5 1.2 −7.6 c0 2 23.8 −18.9 1.1 7.0 c0 3 −19.4 −0.4 −13.3 s0 1 −30.4 −23.7 s0 2 31.6 TABLE XIV. Systematic correlation matrix (%) for K0 SKþK−equal-ΔδD N ¼ 3 binning. TABLE XV. Statistical correlation matrix (%) for K0 SKþK−equal-ΔδD N ¼ 4 binning. c2 c3 c4 s1 s2 s3 s4 c0 1 c0 2 c0 3 c0 4 s0 1 s0 2 s0 3 s0 4 c1 0.8 2.3 0.8 −2.3 0.2 −0.2 0.0 98.6 0.8 2.3 0.2 −2.3 0.2 −0.2 0.0 c2 0.3 −0.8 0.7 −3.0 0.0 0.1 0.8 98.4 0.3 −0.2 0.7 −3.0 0.0 0.1 c3 −0.1 −0.6 0.2 4.3 0.0 2.4 0.3 97.0 −0.0 −0.6 0.2 4.3 0.0 c4 0.0 0.1 0.0 −5.7 0.8 −0.8 −0.1 24.9 0.0 0.1 0.0 −3.6 s1 −10.6 1.5 −0.1 −2.4 0.7 −0.5 0.0 99.7 −10.6 1.5 −0.1 s2 4.2 −2.6 0.2 −3.0 0.2 0.0 −10.6 99.9 4.2 −1.7 s3 0.0 −0.2 0.0 4.2 0.0 1.5 4.2 99.8 0.0 s4 0.0 0.1 0.0 −0.3 −0.1 −2.6 0.0 66.6 c0 1 0.8 2.3 0.2 −2.4 0.2 −0.2 0.0 c0 2 0.3 −0.2 0.7 −2.9 0.0 0.1 c0 3 0.0 −0.5 0.2 4.2 0.0 c0 4 0.0 0.0 0.0 0.8 s0 1 −10.6 1.5 −0.1 s0 2 4.2 −1.7 s0 3 0.0 TABLE XV. Statistical correlation matrix (%) for K0 SKþK−equal-ΔδD N ¼ 4 binning. 052008-24 PHYS. REV. D 102, 052008 (2020) MPROVED MODEL-INDEPENDENT DETERMINATION OF THE … PHYS. REV APPENDIX C: COMBINATION OF BESIII AND CLEO RESULTS ABLIKIM et al. TABLE XIX. Correlation matrix (%) of the combined BESIII and CLEO results for the K0 SKþK−equal-ΔδD N ¼ 3 binning. c2 c3 s1 s2 s3 c0 1 c0 2 c0 3 s0 1 s0 2 s0 3 c1 3.6 0.6 1.8 −13.8 0.6 98.3 3.8 1.1 1.8 −13.8 0.7 c2 4.1 1.9 4.4 1.7 3.7 93.0 2.5 6.1 4.4 1.6 c3 −5.4 1.2 0.9 1.1 1.4 35.8 1.3 1.1 1.9 s1 −1.7 −2.4 2.1 2.4 −1.6 79.0 −1.7 −2.3 s2 7.5 −13.2 4.1 0.6 −1.7 98.4 7.4 s3 0.8 1.7 −1.1 6.1 6.1 89.6 c0 1 3.9 1.3 2.1 −13.1 0.8 c0 2 2.7 2.4 4.1 1.6 c0 3 −1.6 −1.7 −2.3 s0 1 −1.7 −2.3 s0 2 7.5 TABLE XIX. Correlation matrix (%) of the combined BESIII and CLEO results for the K0 SKþK−equal-ΔδD N ¼ 3 binning. XIX. Correlation matrix (%) of the combined BESIII and CLEO results for the K0 SKþK−equal-ΔδD nning TABLE XX. Correlation matrix (%) of the combined BESIII and CLEO results for the K0 SKþK−equal-ΔδD N ¼ 4 binning. c2 c3 c4 s1 s2 s3 s4 c0 1 c0 2 c0 3 c0 4 s0 1 s0 2 s0 3 s0 4 c1 3.1 3.2 2.5 −1.3 −12.8 0.7 3.9 98.6 3.2 3.1 −0.6 −1.4 −12.9 0.7 5.6 c2 3.7 −2.4 5.0 −1.7 −0.3 0.2 2.6 97.3 3.5 −0.2 3.7 −1.7 −0.4 −0.8 c3 −0.6 −2.9 −3.1 −0.4 2.7 2.4 3.6 95.1 −0.4 −2.5 −3.1 −0.4 2.7 c4 3.3 2.6 1.0 −2.6 3.2 −2.2 −0.5 41.0 3.3 2.6 1.0 0.9 s1 −39.4 14.3 10.4 −1.4 4.5 0.8 −2.1 94.2 −39.4 14.3 13.4 s2 −8.0 −3.8 −12.5 −1.7 −3.1 0.2 −38.2 99.6 −8.0 −3.6 s3 0.7 0.7 −0.3 −0.5 −0.7 14.3 −8.0 99.3 2.2 s4 3.6 −1.0 0.8 −0.4 12.1 −3.8 0.6 77.6 c0 1 2.8 2.4 −0.1 −1.4 −12.6 0.8 4.7 c0 2 3.6 −0.4 5.1 −1.7 −0.3 −0.5 c0 3 −0.4 −2.9 −3.1 −0.5 2.8 c0 4 −2.1 0.2 −0.8 −3.1 s0 1 −39.5 14.3 13.3 s0 2 −8.0 −3.6 s0 3 2.3 ) of the combined BESIII and CLEO results for the K0 SKþK−equal-ΔδD N ¼ 4 binning. and systematic covariance matrix related to the CLEO measurements. The values of cð0Þ i and sð0Þ i obtained from the fit including the χ2avg term are given in Table XVII. [3] J. Brod and J. Zupan, J. High Energy Phys. 01 (2014) 051. [4] M. Blanke and A. J. Buras, Eur. Phys. J. C 79, 159 (2019). [5] A. Lenz and G. Tetlalmatzi-Xolocotzi, J. High Energy Phys. 07 (2020) 177. APPENDIX C: COMBINATION OF BESIII AND CLEO RESULTS expression given in Eq. (28). The constraint term is defined as expression given in Eq. (28). The constraint term is defined as The results presented in this paper are compatible with those reported by the CLEO Collaboration [14]. Therefore, it is advantageous to perform an average of the two sets of results to get the best possible estimates of cð0Þ i and sð0Þ i . Following Ref. [13], the average is calculated by adding a multidimensional constraint term to the log-likelihood χ2avg ¼ ðP −PCLEOÞTV−1ðP −PCLEOÞ; ðC1Þ ðC1Þ where P is the vector of 4N parameters in the fit, PCLEO is the vector of corresponding values reported by the CLEO Collaboration and V is the 4N × 4N combined statistical TABLE XVII. Results for cð0Þ i and sð0Þ i from averaging the results from BESIII and CLEO. The uncertainties on the values of parameters are the statistical uncertainties obtained from fit added in quadrature with the systematic uncertainties. VII. Results for cð0Þ i and sð0Þ i from averaging the results from BESIII and CLEO. The uncertainties on the arameters are the statistical uncertainties obtained from fit added in quadrature with the systematic s. N i ci si c0 i s0 i 2 1 0.713 0.032 0.107 0.132 0.737 0.032 0.116 0.132 2 −0.758 0.037 0.394 0.173 −0.782 0.033 0.473 0.174 3 1 0.738 0.030 0.112 0.102 0.765 0.030 0.111 0.102 2 −0.573 0.044 0.550 0.113 −0.503 0.044 0.574 0.113 3 −0.129 0.155 −0.619 0.317 −0.412 0.138 0.089 0.327 4 1 0.796 0.030 −0.082 0.173 0.817 0.030 −0.080 0.173 2 −0.018 0.099 0.393 0.262 0.105 0.098 0.375 0.261 3 −0.691 0.048 0.551 0.200 −0.657 0.048 0.601 0.200 4 0.183 0.182 −0.646 0.415 −0.321 0.185 0.218 0.438 TABLE XVIII. Correlation matrix (%) of the combined BESIII and CLEO results for the K0 SKþK−equal-ΔδD N ¼ 2 binning. c2 s1 s2 c0 1 c0 2 s0 1 s0 2 c1 7.1 −4.3 −0.8 94.4 5.3 −4.4 −0.8 c2 −3.5 −2.8 6.8 65.9 −3.6 −2.9 s1 −4.1 −4.1 −2.5 99.2 −4.0 s2 −0.8 −3.5 −4.0 96.8 c0 1 5.8 −4.1 −0.8 c0 2 −2.3 −3.4 s0 1 −3.9 052008-25 PHYS. REV. D 102, 052008 (2020) M. [1] N. Cabibbo, Phys. Rev. Lett. 10, 531 (1963); M. Kobayashi and T. Maskawa, Prog. Theor. Phys. 49, 652 (1973). [2] M. Gronau and D. Wyler, Phys. Lett. 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https://openalex.org/W2898021514	https://europepmc.org/articles/pmc6211248?pdf=render	English	null	PHO15 genes of Candida albicans and Candida parapsilosis encode HAD-type phosphatases dephosphorylating 2-phosphoglycolate	FEMS yeast research	2,018	cc-by	7,255	Eliˇska Kroˇcov´a1,§,†, Sylva Neradov´a2,†, Rudolf Kupcik1, Sylva Janovsk´a1,‡, Zuzana B´ılkov´a1 and Olga Heidingsfeld1,3,∗,# 1Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, 532 10 Pardubice, Czech Republic, 2Gymnasium, Pardubice, Mozartova, 530 09 Pardubice, Czech Republic and 3Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague, Czech Republic ∗Corresponding author: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Studentsk´a 573 (HB/B), 532 10 Pardubice, Czech Republic. Tel: +420 466 037,779; E-mail: olga.heidingsfeld@upce.cz †These authors contributed equally to this work. y Present address: Department of Epidemiology, Faculty of Military Health Sciences, University of Defence, Hradec Kr´alov´e. One sentence summary: PHO15 genes of Candida albicans and C. parapsilosis encode HAD-type phosphatases, which have no and dephosphorylate 2 phosphoglycolate ‡Present address: Department of Epidemiology, Faculty of Military Health Sciences, University of Defence, Hradec Kr´alov´e. One sentence summary: PHO15 genes of Candida albicans and C. parapsilosis encode HAD-type phosphatases, which have not been characterised as yet and dephosphorylate 2-phosphoglycolate. di l Editor: Carol Munro §Eliˇska Kroˇcov´a, http://orcid.org/0000-0002-4106-3062 #Olga Heidingsfeld, http://orcid.org/0000-0001-9881-8473 ABSTRACT Most of the phosphatases of human fungal pathogens Candida albicans and C. parapsilosis have never been experimentally characterised, although dephosphorylation reactions are central to many biological processes. PHO15 genes of these yeasts have been annotated as the sequences encoding 4-nitrophenyl phosphatase, on the basis of homology to PHO13 gene from the bakers’ yeast Saccharomyces cerevisiae. To examine the real function of these potential phosphatases from Candida spp., CaPho15p and CpPho15p were prepared using expression in Escherichia coli and characterised. They share the hallmark motifs of the haloacid dehalogenase superfamily, readily hydrolyse 4-nitrophenyl phosphate at pH 8–8.3 and require divalent cations (Mg2+, Mn2+ or Co2+) as cofactors. CaPho15p and CpPho15p did not dephosphorylate phosphopeptides, but rather hydrolysed molecules related to carbohydrate metabolism. The preferred substrate for the both phosphatases was 2-phosphoglycolate. Among the other molecules tested, CaPho15 showed preference for glyceraldehyde phosphate and ß-glycerol phosphate, while CpPho15 dephosphorylated mainly 1,3-dihydroxyacetone phosphate. This type of substrate specificity indicates that CaPho15 and CpPho15 may be a part of metabolic repair system of C. albicans and C. parapsilosis. Keywords: Candida albicans; Candida parapsilosis; phosphatase; HAD-family; 2-phosphoglycolate; PHO15 Received: 7 August 2018; Accepted: 8 October 2018 Present address: Department of Epidemiology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove. One sentence summary: PHO15 genes of Candida albicans and C. parapsilosis encode HAD-type phosphatases, which have not been characterised as yet and dephosphorylate 2-phosphoglycolate. g ; p C⃝FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com FEMS Yeast Research, 19, 2019, foy112 doi: 10.1093/femsyr/foy112 Advance Access Publication Date: 10 October 2018 Research Article Received: 7 August 2018; Accepted: 8 October 2018 C⃝FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com C⃝FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com ibuted equally to this work. partment of Epidemiology, Faculty of Military Health Sciences, University of Defence, Hradec Kr´alov´e. INTRODUCTION associated with diseases (Casadevall and Pirofski 2015). How- ever, advanced medical treatment such as chemotherapy, or- gan transplantation or wide use of antibiotics significantly im- proved survival rates for critically ill patients. As a consequence, the number of patients at risk has increased and infections caused by pathogenic Candida species have become a serious The yeasts Candida albicans and C. parapsilosis are among the most clinically relevant Candida species that represent a serious threat to individuals, whose immune system has been weak- ened, mucosal barriers damaged or whose natural microflora has been disturbed. Until the 1950s, Candida had only rarely been 2 FEMS Yeast Research, 2019, Vol. 19, No. 1 public health problem (Pfaller and Castanheira 2016). Superfi- cial infections, e.g. oral or vaginal thrush, can be usually cured by antimycotics available to date (Bondaryk, Kurz atkowski and Staniszewska 2013). However, disseminated invasive candidia- sis, which affects normally sterile body fluids and tissues, is of- ten refractory to treatment and is associated with high mortality rates (Berman 2012; Bilir et al. 2015). Candida albicans and C. para- psilosis are among causative agents of nosocomial bloodstream infections that spread through indwelling medical devices or through the hands of health care personnel (Guinea 2014; Pfaller and Castanheira 2016). ate. Motif IV, (G/S)(D/S)x3–4(D/E), provides two additional Asp or Asp and Glu residues that are involved in coordination of Mg2+, together with aspartates of motif I (Seifried, Schultz and Gohla 2013). Biochemical and structural characterisation of 13 recom- binant HAD-like phosphatases of S. cerevisiae has been re- cently published, and ScPho13p has been included to this panel (Kuznetsova et al. 2015). All of these enzymes required diva- lent cations (Mg2+, Mn2+, Co2+) and pH 6.5–7.5 for their activity. ScPho13p hydrolysed pNPP, but did not dephosphorylate phos- phopeptides. This is in contrast to results obtained with authen- tic ScPho13p isolated from yeast lysates, which was reported to dephosphorylate phosphoserines in casein and histone II-A and displayed highest activity at pH 8.2 (Tuleva, Vasileva-Tonkova and Galabova 1998). Recombinant ScPho13p instead dephospho- rylated 2-phosphoglycolate, glycerol-2-phosphate or phospho- enolpyruvate (Kuznetsova et al. 2015). Since ScPho13p was found to be a part of yeast metabolite repair system acting on poten- tially toxic substrates that arise as side products of glycolytic metabolism, preferential dephosphorylation of small molecules by this enzyme seems more likely (Collard et al. 2016). The dis- cordant data nonetheless called for further investigation. INTRODUCTION The genomes of several Candida species including C. albicans and C. parapsilosis have been sequenced (Butler et al. 2009; Jones et al. 2004) and C. albicans, which is the most frequent human fungal pathogen, has been extensively studied. The number of studies focusing on C. parapsilosis has recently increased as well. Availability of Candida genome sequences accelerated molecu- lar analysis of virulence traits and overall lifestyle of these fungi (Polke, Hube and Jacobsen 2015). Despite the rapid advancement, a large proportion of open reading frames in pathogenic Candida species have not been experimentally characterised and their function is being inferred from electronic annotation, relying mostly on similarities with orthologous genes in baker´s yeast Saccharomyces cerevisiae. This is also the case of PHO15 genes of C. albicans and C. parapsilosis (C1 07230W A and CPAR2 206650, respectively). Proteins encoded by these genes, CaPho15p and CpPho15p, have been annotated as alkaline phosphatases, more specifically as para-nitrophenyl phosphatases, according to a common compound used for colorimetric assay of phosphatase activity. However, they have never been experimentally char- acterised. The annotation is based on homology to alkaline phosphatase Pho13p of S. cerevisiae (ScPho13p), for which de- phosphorylation of para-nitrophenylphosphate (pNPP) has been experimentally confirmed (Tuleva, Vasileva-Tonkova and Gal- abova 1998). Current knowledge of the HAD superfamily phosphatases of C. albicans and C. parapsilosis is restricted to transcription regu- lation of PHO15 genes. Global transcription profiling studies of C. albicans placed CaPHO15 in a subset of 24 genes that were transcriptionally induced by multiple types of stresses, consti- tuting so-called core stress response (Enjalbert et al. 2006), and in a group of genes involved in regulation of switch from de- fault, white cells to opaque, mating competent cells (Hnisz, Schwarzm ¨uller and Kuchler 2009). The data on CpPHO15 are sparse. This gene was slightly upregulated in a C. parapsilosis mutant lacking transcription factor Bcr1p responsible for regu- lation of biofilm formation (Ding et al. 2011). The present study aims at characterisation of CaPho15p and CpPho15p on protein level. The both phosphatases were ex- pressed in Escherichia coli, purified and subjected to substrate specificity testing, which showed clear preference for small molecules related to carbohydrate metabolism over phospho- peptides. Catalytic mechanism of alkaline phosphatases usually involves conserved serine or threonine residue acting as a nu- cleophile that is transiently phosphorylated in the course of catalysis (O’Brien and Herschlag 2002; Pabis and Kamerlin 2016). MATERIALS AND METHODS Construction of PHO15 expression vectors INTRODUCTION However, protein sequence analysis of ScPho13p and the both Candida Pho15p suggested that they differ from majority of well- known alkaline phophatases and categorise into haloacid de- halogenase (HAD) superfamily. Despite the name that has been derived from archetypal enzyme haloacid dehalogenase of Pseu- domonas species, this superfamily includes enzymes catalysing phosphoryl or carbon transfer reactions on a diverse range of substrates, and also a large number of enzymes of unknown function (Burroughs et al. 2006; Motosugi, Esaki and Soda 1982). Purification of recombinant phosphatases where s is the standard deviation, N is the overall number of values, i is an index of a given value of absorbance in the sample, v is the given value, ¯v is the arithmetic average of the adequate values for sample, b is the value of absorbance in the blank and ¯b is the arithmetic average of the adequate values for blank. The standard deviation for each measurement was for the relative statement re-calculated as described above. Five millilitre of Ni-NTA Agarose (Qiagen) was equilibrated with the wash buffer (10 mM imidazole in distilled water). After cen- trifugation at 3000 g for 5 min, the supernatant was removed. Subsequently, solubilised inclusion bodies after dialysis with the addition of imidazole (final concentration 10 mM/l) were added to the Ni-NTA Agarose and the suspension was incubated for 1 h on a rotator at a room temperature. After the incuba- tion, the mixture was centrifuged again and the supernatant was collected. The Ni-NTA Agarose was washed with the wash buffer five times. The phosphatases were left immobilised on the Ni-NTA Agarose. Suspension of Ni-NTA Agarose with bound Pho15p in the wash buffer was stored at 4◦C as a stock solu- tion. The activity of the phosphatases remained stable up to 3 months. chaperones Escherichia coli BL21(DE3) were transformed with pG-KJE8 vector (TaKaRa) conferring chloramphenicol resistance and containing sequences for expression of five different chaperones. Transfor- mants were cultivated in LB broth supplemented with 20 μg/ml chloramphenicol, collected by centrifugation and made compe- tent using the protocol of Sambrook and Russell (2001). These competent cells were then transformed either with pET28- albi-Pho15 or with pET28-para-Pho15. Transformants were se- lected using LB-agar containing 20 μg/ml chloramphenicol and 40 μg/ml kanamycin. To determine the relative values of the absorbance, the repli- cates were arithmetically averaged, the average was reduced by the arithmetic average absorbance of the adequate blank and the highest value was set equal to 1. The other values were cal- culated accordingly. To compare separate measurements, previ- ous step was repeated, except the subtraction of the blank av- erage as its value was already included, and using the gained averages from the separate measurements. Standard deviation was determined according to the following formula: The transformants were inoculated into LB broth con- taining 20 μg/ml chloramphenicol and 40 μg/ml kanamycin. 0.5 mg/ml L-arabinose and 5 ng/ml tetracycline were added to induce chaperone expression. The culture was incubated in a ro- tation shaker at 37◦C up to OD600 = 0.4–0.6. Then, IPTG was added to the final concentration of 1 mM/l. The culture was incubated for another 3 h and harvested by centrifugation at 4000 g for 10 min. s = N i=1 (vi −¯v)2 N −1 − N i=1 bi −¯b 2 N −1 Bacterial expression of CaPho15p and CpPho15p Phosphatases were eluted from Ni-NTA Agarose either with 300 mM imidazole solution or with a 50 mM Bis-Tris Propane, 4 mM MgCl2 buffer of pH 9, 9.5 or 10.4. Alternatively, phos- phatases were released from the Ni-NTA Agarose by cleavage of the His-Tag: 2 U of thrombin in Tris-HCl pH 9.5, 50 mM NaCl, 10 mM MgCl2 were added and the mixture was incubated for 16 h on rotator at room temperature. Escherichia coli BL21(DE3) transformed either with pET28-albi- Pho15 or with pET28-para-Pho15 were cultivated in LB medium supplemented with kanamycin in a rotation shaker at 37◦C up to OD600 = 0.8. Then, Pho15p expression was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG; final concentration 1 mmol/l). The culture was incubated for another 3 h. The cells were harvested by centrifugation at 4000 g for 10 min. Construction of PHO15 expression vectors Candida albicans HE169 and C. parapsilosis P69 that were orig- inally isolated from blood and ear, respectively, were ob- tained from the mycological collection of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic. The yeasts were cultivated overnight in the YPD medium (yeast extract-peptone-dextrose; Sigma-Aldrich) and harvested by cen- trifugation at 4000 g for 10 min. Then, the DNA from the yeasts was isolated as described by Hoffman and Winston (1987). PHO15 sequences were amplified using the primers 5′-gccgcgcggcagccaGTCAATTAAAATTACTTCTAAAGA-3′ and 5′- gtcatgctagccataCTAATTGTTAAGCTCATGGA-3′ for C. albicans; gccgcgcggcagccatATGTCAGTAAAAATAACTG and gtcatgctagc- cataTCAATGGGTAAATTCGTA for C. parapsilosis. The sequences complementary to CaPHO15 and CpPHO15 are capitalised. PCR products were inserted into NdeI site of pET28b vector using the InFusion HD Cloning Kit (Clontech). The resulting vectors pET28-albi-Pho15 and pET28-para-Pho15 enabled expression of CaPho15p and CpPho15p with His-tags at the N-termini. The hallmark of HAD enzymes is the DxD signature, two aspartic residues separated by one amino acid and coordinat- ing Mg2+ ion. The first of the aspartates acts as a nucleophile and forms aspartylphosphate intermediate during the catalysis (Burroughs et al. 2006). Free phosphate is released and catalytic aspartate regenerated upon subsequent nucleophilic attack by a water molecule that has been deprotonated by the second aspar- tate (Asp + 2) of the DxD motif. Mg2+ facilitates correct position- ing of substrates in the active site and stabilises the transition state (Seifried, Schultz and Gohla 2013). The catalytic aspartates are a part of a motif I, which is one of four conserved motifs constituting the active sites of HAD family phosphatases. Motif II contains Ser/Thr after a stretch of hydrophobic amino acids. The role of Ser/Thr residue is formation of hydrogen bond with the phosphoryl group of a substrate. Motif III is poorly conserved, but the key residue is Lys that stabilises the reaction intermedi- Kroˇcov´a et al. 3 Bacterial expression of CaPho15p and CpPho15p Co-expression of CaPho15 and CpPho15 with chaperones In order to determine pH optimum of the phosphatases, pH of the buffer was adjusted to 6, 6.5, 7, 7.5, 7.75, 8, 8.3, 8.5, 9 and 9.5. To determine optimum Mg2+, Mn2+ and Co2+ concentration, 50 mM Tris-HCl buffer was adjusted to pH 8.3 for CaPho15p and to pH 8.0 for CpPho15p, and MgCl2 was added to final concentra- tion ranging from 0.01 to 100 mM. For the Mn2+ and Co2+ optima, the NaOH was not included due to the precipitation of the ions. Optimum temperature was determined using 50 mM Tris-HCl buffer. Isolation and solubilisation of inclusion bodies Activity of Pho15p enzymes was measured using the chro- mogenic substrate pNPP. For free Pho15p, the solubilised Pho15p after dialysis was mixed with 2x concentrated optimal buffer (see below) and with 15.2 mM pNPP in ratio 1:2:1. The sample was incubated for 20 min on an end-over-end rotator. After the incu- bation, 100 μl of the sample was pipetted to a microtiter plate. After addition of 18 μl of 6 M NaOH, absorbance of the sample was measured at 405 nm. Bacterial cells were resuspended in TN buffer (100 mM Tris- HCl, 150 mM NaCl, pH 7.4) containing lysozyme (1 mg/g of wet biomass). The mixture was stirred at room temperature for 30 min and frozen at –20◦C. After thawing, the mixture was treated by sonication and the resulting cell lysate was cen- trifuged at 9000 g for 10 min. The sediment containing inclusion bodies was washed twice with 100 mM Tris-HCl pH = 7.0, 2 M urea, 5 mM EDTA, 2% Triton X-100. The inclusion bodies were solubilised in 50 mM Tris-HCl pH 8.0, 8 M urea, 1 mM glycine, 1 mM EDTA, 100 mM β-mercaptoethanol and dialysed against 20 mM Tris-HCl, 150 mM NaCl, 100 mM MgCl2 pH 7.0. Alterna- tively, the inclusion bodies were solubilised in 60% acetic acid (v/v) and dialysed against distilled water. To perform the assay using immobilised phosphatases, ∼100 μg of Pho15p bound to Ni-NTA resin via the His-tag was mixed with 300 μl of a 50 mM Tris-HCl buffer containing 10 mM MgCl2 and 1.77 mM pNPP. The mixture was incubated for 20 min on an end-over-end rotator at a room temperature and centrifuged for 30 s at 9660 g. The supernatant (100 μl) was pipet- ted to a microtitre plate. After addition of 18 μl of 6 M NaOH, absorbance of the samples was measured at 405 nm. Mass spectrometry analysis of bacterially expressed phosphatases The SDS-PAGE gels containing samples of CaPho15p and Cp- Pho15p were prepared for mass spectrometry as described by Shevchenko et al. (2007) with slight modifications. Briefly, posi- tive bands were excised and destained in 50% (v/v) acetonitrile (ACN) in 100 mM NH4HCO3 at room temperature for 30 min a dehydrated with pure ACN. Proteins were reduced in 10 mM dithiothreitol at 60◦C for 30 min, dehydrated with pure ACN and then alkylated in 55 mM freshly prepared iodoacetamide at RT for 30 min in dark. After alkylation and dehydration, gel pieces were covered with trypsin solution (10 ng/μl in 10 mM NH4HCO3 containing 10% of ACN) for 30 min at 4◦C and then incubated overnight at 37◦C. Supernatant was collected, acidified with 5% trifluoroacetic acid and stored. Peptides from the gel were ex- tracted with 60% ACN/2% formic acid at 37◦C for 15 min. Ex- tracted samples were dried and stored for further analysis. In silico analysis using SignalP has not revealed signal se- quences in these proteins (Petersen et al. 2011). Analysis using WoLF PSORT indicates cytosolic localisation of CpPho15p but it also revealed a DNA-binding motif and a nuclear localisation signal. Nuclear localisation is even more likely in the case of CaPho15p (Horton et al. 2007). The hallmark motifs of HAD super- family are conserved among phosphatases from Candida species and from S. cerevisiae. In order to express the Candida PHO15 genes in E. coli, the whole coding sequences were amplified and inserted into pET28b, so that the resulting proteins have His-tags at the N- termini. Both CaPHO15 and CpPHO15 contain one CUG codon which encodes leucine in a standard genetic code. In Candida species belonging to the so-called CUG clade (or CTG clade), CUG is translated ambiguously, as serine or leucine. However, frequency of serine is ∼97% (Bezerra et al. 2013). In pET28-albi- Pho15 and pET28-para-Pho15, the CTG codons were not replaced with standard codons for serine. Therefore, the phosphatases expressed in E. coli using these vectors contain Leu in corre- sponding positions (Fig. 1). The extracted samples from in-gel digestion were desalted using microcolumns prepared from GELoader tips with reverse- phase POROS R⃝OLIGOTM R3 (Life Technologies) according to Gobom et al. (1999). Samples were directly eluted onto the MALDI plate using solution of α-cyano-4-hydroxycinnamic acid matrix (5 g/l in 60% ACN/0.1% TFA with 2 mM diammonium hydrogen citrate) which serves as a matrix. Bacterial expression and purification of Pho15p Both CaPho15p and CpPho15p were expressed in E. coli BL21(DE3), either using one expression plasmid or in two- plasmid co-expression system with five chaperones. While the use of the sole expression plasmids led to accumulation of phosphatases in inclusion bodies, during co-expression with chaperones the phosphatases remained soluble in cytosol. This, however, did not alleviate problems with solubility of both phos- phatases. During the attempts to purify them from bacterial cy- tosol, both CaPho15p and CpPho15p precipitated and rapidly lost their activity. Mass spectrometry analysis of bacterially expressed phosphatases Mass spectrometric analysis of desalted samples was performed on a Thermo Scientific MALDI LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) and acquired in the positive mode with full MS scan (700–4000 m/z) at 60 000 FWHM at m/z 400. Substrate specificity testing Concentration of free inorganic phosphate released from dif- ferent substrates was measured using the method described by Baginski, Epstein and Zak (1975) with minor alterations. An amount of 310 μl of 10 mM substrate in corresponding buffer (50 mM TRIS-HCl pH 8.3 for CaPho15p or 8.0 for CpPho15p, 4 FEMS Yeast Research, 2019, Vol. 19, No. 1 4 FEMS Yeast Research, 2019, Vol. 19, No. 1 In silico analysis of PHO15 genes and construction of expression vectors In silico analysis of PHO15 genes and construction of expression vectors The predicted alkaline phosphatases CaPho15p and CpPho15p share 75.4% identity and 86.4% similarity. Their identity to S. cerevisiae homologue ScPho13p is 51.2% for CaPho15p and 49.5% for CpPho15p (Fig. 1). The sequences of CaPho15p and CpPho15p differ mostly in the central part of the molecules and at the very C-termini. The regions in the proximity of the motifs I-IV are conserved. Km determination 10 mM MgCl2) was added to ∼100 μg of immobilised protein. The same amount of the chosen substrate was also added to 20 μl of protein-free matrix washed in Wash Buffer (10 mM imidazole, dH2O). These served as blanks. The mixture was incubated at 37◦C on a rotator for 20 min. After the incubation, samples and blanks were centrifuged (45 s, 9960 g) and the su- pernatant was collected. The concentration of the free inorganic phosphate was estimated as described by Baginski, Epstein and Zak (1975) with minor alterations. Instead of arsenite citrate, sodium citrate was used. The working standards included values 0, 0.01, 0.05, 0.1 and 1 mmol of phosphate per litre. The blanks were prepared as described above, and the reagents were added to the blanks in the same order as for the samples. For the Km determination, dilution series of the samples were prepared. The measurements were carried out as described above. The Km values were calculated using SigmaPlot (Systat Software Inc.). RESULTS CaPho15p and CpPHO15p do not dephosphorylate phosphopeptides precipitated under all the conditions tested. Enzyme activity and substrate specificity testing was therefore performed using Pho15 enzymes attached to the Ni-NTA Agarose via His-tag. This setup enabled to preserve enzyme activity, which remained un- changed for several months. As CaPho15p and CpPho15p were predicted to be alka- line phosphatases, phosphopeptides could be their natu- ral substrates. For this purpose, Phosphopeptide Standard Mixture (P33357, Thermo Fisher Scientific), containing phospho- rylated peptides at three different aminoacids (DHTGFLpTEpY- VATR, TRDIpYETDYYRK, VPIPGRFDRRVpTVE and DLDVPIP- GRFDRRVpSVAAE), was used as potential substrates. After incubation with CaPho15p and CpPho15p immobilised on Ni- NTA Agarose, no dephosphorylation was detected using mass spectrometry on either of phosphoserine, phosphothreonine or phosphotyrosine even after 300 min under optimised condi- tions. Substrate specificity of CaPho15p and CpPHO15p To identify potential natural substrates of CaPho15p and Cp- Pho15p, a set of small phosphorylated molecules was selected for dephosphorylation testing. Both phosphatases hydrolysed most efficiently 2-phosphoglycolate, but differed in preferences for other substrates (Fig. 5). While CaPho15p dephospho- rylated readily glyceraldehyde-3-phosphate and β-glycerol phosphate, CpPho15p preferred dihydroxyacetone phos- phate. Phosphoenolpyruvate, phosphorylated sugars, ADP, pyridoxal phosphate, thiamine pyrophosphate and phospho- ethanolamine were poor substrates or were not dephosphory- lated at all. Km values for 2-p-glycolate and 2-glycerolphosphate are in millimolar range for both phosphatases (Table 1, Fig. 6).i Dephosphorylation of para-nitrophenylphosphate by CaPho15p and CpPHO15p Both phosphatases readily hydrolysed pNPP. As illustrated by Fig. 3, pNPP was dephosphorylated most efficiently at pH 8– 8.3 and at 42◦C and 37◦C by CaPho15p and CpPho15p, respec- tively. Both CaPho15p and CpPho15p required the presence of a divalent cation for their activity and were able to use differ- ent cations as cofactors, similarly as S. cerevisiae HAD-family phosphatases (Kuznetsova et al. 2015). Although CaPho15p and CpPho15p showed slightly different profiles of optimal cofac- tor concentration (Fig. 4A–C), pNPP hydrolysis by both enzymes was supported by the cations in a similar order of efficiency: Mn2+ > Co2+ > Mg2+ > Ni2+ (Fig. 4D). CaPho15p was also active in the presence of Ca2+. Cu2+ was accepted as a cofactor by neither of the phosphatases. Mass spectrometry analysis of protein dephosphorylation Aspartate residues of DxD signature are bold and underlined. Residues constituting motifs II, III and IV are highlighted in green. Leucines encoded by the ambiguous CTG codon are boxed and highlighted in blue. CaPho15p and CpPHO15p do not dephosphorylate phosphopeptides Mass spectrometry analysis of protein dephosphorylation Phosphopeptides were dephosphorylated using 20 μl of settled Ni-NTA resin with Pho15p bound to via the His-tag. The resin was resuspended in 130 μl of a 50 mM Tris-HCl buffer (pH 8.3 or 8.0 for CaPho15p and CpPho15p, respectively) containing ei- ther 10 mM MgCl2, 1 mM CaCl2 or 1 mM MnCl2. The sample, 30 pmol of Phosphopeptide Standard Mixture (P33357, Thermo Fisher Scientific), containing peptides phosphorylated on serine, threonine and tyrosine. This mixture was incubated at 37◦C un- der stirring. For analysis, 20 μl of supernatant was collected after 30, 120 and 300 min of incubation and the reaction was imme- diately terminated by addition of 3 μl of 5% trifluoroacetic acid. Samples were desalted and analysed using mass spectrometry as described in previous section, differing only in utilising solu- tion of 2,5-dihydroxybenzoic acid (10 g/l in 50% ACN/0.1% TFA with 1% H3PO4) as MALDI matrix suitable for phosphopeptides (Larsen et al. 2005). Both enzymes were successfully purified from solubilised in- clusion bodies. The highest yield of phosphatases was obtained when the cells were harvested 3 h post induction. Solubilisation of the inclusion bodies in 60% acetic acid and subsequent dial- ysis against water was the only way to obtain soluble protein samples suitable for further purification, which was performed using Ni-NTA Agarose (Fig. 2). Identity of both phosphatases was verified using mass spectrometry analysis (data not shown). Several protocols were tested to release the phosphatases from the Ni-NTA Agarose, including elution with imida- zole solution, with buffer of a high pH, or cleavage of the His-tag using thrombin. However, free phosphatases rapidly Kroˇcov´a et al. 5 Kroˇcov´a et al. 5 Figure 1. Sequence alignment of alkaline phosphatase Pho13p from S. cerevisiae and phosphatases Pho15p from C. albicans and C. parapsilosis. Aspartate residues DxD signature are bold and underlined. Residues constituting motifs II, III and IV are highlighted in green. Leucines encoded by the ambiguous CTG codon are box and highlighted in blue. Figure 1. Sequence alignment of alkaline phosphatase Pho13p from S. cerevisiae and phosphatases Pho15p from C. albicans and C. parapsilosis. Aspartate residues of DxD signature are bold and underlined. Residues constituting motifs II, III and IV are highlighted in green. Leucines encoded by the ambiguous CTG codon are boxed and highlighted in blue. Figure 1. Sequence alignment of alkaline phosphatase Pho13p from S. cerevisiae and phosphatases Pho15p from C. albicans and C. parapsilosis. 6 FEMS Yeast Research, 2019, Vol. 19, No. 1 6 FEMS Yeast Research, 2019, Vol. 19, No. 1 Figure 3. Effect of temperature (A) and pH (B) on the activity of CaPho15p (solid line) and CpPho15p (dashed line). The reactions were performed in triplicate, using immobilised phosphatases and pNPP as a substrate. The details are de- scribed in the Material and Methods section. Figure 2. Bacterial expression and purification of CaPho15p (A) and CpPho15p (B). SDS-PAGE shows the protein composition of cell lysate (lane 1), soluble cyto- plasmic fraction (lane 2), wash of the inclusion bodies (lane 3), inclusion bodies (lane 4), solubilised inclusion bodies after dialysis (lane 5), flow-through fraction after His-tagged protein binding to Ni-NTA Agarose (lane 6), wash of the column (lane 7), purified phosphatase (lane 8). M—Molecular weight marker. Figure 3. Effect of temperature (A) and pH (B) on the activity of CaPho15p (solid line) and CpPho15p (dashed line). The reactions were performed in triplicate, using immobilised phosphatases and pNPP as a substrate. The details are de- scribed in the Material and Methods section. peptides. This was in accordance with the results published by Collard et al. (2016), who described Pho13p as a homolog of mammalian phosphoglycolate phosphatase. These enzymes were found to be a part of the metabolic repair system thanks Figure 2. Bacterial expression and purification of CaPho15p (A) and CpPho15p (B). SDS-PAGE shows the protein composition of cell lysate (lane 1), soluble cyto- plasmic fraction (lane 2), wash of the inclusion bodies (lane 3), inclusion bodies (lane 4), solubilised inclusion bodies after dialysis (lane 5), flow-through fraction after His-tagged protein binding to Ni-NTA Agarose (lane 6), wash of the column (lane 7), purified phosphatase (lane 8). M—Molecular weight marker. Figure 2. Bacterial expression and purification of CaPho15p (A) and CpPho15p Figure 3. Effect of temperature (A) and pH (B) on the activity of CaPho15p (solid line) and CpPho15p (dashed line). The reactions were performed in triplicate, using immobilised phosphatases and pNPP as a substrate. The details are de- scribed in the Material and Methods section. Figure 2. Bacterial expression and purification of CaPho15p (A) and CpPho15p (B). SDS-PAGE shows the protein composition of cell lysate (lane 1), soluble cyto- plasmic fraction (lane 2), wash of the inclusion bodies (lane 3), inclusion bodies (lane 4), solubilised inclusion bodies after dialysis (lane 5), flow-through fraction after His-tagged protein binding to Ni-NTA Agarose (lane 6), wash of the column (lane 7), purified phosphatase (lane 8). 6 FEMS Yeast Research, 2019, Vol. 19, No. 1 M—Molecular weight marker. peptides. This was in accordance with the results published by Collard et al. (2016), who described Pho13p as a homolog of mammalian phosphoglycolate phosphatase. These enzymes were found to be a part of the metabolic repair system thanks to their ability to hydrolyse phosphorylated side products of carbohydrate metabolism. The present work therefore mainly addressed the question, whether C. albicans and C. parapsilo- sis homologs of Pho13p dephosphorylate phosphopeptides or phosphorylated carbohydrates. The results clearly demonstrate that the latter is true. No dephosphorylation of phosphopeptides by CaPho15p and CpPho15p was detected, and the both phos- phatases acted preferentially on 2-phosphoglycolate. Nonethe- less, it should be noted that the experiments were performed with purified, immobilised enzymes and that the CUG codons have not been replaced with non-ambiguous serine codons. Each phosphatase thus contains one leucine residue at the po- sition, which, under the natural conditions, is most probably occupied by serine. Although it is unlikely that these features profoundly change the substrate specificities of the Pho15p en- zymes, minor alterations cannot be ruled out. Availability and usage of different cofactors may also affect the performance of the phosphatases studied. potentially natural substrates differed. Preferences for Mn2+ or Co2+ varied depending on a particular substrate–enzyme combi- nation (data not shown). Mg2+ was therefore chosen because it is the biologically most relevant intracellular divalent cation and because it supported activity of both phosphatases similarly. CaPho15p and CpPHO15p do not display desulphurylation activity HAD family hydrolases are known to display desulphurylation activity. Therefore, hydrolysis of para-nitrophenylsulphate by CaPho15p and CpPho15p was tested. The experiments were per- formed under the optimum conditions required for pNPP de- phosphorylation, either in the presence of Mg2+ or Mn2+, but no desulphurylation activity was detected. Although hydrolysis of pNPP proceeded most efficiently in the presence of Mn2+ for both phosphatases, activity towards DISCUSSION Molecular function of the PHO15 genes of C. albicans and C. parapsilosis, as listed in the Candida Genome Database, was in- ferred from electronic annotation, on the basis of homology to S. cerevisiae PHO13 gene (www.candidagenome.org, 21 June 2018). However, the data on PHO13 reported in literature are contra- dictory. Tuleva, Vasileva-Tonkova and Galabova (1998) published isolation and characterisation of authentic Pho13p phosphatase from S. cerevisiae, showing its histone dephosphorylation activ- ity. Thus, when C. albicans pho15 deletion strain displayed al- terations in epigenetically regulated phenotypic feature known as white-opaque switching, it has been assumed to occur due to altered histone modification (Hnisz, Schwarzm ¨uller and Kuchler 2009). The origin of 2-phosphoglycolate is well described in plants and cyanobacteria, where it is formed as a side product of ribulose-1,5-bisphosphate carboxylase/oxygenase and further metabolised by phosphoglycolate phosphatases producing gly- colate and inorganic phosphate. 2-Phosphoglycolate is toxic, since it inhibits triosephosphate isomerase or phosphofructok- inase (Kelly and Latzko 1976; Norman and Colman 1991). But it is also a signal molecule playing a role in acclimation to low CO2 concentrations and its intracellular level has to be controlled Nevertheless, S. cerevisiae Pho13p phosphatase has later been identified as a HAD-family protein; it was prepared by bacte- rial expression and characterised within a large set of the yeast HAD enzymes (Kuznetsova et al. 2015). Recombinant Pho13p hy- drolysed phosphorylated carbohydrates rather than phospho- Kroˇcov´a et al. 7 Figure 4. Effect of magnesium (A), manganese (B) and cobalt (C) on the activity of CaPho15p (solid line) and CpPho15p (dashed line). The measurements were performed in triplicate, using immobilised phosphatases and pNPP as a substrate. The procedure is described in the Materials and Methods section. Comparison of CaPho15p (dark bars) and CpPho15p (light bars) activities in the presence of different divalent cations (D). For this comparison, 10 mM concentration of each cation was used. Figure 4. Effect of magnesium (A), manganese (B) and cobalt (C) on the activity of CaPho15p (solid line) and CpPho15p (dashed line). The measurements were performed in triplicate, using immobilised phosphatases and pNPP as a substrate. The procedure is described in the Materials and Methods section. Comparison of CaPho15p (dark bars) and CpPho15p (light bars) activities in the presence of different divalent cations (D). For this comparison, 10 mM concentration of each cation was used. Figure 5. Comparison of substrate preferences of CaPho15p (dark bars) and CpPho15p (light bars). 8 FEMS Yeast Research, 2019, Vol. 19, No. 1 8 FEMS Yeast Research, 2019, Vol. 19, No. 1 Table 1. Km and Vmax values for dephosphorylation of 2-phosphoglycolate and 2-glycerolphosphate by CaPho15p and CpPho15p. 2-P-Glycolate 2-Glycerolphosphate Km Vmax Km Vmax mM μmol min−1 ml−1 mM μmol min−1 ml−1 CaPho15p 3.47 ± 0.6 634 ± 48 14.48 ± 4.3 155 ± 22 CpPho15p 3.26 ± 0.4 238 ± 12 6.07 ± 3.3 21 ± 18 Figure 6. Km graphs of CaPho15p (A, B) and CpPho15p (C, D) for 2-P-Glycolate (A, C) and 2-Glycerolphosphate (B, D). The Km and Vmax values are shown in Table 1. The measurements were performed in triplicate, using immobilised phosphatases and corresponding substrate. The procedure is described in the Materials and Methods section. Table 1. Km and Vmax values for dephosphorylation of 2-phosphoglycolate and 2-glycerolphosphate by CaPho15p and CpPho15p. d Vmax values for dephosphorylation of 2-phosphoglycolate and 2-glycerolphosphate by CaPho15p and CpPho15p. Figure 6. Km graphs of CaPho15p (A, B) and CpPho15p (C, D) for 2-P-Glycolate (A, C) and 2-Glycerolphosphate (B, D). The Km and Vmax values are shown in Table 1. The measurements were performed in triplicate, using immobilised phosphatases and corresponding substrate. The procedure is described in the Materials and Methods section. suggests potential role of CpPho15p in adaptation to chang- ing carbon sources available in different host or environmental niches. The type of carbon source was found to influence cell wall properties and remodelling. Metabolic adaptation to avail- able nutrients is often interrelated with expression of virulence factors (Brown et al. 2014). dihydroxyacetone phosphate, an intermediate of glycolysis pathway, which is formed from fructose-1,6-bisphosphate and converted to glyceraldehyde-3-phosphate. Triosephosphate isomerase catalysing reversible interconversion between di- hydroxyacetone phosphate and glyceraldehyde-3-phosphate was found to be inhibited by 2-phosphoglycolate in plants (Norman and Colman 1991). Activity of CpPho15p towards 2- phosphoglycolate and dihydroxyacetone phosphate may thus indicate that this enzyme participates in regulation and scavenging of the glycolytic pathway reactions from fructose- 1,6-bisphosphate to 1,3-bisphosphoglycerate in C. parapsilosis. Dephosphorylation of dihydroxyacetone phosphate yields dihy- droxyacetone, which was found to be toxic for S. cerevisiae. Its toxicity, however, depends on a carbon source and can be sup- pressed by glucose (Molin, Norbeck and Blomberg 2003). This CaPho15 acts on ß-glycerophosphate, as the second best sub- strate of the compounds tested. Its dephosphorylation yields glycerol, which is important for osmoregulation and stress re- sponse. DISCUSSION 2-P-glycolate, 2-phosphoglycolace; glyceraldehyde-P, glyceraldehyde- 3-phosphate; β-glycerol-P, β-glycerophosphate; α-glycerol-P, α-glycerophosphate; 1,3-dihydroxyacetone-P, 1,3-dihydroxyacetone phosphate; ADP, adenosine diphos- phate; O-P-ethanolamine, O-phosphorylethanolamine; pyridoxal-5-P, pyridoxal-5-phosphate; fructose-1,6-bisP, fructose-1,6-bisphosphate; PEP, phosphoenolpyruvate; thiamine pyro-P, thiamine pyrophosphate; fructose-6-P, fructose-6-phosphate; glucose-6-P, glucose-6-phosphate; ribose-5-P, ribose-5-phosphate; P-choline, phospho- choline. Figure 5. Comparison of substrate preferences of CaPho15p (dark bars) and CpPho15p (light bars). 2-P-glycolate, 2-phosphoglycolace; glyceraldehyde-P, glyceraldehyde- 3-phosphate; β-glycerol-P, β-glycerophosphate; α-glycerol-P, α-glycerophosphate; 1,3-dihydroxyacetone-P, 1,3-dihydroxyacetone phosphate; ADP, adenosine diphos- phate; O-P-ethanolamine, O-phosphorylethanolamine; pyridoxal-5-P, pyridoxal-5-phosphate; fructose-1,6-bisP, fructose-1,6-bisphosphate; PEP, phosphoenolpyruvate; thiamine pyro-P, thiamine pyrophosphate; fructose-6-P, fructose-6-phosphate; glucose-6-P, glucose-6-phosphate; ribose-5-P, ribose-5-phosphate; P-choline, phospho- choline. (Haimovich-Dayan et al. 2015). The role of 2-phosphoglycolate in yeast and mammalian cells is not well defined. It is a side prod- uct of pyruvate kinase, which is formed from glycolate (Collard et al. 2016). It is also generated as a result of DNA repair (Pellicer et al. 2003). Nuclear localisation of Pho15p enzymes, which was suggested by the WoLF PSORT algorithm besides the presence in cytosol, would thus make sense. While 2-phosphoglycolate was the best substrate for both phosphatases, CaPho15p and CpPho15p differed in prefer- ences for other compounds tested. CpPho15p hydrolysed 8 FEMS Yeast Research, 2019, Vol. 19, No. 1 Contribution to increase of intracellular glycerol con- centration may be one of the reasons why CaPHO15 is upregu- lated under the stress conditions. Metabolic adaptation to stress conditions is intimately linked to virulence, as it increases the chance of Candida to survive within the host phagocytic cells. CaPHO15 gene is one of ∼30 genes involved in the core stress Kroˇcov´a et al. 9 Kroˇcov´a et al. 9 Casadevall A, Pirofski L. What is a host? Incorporating the mi- crobiota into the damage-response framework. Infect Immun 2015;83:2–7. response of C. albicans. Core stress response of S. cerevisiae is, in contrast, constituted by more than 200 genes, and PHO13 in not among them (Brown et al. 2014). This indicates that the orthologous phosphatases may play different physiological roles in these yeast species. Collard F, Baldin F, Gerin I et al. A conserved phosphatase de- stroys toxic glycolytic side products in mammals and yeast. Nat Chem Biol 2016;12:601–7. Pho13p from S. cerevisiae was found to hydrolyse 4-phospho- erythronate, a side product of glyceraldehyde-3-phosphate dehydrogenase and an inhibitor of 6-phospho-gluconate de- hydrogenase. This important reaction enables coexistence of glycolysis and pentose phosphate pathway (Collard et al. 2016). It has yet to be examined whether this function is conserved also for Candida Pho15p enzymes. Ding C, Vidanes GM, Maguire SL et al. Conserved and divergent roles of Bcr1 and CFEM proteins in Candida parapsilosis and Candida albicans. PLoS One 2011;6:e28151. Enjalbert B, Smith DA, Cornell MJ et al. Role of the Hog1 stress- activated protein kinase in the global transcriptional re- sponse to stress in the fungal pathogen Candida albicans. Mol Biol Cell 2006;17:1018–32. Recombinant phosphatases of two Candida species were rather difficult to work with due to their tendency to precipi- tate. Examination of the substrate specificities of CaPho15p and CpPho15p was made possible only thanks to the attachment of the enzymes to Ni-NTA Agarose via His-tag. Candida albicans and C. parapsilosis phosphatases hydrolyse 2-phosphoglycolate and 2-glycerolphosphate with a Km one order of magnitude higher than Pho13p from S. cerevisiae (Collard et al. 2016). These differ- ences, which may, in part, be due to the different method of mea- surement, presence of the His-tag and enzyme immobilisation, are not likely to affect the overall picture of Candida Pho15p sub- strate specificity. Gobom J, Nordhoff E, Mirgorodskaya E et al. 8 FEMS Yeast Research, 2019, Vol. 19, No. 1 Sample purification and preparation technique based on nano-scale reversed- phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spectrom 1999;34:105– 16. Guinea J. Global trends in the distribution of Candida species causing candidemia. Clin Microbiol Infect 2014;20:5–10. Haimovich-Dayan M, Lieman-Hurwitz J, Orf I et al. Does 2- phosphoglycolate serve as an internal signal molecule of inorganic carbon deprivation in the cyanobacterium Synechocystis sp. PCC 6803? Environ Microbiol 2015;17:1794– 804. In conclusion, Pho15 phosphatases from C. albicans and C. parapsilosis are HAD-family 2-phosphoglycolate phosphatases. They may be a part of metabolic repair system, but their precise role in Candida spp. physiology has yet to be investigated. Hnisz D, Schwarzm ¨uller T, Kuchler K. Transcriptional loops meet chromatin: a dual-layer network controls white- opaque switching in Candida albicans. Mol Microbiol 2009;74:1– 15. ACKNOWLEDGMENTS Hoffman CS, Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transfor- mation of Escherichia coli. Gene 1987;57:267–72. We thank Dr. Jan Sn´aˇsel and Dr. Kvido Stˇr´ıˇsovsk´y (Institute of Organic Chemistry& Biochemistry, Prague) for inspiring dis- cussions on phosphatase substrate specificities and for critical reading the manuscript, respectively. Horton P, Park K-J, Obayashi T et al. WoLF PSORT: protein local- ization predictor. Nucleic Acids Res 2007;35:W585–7. Jones T, Federspiel NA, Chibana H et al. The diploid genome sequence of Candida albicans. Proc Natl Acad Sci USA 2004;101:7329–34. Conflict of interest. None declared. Kelly GJ, Latzko E. Inhibition of spinach-leaf phosphofructoki- nase by 2-phosphoglycollate. FEBS Lett 1976;68:55–58. REFERENCES Baginski ES, Epstein E, Zak B. Review of phosphate methodolo- gies. Ann Clin Lab Sci 1975;5:399–416. Kuznetsova E, Nocek B, Brown G et al. Functional di- versity of haloacid dehalogenase superfamily phos- phatases from Saccharomyces cerevisiae: biochemical, struc- tural, and evolutionary insights. J Biol Chem 2015;290: 18678–98. Berman J. Candida albicans. Curr Biol 2012;22:R620–2. Bezerra AR, Sim˜oes J, Lee W et al. Reversion of a fungal ge- netic code alteration links proteome instability with ge- nomic and phenotypic diversification. Proc Natl Acad Sci USA 2013;110:11079–84. Larsen MR, Thingholm TE, Jensen ON et al. Highly selective en- richment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 2005;4:873–86. Bilir SP, Ferrufino CP, Pfaller MA et al. The economic impact of rapid Candida species identification by T2 Candida among high-risk patients. Future Microbiol 2015;10:1133–44. Molin M, Norbeck J, Blomberg A. Dihydroxyacetone kinases in Saccharomyces cerevisiae are involved in detoxification of di- hydroxyacetone. J Biol Chem 2003;278:1415–23. Bondaryk M, Kurz atkowski W, Staniszewska M. Antifungal agents commonly used in the superficial and mucosal can- didiasis treatment: mode of action and resistance develop- ment. Adv Dermatol Allergol Dermatol Alergol 2013;30:293–301. Motosugi K, Esaki N, Soda K. Purification and properties of a new enzyme, DL-2-haloacid dehalogenase, from Pseudomonas sp. J Bacteriol 1982;150:522–7. Brown AJP, Budge S, Kaloriti D et al. Stress adaptation in a pathogenic fungus. J Exp Biol 2014;217:144–55. Norman EG, Colman B. Purification and characterization of phosphoglycolate phosphatase from the Cyanobacterium Coccochloris peniocystis. Plant Physiol 1991;95:693–8. Burroughs AM, Allen KN, Dunaway-Mariano D et al. Evolution- ary genomics of the HAD superfamily: understanding the structural adaptations and catalytic diversity in a super- family of phosphoesterases and allied enzymes. J Mol Biol 2006;361:1003–34. O’Brien PJ, Herschlag D. Alkaline phosphatase revisited: hydrolysis of alkyl phosphates. Biochemistry 2002;41: 3207–25. Butler G, Rasmussen MD, Lin MF et al. Evolution of pathogenic- ity and sexual reproduction in eight Candida genomes. Nature 2009;459:657–62. Pabis A, Kamerlin SCL. Promiscuity and electrostatic flexibility in the alkaline phosphatase superfamily. Curr Opin Struct Biol 2016;37:14–21. Sambrook J, Rusell DW. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY, USA: Cold Spring Harbor laboratory Press, 2001. 10 FEMS Yeast Research, 2019, Vol. 19, No. 1 Pellicer MT, Nu ˜nez MF, Aguilar J et al. Role of 2-phosphoglycolate phosphatase of Escherichia coli in metabolism of the 2-phosphoglycolate formed in DNA repair. J Bacteriol 2003;185:5815–21. 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https://openalex.org/W4280605263	https://zenodo.org/records/6550985/files/THERMAL%20ANALYSIS%20OF%20THERMO%20ELECTRIC%20GENERATOR%20FOR%20WASTE%20HEAT%20RECOVERY%20(3).pdf	English	null	Thermal analysis of thermo electric generator for waste heat recovery	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,834	Sustainable Engineering Science and Research Journal Volume: 01: 01 - 05 ABSTRACT The thermoelectric generators (TEGs) are used to generate electric power. Thermo electric modules which are used in TEGs are utilized to change the thermal energy into electrical energy and it operates on the basic concept of See beck effect. Power generation by Thermo- Electric (TE) materials is an interesting method for the direct translation of thermal energy into electrical energy. This work discovers a method of analysis on the performance of TEGs with different materials for recovering the waste heat and converts it into electric power. For this, an experimental setup for waste heat recovery and energy conversion equipment was designed and fabricated. The TEGs made by Bismuth Telluride (Bi2Te3), Lead Telluride (PbTe), and Silicon Germanium (SiGe) are taken for the performance analysis. From these above-mentioned TE materials, the Bismuth Telluride (Bi2Te3) has better performance when compared with others within a temperature span that is most appropriate for cooling and heating utilities. Ragupathi P Assistant Professor, Department of Mechanical Engineering, Karpagam Academy of Higher Education, Coimbatore-641021 Received: 11 April 2022 Accepted: 04 May 2022 Published Online: 16 May 2022 © The Author(s) 2022 Sustainable Engineering Science and Research Journal Sustainable Engineering Science and Research Journal Heading Editor: Satheesh Kumar Address Correspondance to E-mail: ponragupathi@gmail.com https://doi.org/10.5281/zenodo.6550985 Heading Editor: Satheesh Kumar Address Correspondance to E-mail: ponragupathi@gmail.com https://doi.org/10.5281/zenodo.6550985 Heading Editor: Satheesh Kumar Thermo electric generator (TEG), Waste heat recovery (WHR), Lead Telluride (PbTe), Silicon Germanium (SiGe), Bismuth Telluride (Bi2Te3) Keywords Thermo electric generator (TEG), Waste heat recovery (WHR), Lead Telluride (PbTe), Silicon Germanium (SiGe), Bismuth Telluride (Bi2Te3) Thermo electric generator (TEG), Waste heat recovery (WHR), Lead Telluride (PbTe), Silicon Germanium (SiGe), Bismuth Telluride (Bi2Te3) I. INTRODUCTION The machineries which do work or other energy transformation produce heat along with the output power. This heat is termed as waste heat as it is discharged in the environment. This waste heat after being discharged to the environment causes the rise in temperature of the environment. Due to this an adverse effect is created on the environment [1-3]. The heat generation shows that a partial input energy is wasted and not transferred into useful work. Preferably there should be no waste heat but it is not almost likely to transfer all the input energy into work [4, 5]. It can be tough or intricate to improve the effectiveness of any machine directly but the efficiency is indirectly increased if the produced waste heat is utilized for some other purposes [6-9]. Conversion of the heat energy into electric power is a one of the methods to use the waste heat.TEGis a device which can be utilized to transfer heat into electricity and it operates on the concept of See beck effect i.e., it converts the change in temperature into electric power [10-12]. The change in temperature is needed at both ends of a TEG to generate a potential difference (voltage). More in temperature difference will create more in the degree of the power generated [13-16]. TEGs are generally made up of different materials like Bismuth Telluride (Bi2Te3) alloys, Lead Telluride (PbTe) alloys and Silicon Germanium (SiGe) alloys. The performance of these different material TEGs was analyzed and best one was found. Fig. 1. Experimental Setup Electric Heater TEG Cooling Water Output Data Measuremen t Input Data Measurement Input Data Measurement Output Data Measuremen Electric Heater S E S R J ( 2 0 2 2 ) 0 1 \| 2 \| 2 I. INTRODUCTION processes. TEGsare used for this purpose. Heaters serve as heating devices and TEGswith a size of 40 mm x 40 mm x 4 mm are used to produce electricity from the change in temperature across its junctions. This experiment was conducted by fixing the TEG between an electrical heaters and water-cooling system. The heat input to the electric heater was changed between 20W and 120 W. The measurement of temperature was carried by thermocouple. TEGs work on the Seebeck’s effect principle. TEGshave a several merits than the other electric power generators such as free in pollution, solid state, no maintenance needed, light in weight, noiseless operation and it is a passive device. The heater is connected to a dimmer stat of 600W.The ampere and the voltage is monitored using an ammeter and the voltmeter. The output of the TEG is connected to the voltmeter and the ammeter to measure the output power generated. Now when the electric heater is switched on and at the same time the cooling water is fed through the block. The TEG produces the electric power by the Seebeck’s effect. Then three different kinds of TEGs are fixed one by one and the experiment was carried. The work flow is mentioned in Fig. 1. II. METHODOLOGY Waste heat is generated in nearly every energy translation process. This waste heat is passed to the atmosphere and has an adverse result on it. Investigations express that practically 33% of the total expended energy is goes as waste heat. Present days it is very much needed to recover and utilize this energy as the fossil fuels are exhausting rapidly. The waste heat can be re-claimed in several ways thereby improving the performance of the Fig. 1. Experimental Setup Fig. 1. Experimental Setup S E S R J ( 2 0 2 2 ) 0 1 \| 3 ηTEG = Po Qi ---------------- (1) Fig. 2 Maximum power generated versus TEG Material III. RESULTS AND DISCUSSIONS The experiments were carried out for three different TEGs by changing the heat input value from 20W to 120W with a step of 20W raise. During the experiments, the steady state was attained after 5 minutes of continuous running of the heater. The TABLE I shows the experimental results of the TEGs. An effort has been taken to conserve the heat of heater with TEG and electricity was produced by using TEGs. It was observed that, if there is an increase in the heater input power, there is a rise in temperature difference between hot and cold end of TEGs and then the rate of electricity generation is also increased. The energy conversion efficiency of TEG was determined by utilizing equation (1). Fig. 2 Maximum power generated versus TEG Material Fig. 2 Maximum power generated versus TEG Material S E S R J ( 2 0 2 2 ) 0 1 \| 4 Fig. 3 Conversion Efficiency versus TEG Material 2. Johnson, Ilona, William T. Choate, and Amber Davidson. Waste heat recovery. Technology and opportunities in US industry. BCS, Inc., Laurel, MD (United States), 2008. 3. Fleurial, Jean-Pierre. "Thermoelectric power generation materials: Technology and application opportunities." Jom 61, no. 4 (2009): 79-85. 4. Law, Richard, Adam Harvey, and David Reay. "Opportunities for low-grade heat recovery in the UK food processing industry." Applied thermal engineering 53, no. 2 (2013): 188-196. 5. Ragupathi, P., Barik, D., Pradeep, S. and Lakshan, L., 2018. A Review on Waste Heat Recovery Technologies in Internal Combustion Engines. International Journal of Research in Advent Technology, pp.270-276. 6. Rowe, David Michael. "Thermoelectric waste heat recovery as a renewable energy source." International Journal of Innovations in Energy Systems and Power 1, no. 1 (2006): 13-23. Fig. 3 Conversion Efficiency versus TEG Material 7. Riffat, Saffa B., and Xiaoli Ma. "Thermoelectrics: a review of present and potential applications." Applied thermal engineering 23, no. 8 (2003): 913- 935. As the load on the heater increases the heater consumes more quantity of current to compensate the load, hence heating of more heater results in an increase temperature. High temperature led to elevate the performance of the TEG and produces more electricity. In these three TEGs the Bi2Te3 gives better performance when compared with the others. It gives the maximum temperature difference of 95OC, maximum generated power of 1.72 W and the maximum conversion efficiency of 1.43% as shown in Fig. 2. 8. Ismail, Basel I., and Wael H. Ahmed. "Thermoelectric power generation using waste-heat energy as an alternative green technology." Recent Patents on Electrical & Electronic Engineering (Formerly Recent Patents on Electrical Engineering) 2, no. 1 (2009): 27-39. 9. Bell, L.E., 2008. Cooling, heating, generating power, and recovering waste heat with thermoelectric systems. Science, 321(5895), pp.1457-1461. 10. Singh, Randeep, Sura Tundee, and Aliakbar Akbarzadeh. "Electric power generation from solar pond using combined thermosyphon and thermoelectric modules." Solar Energy 85, no. 2 (2011): 371- 378. IV. CONCLUSION The energy conversion efficiency of the TEGs will be subject to the change in temperature across its junctions. The efficiency of different TEG material was found using the experimental setup and the results are summarized as follows: 11. Ragupathi, P., Barik, D., Vignesh, G. and Aravind, S., 2020. Electricity Generation from Exhaust Waste Heat of Internal Combustion Engine Using Al2O3 Thermoelectric Generators. Journal of Applied Science and Engineering, 23(1), pp.55-60. 12. Kim, Shiho, Soonseo Park, Sunkook Kim, and Seok-Ho Rhi. "A thermoelectric generator using engine coolant for light-duty internal combustion engine-powered vehicles." Journal of electronic materials 40, no. 5 (2011): 812-816. • It is found that if the heat input value increases, then the temperature difference increases. • Due to the increase in temperature difference the power generated and conversion efficiency also increases. 13. Kim, Sun-Kook, Byeong-Cheol Won, Seok-Ho Rhi, Shi-Ho Kim, JeongHo Yoo, and Ju-Chan Jang. "Thermoelectric power generation system for 28 future hybrid vehicles using hot exhaust gas." Journal of electronic materials 40, no. 5 (2011): 778-783. • The Bi2Te3TEG produced maximum power of 1.72W with the conversion efficiency of 1.43% when compared with PbTe and SiGe as shown in the Fig. 3. 14. Goncalves, L. M., Jorge Martins, Joaquim Antunes, Romeu Rocha, and Francisco P. Brito. "Heat-pipe assisted thermoelectric generators for exhaust gas applications." In ASME International Mechanical Engineering Congress and Exposition, vol. 44298, pp. 1387-1396. 2010. TABLE I PERFORMANCE OF TEGS TEG Material Input Heater Power (Qi) (W) Average ΔT (°C) Maximum Power Generated (Po) (W) Conversion Efficiency ηTEG(%) Bi2Te3 20 16 0.06 0.30% 40 28 0.24 0.60% 60 52 0.52 0.87% 80 68 0.84 1.05% 100 85 1.37 1.37% 120 95 1.72 1.43% PbTe 20 15 0.05 0.25% 40 29 0.23 0.58% 60 50 0.51 0.85% 80 67 0.84 1.05% 100 83 1.34 1.34% 120 95 1.70 1.42% SiGe 20 15 0.05 0.25% 40 28 0.23 0.58% 60 50 0.51 0.85% 80 66 0.82 1.03% 100 82 1.32 1.32% 120 94 1.68 1.40% S E S R J ( 2 0 2 2 ) 0 1 \| 4 16. Nuwayhid, Rida Y., Alan Shihadeh, and Nesreen Ghaddar. "Development and testing of a domestic woodstove thermoelectric generator with natural convection cooling." Energy conversion and management 46, no. 9-10 (2005): 1631-1643. REFERENCES 1. Rowe, D. M. "Thermoelectrics, an environmentally-friendly source of electrical power." Renewable energy 16, no. 1-4 (1999): 1251- 1256. 15. Ragupathi, P., Barik, D., Aravind, S. and Vignesh, G., 2021, February. Performance Optimization of Thermoelectric Generators using Taguchi Method. In IOP Conference Series: Materials Science and Engineering (Vol. 1059, No. 1, p. 012053). IOP Publishing. S E S R J ( 2 0 2 2 ) 0 1 \| 5
https://openalex.org/W3005100007	https://dspace.spbu.ru/bitstream/11701/16663/1/321-344.pdf	Kirghiz, Kyrgyz	null	The Persian Mirrors for Princes Written in the Saljuq Period: the Book Series	Vestnik Sankt-Peterburgskogo universiteta. Vostokovedenie i afrikanistika/Vestnik Sankt-Peterburgskogo universiteta. Seriâ 13, Vostokovedenie, afrikanistika	2,019	public-domain	16,114	The Persian Mirrors for Princes Written in the Saljuq Period: the Book Series A. A. Khismatulin Institute of Oriental Manuscripts of the Russian Academy of Sciences, 18, Dvortsovaya nab., St. Petersburg, 191186, Russian Federation A. A. Khismatulin For citation: Khismatulin A. A. The Persian Mirrors for Princes Written in the Saljuq Period: the Book Series. Vestnik of Saint Petersburg University. Asian and African Studies, 2019, vol. 11, issue 3, pp. 321–344. https://doi.org/10.21638/spbu13.2019.306 The article begins with a concise description of the genre, period, and classical Persian texts covered by the announced book series of three books: 1) Amir Mu‘izzi Nishapuri. The Siyasat- nama/Siyar al-muluk: A Fabrication Ascribed to Nizam al-Mulk — this text is still being pub- lished and reprinted under the authorship of Nizam al-Mulk, an outstanding Prime Minister of the Saljuqids. However, the results of historical, codicological and textual analyzes show that the text was compiled by Muhammad Mu‘izzi Nishapuri, the head of poets department under the Saljuqid rulers Malik-shah and his son Sanjar, and then attributed by him to the dead Nizam al-Mulk with completely definite goals; 2) The Writings of Imam al-Ghazali is a book that includes six texts. Three of them are authentic: a student manual entitled by the author as the Zad-i Akhirat (Provisions for the Hereafter); an authentic part of al-Ghazali’s epistle to Sultan Sanjar entitled the Nasihat al-muluk (Counsel for Kings) and a medieval collection of letters addressed by the Imam to various recipients and entitled the Fada’il al-anam min rasa’il Hujjat al-Islam (The Virtues of People [drawn] from the Epistles of the Proof of Islam). The remaining three texts are fabrications; 3) Kay Kawus b. Iskandar b. Qabus. Qabus-nama (The Book of Qabus) and Nizami ‘Aruzi Samarqandi. Chahar maqala/Majma‘ al-nawadir (Four Discourses/The Miscellany of rarities) is a book that includes two authentic texts. After this, the article touches upon the problem of existence of literary and physical forgeries in medieval Islamic literature, their categories and methods of their identification. Keywords: Mirrors for Princes genre, advice literature, medieval Islamic forgeries, literary fakes, identification of literary fabrications, Nizam al-Mulk, Siyar al-muluk, Siyasat-nama, Amir Mu‘izzi, Saljuqs, Saljuqides, false attribution, talbis, tazwir, muzawwir, kitab maj‘ul. Literary works written in the Great Saljuq era (11th–12th centuries) in Persian in the didactic genre have recently been lively discussed by both Western Iranists and Iranian experts on this period, not to mention Turkish researchers. UDC 82-342; 821.222.1 UDC 82-342; 821.222.1 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 © Санкт-Петербургский государственный университет, 2019 The Persian Mirrors for Princes Written in the Saljuq Period: the Book Series They have published numer- ous articles and monographs on this theme, which can be easily found on the Internet, if required. The issues addressed by these works regained importance, most obviously in connection with what exactly is meant by the ideal political model of the organization of Islamic society under challenging modern conditions. The same complicated political conditions were observed in the Saljuqid period also. If viewed in a schematic and very simplified way, they can be described as follows: the no- madic Turks, who had traditionally kept a well-trained and powerful army, conquered the entire realm of Iran and a large part of the Arab world; the Iranian elite, well experienced 321 https://doi.org/10.21638/spbu13.2019.306 in governing the settled population and collecting taxes from it, served the interests of these Turks and with a good reason considered themselves the actual rulers of the Saljuqid Empire; the Arab caliphs in Baghdad from the Abbasid dynasty essentially acted as the outside observers, being involved in the problems of religion and legalizing the enthrone- ment of the Saljuqid rulers (persian language has already gotten the official status, largely due to the Iranian administration, and gradually became the language of science). In terms of religion, this period represented the case of a multi-vector religious com- petition between different creeds (madhhab). The competitive advantage in the rivalry between the Hanafites and the Shafiites was received by those supported by the power in the authoritarian governance system, in the person of the Prime Minister and the Sultan. The predominantly Sunnite administration was tolerant, in general, to the Shiite Muslims, despite the serious opposition of two administrative bureaucracies — the Khurasan and the Iraqi groups. The use of such terminology implied geo-confessional referencing. The Khurasan officials meant the Sunnites of the Khurasan origin, while the Iraqi group in- cluded the pro-Shiite descendants from Persian Iraq (Iraq al-‘ajam), i. e., the central and northern regions of modern Iran. g The official authorities were not fully loyal to the Shiites. At that time, any of them could first be accused of sympathizing with the Batinites-Ismailites, the ideological alli- ances of the Shiites and serious opponents of the current administration, and then put to death. Such danger sometimes forced the Shiites to take to hiding their madhhab by using the principle of taqiyya and disguising themselves for the Shafiites, the most Shiite-loyal madhhab among the four Sunni. Literary works included in the series The tradition of creating didactic writings goes back to the pre-Islamic era and is ana- lyzed in detail in modern studies [4]. However, in Islamic literature, it acquires its charac- teristic features. These are directly related to the development of the bureaucratic system and to the facts, by whom traditionally, for whom, and for what purpose were written the texts, which today are referred to the didactic genre. To get a relatively objective view of their specifics at the Saljuqids time, with their branched bureaucratic apparatus, several such texts will be presented in this series of three books. For some of them, Russian trans- lations were published in the mid-20th century, but these are already outdated by many criteria. Other texts will be published for the first time. However, all of them are part of the classical heritage of Persian literature and its reinterpretation is a natural and unavoidable process, which concerns not only the bearers of Persian culture, who republish these texts with amazing regularity. The first book of the series — Amir Mu‘izzi Nishapuri. The Siyasat-nama/Siyar al- muluk (The Book of Government/The Vitae of Rulers): a fabrication ascribed to Nizam al-Mulk — is a classic text well known to every Iranist by the two cited titles. This text was translated into at least 11 languages, including Russian [6], is still published and re- printed in Iran under the authorship of Nizam al-Mulk. However, as demonstrated by the results of historical, codicological, textual and stylometric analysis, the text was compiled by Muhammad Mu‘izzi Nishapuri (d. between 518–522/1124–1128) [7], Head of the De- partment of Poets (the Amir al-shu‘ara), under the Saljuqid Sultan Malik-shah (poisoned in 485/1092) [8], and then intentionally ascribed to the murdered Nizam al-Mulk, with a very specific purpose — to obtain a high-status position at the Court of the new Saljuqid ruler. Mu‘izzi’s innovative idea was to comment on the articles of the legal document, i. e., the labor agreement (muwada‘a) of Nizam al-Mulk with the Sultan-employer, with vari- ous stories, legends, tales, etc. The main method of compilation was to add this comment whenever possible to every article of the labor agreement. This was how the first redaction of the text appeared. After that the second redaction followed, created by an unknown medieval editor. The Persian Mirrors for Princes Written in the Saljuq Period: the Book Series Therefore, the opponents of both Shiites and Shafiites of- ten used in their religious debates the dual-purpose label “pure/refined” (pakiza) Shafiite. g The Batinites-Ismailites led by Ahmad ‘Attash (executed in 500/1107) and Hasan b. al-Sabbah (d. 518/1124) [1] became another key player for quite a long time, openly op- posing the Saljuqid vertical of power. Sometimes they were subjected to persecution and elimination, sometimes were used as a tool in the internal struggle for power among the top administration officials. According to Jalal al-Din Muhaddith Urmawi (d. 1358/1979), the editor and commentator of the Persian work Ba‘d mathalib al-nawasib fi-naqd “Ba‘d fada’ih al-rawafid”, written between 556–566 / 1161–1170 by ‘Abd al-Jalil Qazwini Razi, a Shiite author of 6th / 12th century, to demolish the defamation of the Shiites: Each of the Saljuqid sultans, vazirs and military leaders-amirs, intending to remove his competitor and destroy his rival, conspired with the Ismailites—Hasan Sabbah and his follow- ers — and threw him down with their help [2, i, p. 285]. The conclusion of Jalal al-Din Muhaddith Urmawi cannot be extended unexcep- tionally over the entire Saljuqid top administration, at least because Nizam al-Mulk (k. 485/1092), the prominent Prime Minister of the Saljuqids, and his sons did not con- spire with the Ismailites. Therefore, the findings of fact analysis given in both historical chronicles and in the writings of authors belonging to different madhhabs seem more objective. These [= religious] discords were manifested in various planes. In the cultural plane, there was a struggle that came with the arrangement of dispute meetings, compilation of books on theology and religious ideology for approving or denying of a certain madhhab, establishing educational institutions intended for the followers of specific madhhabs, while the cultural and scientific activity was limited to religious areas and the development of rational sciences was terminated. In the 322 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 political plane, the practice of arranging conspiracies in ministries and at the Court against rivals in the madhhab went at full swing, which made the prerequisites for terror and exile of political opponents. This deprived the society of unity and political independence. In the military plane, the practice of eliminating opponents in the madhhab and terror against political and religious leaders became common, and social security went away from society [3, p. 72]. The Persian Mirrors for Princes Written in the Saljuq Period: the Book Series This made an extremely intricate religious and political background for the appear- ing didactic texts, which sometimes were actually written by well-known religious and political figures, but more often were attributed to them with quite specific goals. Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Literary works included in the series He reduced the original text, partially edited it and made changes to the foreword, presenting it to be authored by Nizam al-Mulk. These summarized conclusions are drawn from the analysis performed in the Introduction to the translation of the first redaction, in the following sequence: • a brief overview of previous editions and research findings before and after the discovery of Muhammad Nakhjavani ’s copy (Part 1); • a brief overview of previous editions and research findings before and after the discovery of Muhammad Nakhjavani ’s copy (Part 1); brief overview of previous editions and research findings before and after the • a codicological view of the earliest copies of the Siyar al-muluk (Part 2); 323 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 • a historical context of the appearance of the Siyar al-muluk (Part 3); • references of independent sources related to the Siyar al-muluk in the authorship of Nizam al-Mulk (Part 4); • references of independent sources related to the Siyar al-muluk in the authorship of Nizam al-Mulk (Part 4); • textual analysis of two main redactions of the text (Part 5); • selection of the articles of the Nizam al-Mulk’s labor contract, which formed the basis of the first redaction of the Siyar al-muluk, and were safely retained in the second one, as well. Here, attention should be paid to the following fact. In the early 1960s, the first report of the unexpected discovery of the Siyar al-muluk oldest copy appeared. This happened amid the international scandal with physical forgeries of ancient manuscripts (see below). According to Hubert Darke, an English publisher of a redaction represented in this copy, it nearly goes back to the autograph of Nizam al-Mulk. Since my first edition of this text was published in 1962, or rather during the closing stages of its printing, I became aware of the existence at Tabriz, of a manuscript older than any hitherto known. This manuscript, which is preserved in the Nakhjavani collection and housed in the National Library, is dated 673/1274 and in correctness, that is to say credibility, far surpasses all other manuscripts [9, p. v]. The discovered manuscript was later entitled a copy of Nakhjavani by the name of its first owner Muhammad Nakhjavani (d. 1341/1962), a famous Iranian philanthropist, collector and bibliophile [10]. His copy represents the second redaction of the text in the authorship of Nizam al-Mulk. Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Literary works included in the series Since then, this redaction has played a leading role in fur- ther research, both within Iran and beyond. y The second book of the series — The Writings of Imam al-Ghazali (d. 505/1111). For objective reasons, it was published slightly earlier than the first book [11] but I hope it will be republished together with the first book in the series. First, it presents three gen- uine texts: a student manual under the author’s title the Zad-i akhirat (Provisions for the Hereafter); the authentic part of al-Ghazali’s epistle to Sultan Sanjar (d. 552/1157) entitled the Nasihat al-muluk (The Counsel for Kings); a collection of Imam’s letters to various ad- dressees under the title of the Fada’il al-anam min rasa’il Hujjat al-Islam (The Excellencies of People from the Epistles of the Proof of Islam). Next to the originals, the book includes three fabrications. The first and the second were created by compiling text blocks from al-Ghazali’s original writings with non-au- thentic inserts. The first fabrication is al-Ghazali’s letter to his already consummate dis- ciple, previously considered as genuine, which became known in the Persian original and Arabic translation under two titles — the Ey farzand/Ayyuha al-walad (O child). The sec- ond is a compilatory letter to some ruler entitled the Pand-nama (The Advice Epistle). The book is concluded with a text addressed to a certain Saljuqid sultan, which became known both under its own title — al-Farq bayna al-salih wa ghayr al-salih (The Difference between the Pious and not Pious), and as the “second part” of the Nasihat al-muluk, at- tached to the authentic text either intentionally or mechanically. y y The third book to be published in this series will contain two newly translated works of two authors. These are the Qabus-nama (The Book of Qabus) by Kay Kawus b. Iskandar b. Qabus (d. 462/1069–70) and the Chahar maqala (The Four Discourses), or the Majma‘ al-nawadir (The Miscellany of Rarities), penned by Nizami ‘Aruzi Samarqandi (d. the sec- Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 324 ond half of the 6th/7th century). Throughout the 19th/20th centuries, the Qabus-nama was translated into the main European languages from both the Persian original and medieval Turkic translations. The book has been published twice in Russian translation: translated from Tatar by O. S. Lebedeva (1886) and translated directly from Persian by E. E. Bertels (1953). Literary works included in the series The inclusion of the Chahar maqala, also published earlier in the Russian trans- lation by S. I. Baevsky and Z. N. Vorozheykina (1963), in this book is determined by the small size of this text and inexpedience of a separate book publication under the chosen series format. The reasons for the re-translation and reprint of both works are explained in research introductions to translations in all the books of this series. Since the announced series deals with both originals and fabrications, it makes good sense to give a clear definition of what is meant by fabrications and whether such under- standing was characteristic of Islamic literature of the Saljuqid period, as well as to raise the issue of categories of fabrications and ways of their identification. Definitions of fabrications In modern scholarly publications, the reader can occasionally come across a work, which is attributed to some author by the researchers, implying the probability of false attribution. This wording suggests that the authenticity, or originality of the authorship is not identified, or not proven, or uncertain. To emphasize the fact of uncertain or yet still unclear authorship, the prefix “pseudo-” is sometimes added to the author’s name. Falsely attributed works are found in every cultural tradition. It can be even said that their presence is an integral element of culture, arising, as a rule, for two reasons. First, when in the absence of a direct indication of authorship, historians and literary scholars, relying on some indirect signs, begin to consider a particular text to be written by a certain author. Secondly, when someone, having written a certain text, intentionally concealed his authorship and attributed it to another person, pursuing his goals. As a result, a text with false attribution emerges, which in the second case is burdened by the fact of forgery. It is not uncommon that a comprehensive text analysis reveals both causes, i. e., research- ers attribute the text to the same person the forger attributed it. Therefore, in this series, forgeries, or fabrications denote texts with false attribution, which were accidentally or intentionally attributed to persons uninvolved in creating these texts. y p g It is well known that the creator of any forgery (paintings, jewelry, banknotes, etc.) follows the already preset and recognizable sample of the original, trying to bring it as close as possible to the latter with minimized costs of production. At the same time, the methods used to manufacture fabrications are very similar in appearance to those of creat- ing originals and the costs are minimized through falsification of uniqueness by copying it in order to extract the maximum profit. To produce successful falsification, the creator of a fabrication should have expertise in his sphere: to study the form and genre of the author, his manner, stylistic features, etc., in order to be able to copy them further and present them as genuine. p g The same is true for fabrications of medieval scholarly writings. The main objective in identifying such fabrications is to detect alien fragments, to separate them from those original (if any), to prove their inauthenticity and ultimately to determine the purpose of creating a fabrication. 325 Вестник СПбГУ. Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Definitions of fabrications Востоковедение и африканистика. 2019. Т. 11. Вып. 3 A fabrication is the same cultural artifact as any other, but made with special purposes. That is why, in order to definitively prove the falsification of an artifact, it is necessary to show absolutely clearly and convincingly the purpose, for which this fabrication was done [12, p. 341]. A fabrication is the same cultural artifact as any other, but made with special purposes. That is why, in order to definitively prove the falsification of an artifact, it is necessary to show absolutely clearly and convincingly the purpose, for which this fabrication was done [12, p. 341]. Taking a fabrication for the original in scholarly research, making conclusions and building the entire theories upon it, many modern authors prefer sometimes to keep si- lent about the fact that they deal with a fabrication, made intentionally by someone for mercenary, ideological or other reasons. The longer this fact is concealed and the more researchers do prefer it, the higher the probability of the future unspoken consensus about the authenticity of the fabrication is. Quantity is transformed into quality. In the future, a noncritical attitude towards it will eventually form a historical myth, with many gen- erations of scholars believing it, defending their MS, PhD and doctoral research works, publishing articles and monographs. It is good if a fabrication was unmasked without delay, shortly after the publication. It has no time to enter the mass circulation and to fool many innocent people. But more often the opposite happens. A sensational document gets widespread, receives coverage from the sensation-seeking mass media, thereby rooting it in the mass consciousness. To discredit such a time-tested “source” is very difficult [13, p. 9]. Refusing the established myths is not only “very difficult”, but is always extremely painful, and sometimes just uncomfortable. However, as practice shows, the facts of fabri- cations, both modern and medieval, are convincingly revealed sooner or later, and myths have to be refused. There is no doubt that with evolving digital technologies and digital humanities, the number of identified fabrications will only increase, leading to a revision of previous ideas, as well as the inevitable fall of many myths that still exist in any culture. This trend is already well traced in identifying fake news in modern mass media. Medieval fabrications There are enough facts that suggest that the fabrication of texts with their ascribing to famous people was not a rare phenomenon in medieval Islam. An illustrative example of circulation of deliberate fabrications and false attributions in the Islamic written tradition is the presence of a vast corpus of statements-hadiths included in the category of forgery — al-hadith al-mawdu‘. For various reasons and for different purposes, their authorship was attributed (sometime and by someone) to the Prophet, and their definition was made ex- actly by Muslim researchers. The same can be said about the so-called 115th Surah of the Quran created by someone. If the end justified the means for the Prophet’s statements and even for the Quran, then what can we say about other authors and their writings. The most remarkable cases of medieval fabrications in the Persian written tradition are related to the work of poets, whose names are familiar not only to the Iranians. These are: a large number of quatrains attributed to ‘Umar Khayyam (d. 510/1131) [14], the seventh daftar of the Mathnawi created by someone and attributed to Jalal al-Din Rumi (d. 672/1273), etc. Almost every popular medieval Persian poet became a posthumous owner of poems, which he had never composed and whose authors could hardly ever be identified. Modern scholars will have to look more closely and systematically at what their me- dieval colleagues wrote about fabrications and do not limit themselves to addressing this Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 326 topic only in connection with physical fabrications1. For instance, Imam Muhammad al- Ghazali tells of three attempts to forge his works. On the frontispiece of the first, authentic part of the Nasihat al-muluk addressed to Sultan Sanjar, the Imam makes a written note, which is further included as the third letter in his epistle collection entitled the Fada’il al- anam min rasa’il Hujjat al-Islam, telling as follows: Envy leaped in the envious. They found no other acceptable [way of] defamation, except for making falsification (talbis): in the book al-Minqidh min al-dalal and in the book Mishkat al-anwar, they changed several phrases, added phrases of disbelief and sent me to write a written permission (ijaza)2 on their frontispieces. The Exalted and Holy Worshipped, by His mercy and generosity, honored me the intuition to study them and realize their falsification. Medieval fabrications g y yi Later, this case became known to the Head of Khurasan. Having arrested the counterfeiter (muzawwir), he eventually sent him out of Nishapur, after which he went to the headquarters of the Lord of Islam and wagged his tongue of defamation, but was powerless. Then he took the synopsis, which I had done in my youth and wrote al-Mankhul min ta‘liq al-usul on its frontispiece. As early as thirty years before, those envious added to this synopsis a number of phrases condemning Imam Abu Hanifa [11, pp. 116, 184]. As seen from this passage, one of the three attempts was successful. If such attempts were made during the life of al-Ghazali, we can imagine what happened after his death. In the words of the Imam, the counterfeiter was arrested but soon released. In other words, creating fabrications was considered a wrongful act already in those times. Even then, it was condemned and entailed punishment in terms of the Muslim law. Under the legisla- tion, fabrications were called by the terms talbis (‘falsification’) by editing the author’s text and tazwir (‘fabrication’), and their creators — by the term muzawwir (‘counterfeiter’). In their writings, our medieval colleagues also mention rather often the cases of out- right plagiarism that took place in their surrounding. Plagiarism was defined by the Arabic term al-sirqa (‘theft’). So, al-Hujwiri, the author of the pioneer Persian compositions on Sufism, the Kashf al-mahjub (The Revelation of the Veiled) dated the late 5th / 11th century, complains that the only copy of his collection of poems was stolen by his contemporary, who appropriated the authorship to himself. Al-Hujwiri also speaks of the fortunately failed attempt to replace his name as an author with another in the work the Minhaj al-din (The Highway of religion). The desire to put on my name at the beginning of the book was twofold: one is a lot of the experienced, the other is a common lot. As regards the common lot, it is that when persons ignorant of this knowledge see a new book, in which the author’s name (musannif) is not set down in several places, they attribute his work to themselves, and thus the author will not reach his goal. 2 I j a z a, or k h a t t - i i j a z a, is a written permission from the author, verifying a copy of his work and actually turning it into the original (asl). This procedure is somewhat similar to the modern notarization of document copies; in the Middle Ages, it was made on the frontispiece of a manuscript — a space reserved for various inscriptions. 1 An overview of the problem in the Persian cultural tradition, with reference to particular cases of physical forgery, see the collective article Forgeries on the Encyclopædia Iranica website [15]. 2 I j a z a, or k h a t t - i i j a z a, is a written permission from the author, verifying a copy of his work and actually turning it into the original (asl). This procedure is somewhat similar to the modern notarization of document copies; in the Middle Ages, it was made on the frontispiece of a manuscript — a space reserved for various inscriptions. 3 An English translation: The Kashf al-mahjub. The Oldest Persian Treatise on Sufism by ‘Ali b. ‘Uth- man al-Jullabi al-Hujwiri, transl. by Reynold A. Nicholson, Leiden, Brill, 1911; a Russian translation from the English translation: Ali ibn Uthman al-Hujwiri. Raskrytie skrytogo za zavesoi. Stareishii persidskii traktat po sufismu, tr. from English by Orlov A., Moscow, Edinstvo, 2004. Medieval fabrications After all, the desire to collect (jam‘), compile (ta’lif) and compose (tasnif) is only that the author’s name would be kept alive and that readers and students would pray for his good thanks to his book. It happened to me twice. Once a man asked me for a collection of my poems (diwan) and kept it in his possession, and its original was in a single copy. He rewrote everything in it, struck out my name from the title and nullifying my labor. May Allah forgive him! I also wrote a book overview of the problem in the Persian cultural tradition, with reference to particular cases of gery, see the collective article Forgeries on the Encyclopædia Iranica website [15]. 2 I j a z a, or k h a t t - i i j a z a, is a written permission from the author, verifying a copy of his work and actually turning it into the original (asl). This procedure is somewhat similar to the modern notarization of document copies; in the Middle Ages, it was made on the frontispiece of a manuscript — a space reserved for various inscriptions. 327 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 about a method in Sufism entitled the Minhaj al-din. A shallow pretender, which is not worth talking about, erased my name from its title and presented it to the public as if he had written it himself, although connoisseurs laughed at his words. [17, pp. 1–2]3. The cases of such illegal actions can be found in every epoch. The famous Shafii scholar Jalal al-Din al-Suyuti (d. 911/1505) was struck by the case of open plagiarism that had happened to him and even wrote a short essay on this topic entitled al-Fariq bayna’l- musannif wa’l-sariq (The Differentiator between the Сomposer and Plagiarist). f g These facts clearly indicate that deliberate forgery and plagiarism were common in medieval Islam, referring to illegal, blamed and punishable acts the same way, as it is now. Despite the formal resemblance, plagiarism should be considered separately from fabrications. After all, in the case of plagiarism, we face a trivial theft, while in the case of intentional fabrication we still encounter creativity, albeit specific. The persons involved in such creativity used either the name of a famous author, or his intellectual property, or both, resorting to various methods and techniques. Medieval fabrications To identify fabrications, first of all, we need to know the traditional forms of creativ- ity in the scholarly literature of the Islamic Medieval Ages. Knowing the form of creating a work with doubtful authorship, it can be subjected to the textual analysis, comparing its content, logic, presentation style, etc., with a reference work, i. e., a guaranteed authentic text (or better, texts) written by the author in the same form and genre. Forms of medieval scholarly literature The same way as there are modern templates and recommendations for writing MA, PhD and doctoral theses, the main forms of writing scholarly books are identified in the texts of medieval Muslim scholars. There are three of them: collecting (jam‘) of material, its compiling (ta’lif) and composing, or systematization/classification (tasnif). These three terms are mentioned in the above passage from the Kashf al-mahjub by al-Hujwiri. These forms were common in almost all genres of scholarly literature of the time: hagiographi- cal, historical, theological, etc. Sometimes the first two terms, jam‘ and ta’lif, are found together in the author’s preface. However, the collection of material was often meant as a natural preparatory process prior to its compilation and in this case, only ta’lif is men- tioned in the preface or across the text. Tasnif represents an entirely different, independent form of scholarly creativity. The same applies to the author, who chose one of the forms to write his text. He was called either the collector — jami‘, or the compiler — mu’allif, with- out any negative connotation, or the composer/systematizer — musannif, respectively. The key difference between these forms lies in the proportion of synthesis and analysis. y p p y y T h e p r i m a r y t a’ l i f : the source for the compilation is mostly oral tradition, i. e., the compiler collects material for his work mainly from living informants or from his notes and memories— reminiscent of the modern field research associated with inter- viewing respondents. Then he has to allot (takhsis) the collected material and arrange it, following the genre pattern preset by the existing literary tradition. After that, he is quali- 328 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 fied for giving a title to his text and to write a foreword. The main objective of the primary ta’lif is to collect, compile, minimally arrange the material according to a given pattern and introduce it in this form into the written tradition. It holds the maximum information transfer and the minimum of its analysis and own comments. That is why primary ta’lifs are mostly characteristic of the hagiographic, or saints’ vitae, literary genre — maqamat, tadhkira, manaqib, etc. Forms of medieval scholarly literature T h e s e c o n d a r y t a’ l i f : the working algorithm is the same as in the case of the primary ta’lif, but with one fundamental difference: the source of the material for com- pilation is mostly the written tradition, i. e., ta’lifs and sometimes the tasnifs of the prede- cessors. The main objective of such a ta’lif is a thematic collection of information taken mostly from written sources. That is why the secondary ta’lif is the most common form of medieval scholar creativity. It covers almost the entire genre spectrum. In some genres (for instance, hadith studies) a compilation can include not a single word from the author except for the preface, sometimes formal, and sometimes even lacking. p p g The originality of such ta’lifs is represented, firstly, by the author’s selection of sourc- es. This collation is intended to clearly demonstrate the reader that the compiler belongs to a particular religious law school (madhhab). For instance, a Sunni hadith scholar will not use the Shiite collection of hadiths for his ta’lif save for the genre of refutation (radd). Secondly, the minimum analytics appears in the secondary compilation — the compiler arranges the selected material, following the preset logic and the genre pattern, and de- termining its vector. For example, two vectors are distinctly traced in the hadith litera- ture — either uniting all multi-thematic statements from each hadith carrier, or arranging statements from different carriers according to the thematic principle. In the historical compilation (tarikh), there are also two vectors: fixing the chronology of events with a tendency to expansion and adding current events by the subsequent historians, or con- versely, a tendency to reduce the material presented by the predecessors; in the genre of commentary (sharh) or refutation (radd) — expansion of the primary text by consecutive commenting on each thematically complete statement with quoting other texts and state- ments on the same theme, etc. In other words, the literary template of the secondary com- pilation has already its own established genre vector to be followed by the compiler [19]. Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Forms of medieval scholarly literature p y g y p The emergence and evolution of the secondary ta’lif as a form of scholarly creativity should be linked, apparently, to the system of traditional Muslim education, where the key feature was studying, note-taking, or even memorizing the books of particular scholars under the guidance of experienced mentors. Such ta’lif resembles collecting mosaic ele- ments, when each compiler can compose his own pattern from the material studied (lec- ture), and/or material available or newly-entered the intellectual market (books), accord- ing to the template already defined by the tradition. In today’s reality, this ta’lif is similar by its structure and approach to the MA work in humanities, with a series of quotations from various studies and bits of the author’s own thoughts, or a PhD thesis with the same bulk of quotes and minimal analytics. Ta s n i f represents a top performance of analytical thought that requires an ex- traordinary level of training and, in the context of medieval science, a high religious status of the author (musannif). In the modern reality, tasnif is an ideal version of a doctoral thesis. Not surprisingly, this term is used as part of nominal predicate to refer to music and verses — ‘to compose verses, music’, i. e., to create something original. Even with the abundant quotations cited as illustrative and evidentiary base, the analysis holds a promi- 329 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 nent place in tasnif. Sometimes it can occupy up to one third of the text, which is quite consistent with the ratio between synthesis and analysis in modern analytical studies. The main objective of tasnif is to propose a new idea, break down the existing patterns and show a new facet of the object under study by applying an innovative approach (declara- tive or actual), which is unfailingly mentioned by the author in the preface or in the text of the book. At the same time, the author’s references to the tasnif written by another author should be taken critically. They were often made just to show respect or to emphasize his high religious status [20]. The main categories of medieval fabrications Fabrications can be classified into three categories, which were made in three main ways and appeared periodically on the intellectual market of medieval literature. The meth- ods of their production are very similar to the forms of scholarly creativity described above. 1. Intentional attaching a non-authentic text to the genuine text is the most common and easily recognizable method. It was mentioned by al-Ghazali in the case of al-Mankhul min ta‘liq al-usul, it is also reviewed in the first book of this series in connection with the addition of a non-author comment to the Nizam al-Mulk’s labor contract and the addi- tion of 11 new chapters in the end, as well as in the second book of the series in connec- tion with the “second part” of the Nasihat al-muluk. The cases with the seventh daftar of Mathnawi, which once was attempted to be added to the six genuine daftars, and the 115th surah of the Quran also refer to this category. This method goes together with the quite traditional and legal manner of writing sec- ondary ta’lifs, in particular, historical chronicles and anthologies. The text was frequently updated on a chronological basis by another author taking from the place where his pre- decessor had stopped. If apply culinary definitions, a fabrication created by this method can be compared with a two-layer pie, where each layer is created by a separate baker. Also, here are occasional copyist errors, when the title is skipped and a colophon is absent. As a result, when the text of one author was unintentionally, due to the human factor, add- ed to the text of another author and was passed to further copying or translation in such an “updated” form. Therefore, when identifying a non-authentic part attached at the end, which sometimes is not even styled as authentic, the key question for further investigation is whether the attaching occurred accidentally or intentionally. 2. Purposeful compilation of text blocks taken from the original writings of a famous author, with non-authentic text blocks, in order to create the illusion of authenticity and achieve personal goals by appealing to the author’s influence. Such fabrications were almost always created deliberately, followed a specific plan and pursued specific goals. The illustra- tive cases are given in the second book of this series, two forgeries attributed to al-Ghazali: the Ey farzand/Ayyuha al-walad (O child) and the Pand-nama (The Advice Epistle). 2. Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 The main categories of medieval fabrications Purposeful compilation of text blocks taken from the original writings of a famous author, with non-authentic text blocks, in order to create the illusion of authenticity and achieve personal goals by appealing to the author’s influence. Such fabrications were almost always created deliberately, followed a specific plan and pursued specific goals. The illustra- tive cases are given in the second book of this series, two forgeries attributed to al-Ghazali: the Ey farzand/Ayyuha al-walad (O child) and the Pand-nama (The Advice Epistle). Fabrications included in this category are similar to multi-layered pie and are the most difficult in terms of validating their inauthenticity. After all, it can be argued with good rea- son that the author changed his point of view to a certain subject over time, or completely revised his views, especially when there is an underlying formal reason to do that. Thus, the authenticity of many texts attributed to al-Ghazali, or are deliberate fabrications, is validated only upon the revision of the Imam’s views caused by his spiritual crisis and the subsequent withdrawal from active social life, without giving more weighty arguments. 330 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Obviously, if such a multi-layer compilation is not attributed to anyone and has a unique author’s title and preface, it is completely legal and created in the form of a second- ary ta’lif, full of quotations from various sources. At the same time, it cannot be ruled out that this was made by the forger — he, instead of the author, gave the title, wrote the pref- ace and filled the text with quotations he needed. This similarity of methods of creating original compilations in this category and their fabrications is the reason, why so many medieval fabrications survived to the present day and remained undetected. p y Apart from the typical compilations, private correspondence and official documents were most prone to this way of falsification. After all, the forger did not need to name them and write author’s prefaces. The epistolary genre did not assume this and the docu- mentary genre in every epoch and under every Muslim dynasty had its own strict tem- plate to be only followed. Moreover, the texts created in these genres, for obvious reasons, seldom entered the book market and did not require written verification from the author (ijaza), i. The main categories of medieval fabrications e., they had no barrier to protect them somehow against fabrications. j y p g 3. Deliberate editing of the text content, when adding or removing the negation is enough to change the meaning to the opposite, not to mention the more complicated intellectual editing, as described by al-Ghazali in the case of al-Minqidh min al-dalal and Mishkat al-anwar. Such fabrications resemble a pie with an inauthentic filling. This also includes casual errors of copyists. 3. Deliberate editing of the text content, when adding or removing the negation is enough to change the meaning to the opposite, not to mention the more complicated intellectual editing, as described by al-Ghazali in the case of al-Minqidh min al-dalal and Mishkat al-anwar. Such fabrications resemble a pie with an inauthentic filling. This also includes casual errors of copyists. py To identify fabrications made this way, it is sometimes enough to prepare a critical text based on several versions, which will make it possible to identify non-author’s editing. Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Physical forgeries The issue of physical forgeries of manuscripts would not have been the subject of dis- cussion here, unless it had affected two texts from the announced series: partially — the text presented in the first book, and directly — the text scheduled for publication in the third book. That is why this problem could not be ignored. The specifics of any forged manuscript is not only in claiming its physical uniqueness (the most ancient, an autograph, or a copy of an autograph copied by a famous calligrapher, etc.) but the fact that it always represents some redaction of text. This makes the major schol- arly problem of physical forgery. The text of the “unique” manuscript is considered as more reliable after its publication and presentation, and noticeably influences the nature of further research until it turns out that the manuscript where it was presented was fabricated. The problem of physical forgeries in the Iranian cultural tradition in general, and in manuscript studies in particular, is under the scrutiny of the Iranian scholars. It stood out most acutely by the late 1950s. At that time, several allegedly ancient manuscripts were purchased for a large amount of money by well-known manuscript repositories in the USA, Europe, and Iran itself. Additional expert examinations revealed that the manu- scripts had been made in contemporary workshops in Iran. A scandal broke out, in which well-known Iranian, Western and even Soviet scholars were involved and harmed, one way or another. Discovery of numerous (though, obviously, not all) fabrications also re- vealed that people involved in their production had been active in this trade for over twenty years, i. e., approximately from the beginning of World War II. At that time, an increased demand for antique manuscripts emerged in Iran. Here, the capital law should be emphasized: the forgery business would not have achieved such 331 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 a huge scope without market demand. It was fueled by both collectors and the scholarly community. From the end of the 19th century, the latter experienced an acute shortage of available medieval texts in need to be published. Market demand begot local craftsmen. They started to professionally satisfy it both within the country and abroad, until the early 1960s. Physical forgeries At first, it was more domestic-oriented, apparently triggered by local collectors and the need to fill up the manuscript collections of recently opened libraries (e.g., the Nation- al Library of Iran, 1316/1937) with valuable copies. Later, the forgery business expanded, going beyond the borders of Iran and starting to meet the external demand for rare books. g g y g To represent the scale of disaster from physical forgeries, which seized at that time international Iranian studies, a brief critical note by Mujtaba Minuwi (d. 1355/1977), a well-known Iranian specialist in textual and manuscript studies is given below [21]. The reason for its publication originated from the publication of the Ruba‘yyat (Quatrains) by ‘Umar Khayyam in the USSR. As far as I am aware of, this article has never been translated into Russian or English; therefore, here its full text is provided. A note by the expert Mujtaba Minuwi on the number of the forged manuscripts [22] A critical note … about the printed version of Ruba‘yyat (Quatrains) by Khayyam, which was published in Soviet Russia [23], provides an opportunity to write and communicate to the public a few words regarding the copy, which was published as a facsimile together with this publication. In the past twenty years, a number of manuscripts have been put for sale to public libraries and individuals in different cities, which were sold for large sums as “ancient manuscripts”. They have dates and colophons that indicate their antiquity, but actually they are fabrications and for- geries that gained publicity and led to the deception of a group of individuals. 1. A copy of the Andarz-nama (The Book of Advice) by Kay-Kawus, about which I published an article entitled “Kapus-nama-yi Frye” in the journal Yaghma [24]. 1. A copy of the Andarz-nama (The Book of Advice) by Kay-Kawus, about which I published an article entitled “Kapus-nama-yi Frye” in the journal Yaghma [24]. 2. A copy of Diwan of Qatran Tabrizi, “copied by Anwari,” about which Mr. Dr. Mahdi Bayani wrote an article published in the journal Yaghma [25]. 2. A copy of Diwan of Qatran Tabrizi, “copied by Anwari,” about which Mr. Dr. Mahdi Bayani wrote an article published in the journal Yaghma [25]. 3. The book al-Hidaya wa’l-dalala (The Right Guidance and Deviancy), a work by Sahib Ibn ‘Ab- bad, published as a separate risala by Mr. Husayn ‘Ali Mahfuz [26]. 3. Physical forgeries The book al-Hidaya wa’l-dalala (The Right Guidance and Deviancy), a work by Sahib Ibn ‘Ab- bad, published as a separate risala by Mr. Husayn ‘Ali Mahfuz [26]. 4. A copy of the Ruba‘yyat by Khayyam, dated 658 AH and owned by Chester Beatty library. Mr. Professor Arthur J. Arberry published the Ruba‘yyat upon this copy [27]. 4. A copy of the Ruba‘yyat by Khayyam, dated 658 AH and owned by Chester Beatty library. Mr. Professor Arthur J. Arberry published the Ruba‘yyat upon this copy [27]. 5. A copy of the Ruba‘yyat by Khayyam, dated 604 AH and kept in the library of the University of Cambridge. Initially, this copy belonged to the late ‘Abbas Iqbal Ashtiyani and was presented in the pages of Yadgar journal. Its photocopy supplemented with a French translation was published by Pierre Pascal [28], and a photocopy from it was also published in the Russian edition. It was also used in the German translation of the Ruba‘yyat by Khayyam, recently published in East Germany. 6. A copy of the Ruba‘yyat by Khayyam belonging to the library of Mr. Engineer ‘Abbas Mazda and dated 654 AH, a complete photocopy of which I have seen. 7. A copy of the Ruba‘yyat by Khayyam, dated 619 AH and kept with an American antiquarian in New York, a photocopy of a page of which I have seen. 8. A copy of Diwan by Mu‘izzi, which was sold as “a copy of a contemporary of the poet” and taken to the US. Later, they complained it was a fabrication. 9. A copy of the Mi‘raj-nama (Book of the Ascension) attributed to Ibn Sina, dated 584 AH and located among the books of Dr. Mahdi Bayani, a facsimile of which was published by him under the title “Fakhr Razi’s handwriting” [29]. 10. Al-Munajat al-ilahiyyat ‘an Amir al-mu’minin (Divine intimate conversations by the Amir of believers), which were published by Fakhr al-Din Nasiri in offset printing in 1340/1961. 332 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Three or four more copies of the Ruba‘yyat by Khayyam, the Khalas by Natanzi and some- thing else that was sold to the National Library about twenty years ago and is now there should also be added to this list. Physical forgeries The calligrapher who made these copies, or most of them (dated from 485 to 658 AH), is the one and the same person who is still alive. All these manuscripts are products of a workshop that has been operating in Tehran for twenty years. One or two calligraphers, one paper maker, a broker and a salesman responsible for selling antiques managed the workshop, making their craft a fraud of a group of gullible people: they once earned seventy thousand dollars at one American, once fifty thousand tumans at Teh- ran University, offering falsehood, a fabrication and paper making. If they hadn’t been brought to clean water, they would have continued to steal and cheat. I know nothing about the laws related to the forgery of such documents. I do not know whether the Prosecutor or private prosecutors can sue and punish the sellers, copyists and manu- facturers of these books. But I know that their activity will result in enormous damage to Iranian literature. It has already resulted . The culture authorities should take serious measures, publicly and openly inform everybody about the falsification of these books and oversee that manuscripts of this kind are not bought in the future, and this structure vanished. I know nothing about the laws related to the forgery of such documents. I do not know whether the Prosecutor or private prosecutors can sue and punish the sellers, copyists and manu- facturers of these books. But I know that their activity will result in enormous damage to Iranian literature. It has already resulted . The culture authorities should take serious measures, publicly and openly inform everybody about the falsification of these books and oversee that manuscripts of this kind are not bought in the future, and this structure vanished. I am not afraid to publicly disclose without reserve the names of several falsifiers, deceiv- ers and fraudsters engaged in such activity earlier and at the moment.4 However, if, after a huge scandal and a court trial they are acquitted, I am presented as a slanderer, and the books they have fabricated are meanwhile recognized as uninvolved in the slander, then it is better they remain unknown and people only get familiarized with the books. 4 The names of the organizers of this family business have already been named in the Minuwi article [24, 456–457], but, apparently, his first exposure did not harm the falsifiers, who are also mentioned in the article by Francis Richard [15]. Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Physical forgeries Mujtaba Minuwi Mujtaba Minuwi 27 Urdibihisht 1342 [= April 18, 1963] * The Society of National Monuments has already made a number of errors in the epitaphs on the pedestal of Khayyam’s tomb. It was redirected to a copy of Khayyam published in Russia. The appearance and handwriting of these epitaphs is similar to the appearance and handwriting of a large format in a forged copy of the Ruba‘yyat by Khayyam, dated nine-hundred-odd AH and sold twenty years earlier to the National Library. Details of the emergence and development of the crisis related to physical forgeries are given in one of Mujtaba Minuwi’s letters. The letter was first published in the Khandaniha (Worthy Fiction) publicistic magazine which was issued intermittently from 1319/1940 to 1358/1979. Clippings from this magazine were found in the personal archive of Mas‘ud Farzad (d. 1981), researcher of Hafiz and Khayyam works, literary scholar and translator. The archive consisting of materials for research, letters from colleagues and diaries was found after the owner’s death in the cell of the National Bank of Iran, in Shiraz branch. Part of the materials was published in 1999, including an excerpt from Minuwi’s letter. Minuwi wrote his open letter after the publication of the above-mentioned “Critical note” in response to the accusations of his colleague that it was because of him that Iranian orientalists suffered image losses, in particular, in the eyes of Soviet Iranists, and that since he himself initially took the first fabrication for the original, he himself should compen- sate for material damage to the University of Cambridge. Here some discrepancies in the dating of individual fabrications compared to that mentioned in the “Critical note” should be disregarded, because Minuwi obviously wrote on a topic that was emotionally colored for him and such circumstances sometimes lead to inevitable errors. 4 The names of the organizers of this family business have already been named in the Minuwi article [24, 456–457], but, apparently, his first exposure did not harm the falsifiers, who are also mentioned in the article by Francis Richard [15]. 333 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 You have touched on the matter with the copy of the Ruba‘yyat by Khayyam. As you may know, this copy belonged to the late ‘Abbas Iqbal. It was presented to him by one of his friends (perhaps the one who made and fabricated it). Physical forgeries Iqbal personally presented it in the Yadgar journal, and then, having arrived to Paris on a business trip, offered it to customers in London with my mediation. Without assuming any responsibility and not acting as a guarantor of its genuineness and authenticity, I handed it to the buyer, becoming an intermediary in delivering money to the late Iqbal. Here it is necessary to give an explanation: at that time I considered this copy genu- ine, ancient and authentic. But when the number of such copies increased further (Khayyam, dated 613 AH, in New York; Khayyam, dated 653 AH, with Mr. ‘Abbas Mazda; Khayyam, dated 658 AH, in the Chester Beatty library; the Qabus-nama, dated 483 AH, and seventeen-eighteen other copies — all written by one person and all came from the same workshop), I was convinced that they were fabrications. I openly announced their fabrication and forgery in 1960 in Washington. In the same year, at the Congress of Orientalists in Moscow, I saw that Mr. Aliyev published the same copy of Khayyam in the form of a facsimile, issuing a typesetting text and a Russian translation on its ba- sis, because considered it authentic and genuine. Being there, one day at the ad hoc meeting I told the sixteen or seventeen attending Tajik, Russian and Caucasian Iranists for two hours and forty- five minutes about a copy of Khayyam and other forged copies that appeared on the market over the past twenty-plus years and recognized by a number of persons, proving with hard-hitting arguments and evidence that these copies could not be taken as a sample when publishing texts. One of those present was the same Mr. Professor Aliyev, who is now in Tehran. You can ask him. y Therefore, I was the first to announce the forgery of this copy. Thanks to the article I pub- lished about a fabricated copy of the famous Qabus-nama, everyone’s attention was drawn to this copy-making workshop. If Mr. Aliyev said anything about this, he should have said in such ex- pressions: “I considered this copy [of the Ruba‘yyat by Khayyam] authentic, but one expert from Tehran University dispelled my misconception”. Physical forgeries After that, this issue was again discussed in the Rahnama-yi kitab (A Guide to Books) jour- nal, where I gave a list of ten to twelve fabrications, indicating that part of these books were pur- chased for a considerable price by the Ministry of Culture and the other part — by the University of Tehran [see above] … [30, pp. 192–193]. Here a number of historical references should be given. Here a number of historical references should be given. 1. In August 1960, the 25th International Congress of Orientalists was held in Mos- cow. 1. In August 1960, the 25th International Congress of Orientalists was held in Mos- cow. 2. Minuwi took for the original not only the copy of the Ruba‘yyat by Khayyam, but also the forgery of the Qabus-nama [31], dated 483/1090, illustrated with minia- tures and mentioned as the first item in his “Critical note”. This happened back in 1950. Here is what he writes: In 1950, I personally saw two pages from this book in Paris with the late ‘Abbas Iqbal Ash- tiyani, who brought them perhaps to sell in Europe. He said that the copies belonged to one of his friends, who asked five thousand GBR for them. I saw these two pages for some ten minutes and, without bothering to analyze them somehow, was deceived by their appearance and did not doubt their authenticity. A few weeks later, Mr. Prof. Reuben Levy made a presentation about Qabus-nama to the Iranian Society in London. Upon the end of his presentation, I openly an- nounced that a copy of Qabus-nama, dated 483 AH, was found and that it would be proper if Mr. Levy puts it as the core of his publication [24, p. 451]. 3. Later, Minuwi became doubtful about the authenticity of the “ancient” manu- scripts, which popped up in Iran like mushrooms after a rain. In 1952, he openly warned Richard N. Frye (d. 2014), a relatively young American Iranist with mini- 3. Later, Minuwi became doubtful about the authenticity of the “ancient” manu- scripts, which popped up in Iran like mushrooms after a rain. In 1952, he openly warned Richard N. Frye (d. 2014), a relatively young American Iranist with mini- 334 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. Physical forgeries The experts failed to issue an unambiguous verdict upon the chemical composition of the paper but the miniatures and the blue ink color used in the manuscript were recognized as fabrication with confidence [33, pp. 87–91]. Export permit for the copy was issued by Dr. Mahdi Bayani (d. 1346/1967), the founder and first director of the National Library of Iran and a part-time head of the expert council on values at the Ministry of Culture. In 1331/1952 he already knew for certain that this manuscript was a fabrication, otherwise he would not have given export permit, which he wrote in response to Frye’s written claim: You know, if we considered the copy of the Diwan by Mu‘izzi to be genuine and ancient, we would not be able to give permission for its export. You did not ask our opinion on its authenticity or forgery, but only asked export permit, which we gave you. You will have to take responsibility by yourself, whether it is an original or fabrication [24, 453]. 5. The fabrication of the Qabus-nama was sold by forgers by parts and in 1953 was already smuggled into the US by the authorized agents of two buyers. One part went to the collection of the Museum of Cincinnati, the second — to the collector, Hagop Kevorkian (d. 1962) to New York. Richard Frye became famous for buying a “rare” manuscript for Harvard and later, following the instructions from Kev- orkian, toured across the Middle East countries in search of antiques and came again to Iran in 1953. That is why Minuwi at first was mistaken that the Qabus- nama was smuggled out by Frye. 6. In 1954–55, Walter Bruno Henning (d. 1967), one of the leading experts in the pre-Islamic history of Iran [34] and Minuwi’s teacher revealed the forgery of the Qabus-nama upon the philological text analysis. He wrote a letter to Minuwi with the results, asking him to publish these findings without mentioning his name. Minuwi did just as requested in a famous article, “Kapus-nama-yi Frye”, taking Henning’s letter as a baseline. 7. Following numerous articles published in Iran, mutual accusations and excuses, as well as the publication of official documents in the international scientific jour- 7. Following numerous articles published in Iran, mutual accusations and excuses, as well as the publication of official documents in the international scientific jour- 335 Вестник СПбГУ. Востоковедение и африканистика. 2019. Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Physical forgeries 3 mal experience in manuscript studies, that he should not buy manuscripts from a famous Iranian family with whom Minuwi was personally acquainted and knew about their trade. But Frye really should not hold a grudge against anybody but himself. Because in 1952 and 1953, when as a guest of the University of Tehran he lived as a student in this city and, incidentally, was looking for rare manuscripts — I guarded him against, discouraged and warned him to refrain from manuscripts that come out of the hands of certain people and a famous family, God forbid, he would be deceived. Instead of displaying vigilance, Frye just conveyed my words openly to the very person I had warned him against, putting me in a difficult position to respond to his claims. One or two months after, Mr. Frye returned to the New World with a copy of Diwan by Mu‘izzi, for which the sellers took a significant amount from him [24, p. 452]. 4. After a forged copy of the Diwan by Mu‘izzi, dated 551 AH, richly illustrated with miniatures, was officially purchased by the Houghton Library at Harvard University (MS Typ 1016 at Houghton Library), upon the advice from Frye, the examination of miniatures and chemical composition of paper and ink was completed there by 1954. The experts failed to issue an unambiguous verdict upon the chemical composition of the paper but the miniatures and the blue ink color used in the manuscript were recognized as fabrication with confidence [33, pp. 87–91]. Export permit for the copy was issued by Dr. Mahdi Bayani (d. 1346/1967), the founder and first director of the National Library of Iran and a part-time head of the expert council on values at the Ministry of Culture. In 1331/1952 he already knew for certain that this manuscript was a fabrication, otherwise he would not have given export permit, which he wrote in response to Frye’s written claim: 4. After a forged copy of the Diwan by Mu‘izzi, dated 551 AH, richly illustrated with miniatures, was officially purchased by the Houghton Library at Harvard University (MS Typ 1016 at Houghton Library), upon the advice from Frye, the examination of miniatures and chemical composition of paper and ink was completed there by 1954. Physical forgeries Т. 11. Вып. 3 nal Nama-yi Baharistan, the latest purchase details of the fabricated Qabus-nama and Diwan by Mu‘izzi surfaced in Frye’s autobiographical memoirs [35]. nal Nama-yi Baharistan, the latest purchase details of the fabricated Qabus-nama and Diwan by Mu‘izzi surfaced in Frye’s autobiographical memoirs [35]. nal Nama-yi Baharistan, the latest purchase details of the fabricated Qabus-nama and Diwan by Mu‘izzi surfaced in Frye’s autobiographical memoirs [35]. 8. ‘Abbas Iqbal Ashtiyani, a distinguished historian and specialist in textual studies, died in Rome in February 1956 [36], did not see either the first or the subsequent Minuwi’s revelations, but he was probably aware of news from the US. Appar- ently, the forgers used him “blindly” to their advantage, without his knowledge, to search for buyers abroad. Most likely, he, just like Minuwi, took the fabricated copy of the Ruba‘yyat by Khayyam presented to him for the original, which speaks only of the high level of fabrication manufacturing. 9. Relying on in-house observations and the available works of foreign colleagues, Iranian scholars from the National Library of Iran carried out studies on the clas- sification of the main external signs of physical forgeries. The research results, supported by illustrative examples, were recently published [37, 265–291]. Unfor- tunately, certain external features of fabrications listed in the study, can no longer serve as indisputable evidence for their identification, though are quite capable of attracting attention and transferring the manuscript to the category of “uncertain”. Operational aspects of a forgery workshop The bare facts below provide an opportunity to note some business details of forgers of medieval manuscripts in the mid-20th century. List of productions Calligraphers of this team felt more confident in forging the manuscripts of mainly Persian-language writings of the Great Saljuqs reign (431–552/1040–1157): the Qabus-na- ma, allegedly copied in 483/1090, the Ruba‘yyat by Khayyam (d. 510/1131), part of the Diwan by Mu‘izzi (d. between 518–522/1124–1128), part of the Diwan by Qatran Tabrizi (d. 465/1072), allegedly copied by Awhad al-Din Anwari (d. 585/1190), al-Khalas by Adib Natanzi (d. 497/1103 or 499/1105) [38]. They obviously preferred the Saljuqid epoch be- cause of the high cost of literary works of this period in the book market, which does not exclude deviations in one direction or another. Dates of the copied writings in the discovered fabrications cover a period of slightly less than two centuries, from 483/1090 to 658/1259–60. Open questions On what grounds and who made a decision to manufacture this or that fabrication? Has the demand, potential victims and their preferences been investigated? Who else was involved in the forgery business, apart from those directly involved in manufacturing fab- rications? All of them all were members of an educated elite of the Iranian society, knew each other well, were organized in various associations, were part of the administration of state organizations and national libraries. And, the top question: how many fabrications were made? After all, none of the forgers has never (sic!) provided a full list of fabrica- tions made by them for over 20 years, from the late 1930s to the early 1960s. And by all accounts, their performance was pretty good. The appearance of a Siyar al-muluk copy from the Nakhjavani collection A copy of Siyar al-muluk from the Nakhjavani collection appears in the interval of maximum range between 1334–1337/1955–1958. The first date refers to the time when the third edition of Siyar al-muluk was released in Iran, without reference to this copy, which nobody knew of before and six or seven years after that release. The second date is the time when Muhammad Nakhjavani donated his collection of manuscripts and books to the newly established Tabriz National Library in the late April 1958. In the period be- tween these two dates the copy enters the collection of Muhammad Nakhjavani, i. e., a year before the publication of Minuwi’s “Kapus-nama-yi Frye” (1956) and five years before the release of his “Critical note” (1963). The discovery of a new copy in the midst of a physical forgery scandal is not in the least indicative in itself. Also, nothing is proved by the fact that its existence first became known to a foreigner rather than Iranian specialists, who had previously published Siyar al-muluk, including Mujtaba Minuwi. It suggests analogies with the Diwan by Mu‘izzi and the Qa- bus-nama, for which the existence of the “oldest” copies became known to Iranian experts only after they were moved abroad, completely or partially. However, all this could be a sim- ple coincidence. But in combination with the facts cited in the Introduction to the first book of the announced series, the physical authenticity of this manuscript raises grave doubts. Distribution Three features of finished products distribution are seen most clearly: Three features of finished products distribution are seen most clearly: • a fabricated copy could be sold both as a single piece and by parts; • the main customers, both domestic and from abroad, were private collectors and manuscript repositories; • since the early 1950s, when the workshop business entered the international level, the forgers took to conspiracy, when foreigners were the first to get news about the workshop products, instead of Iranian scientists; • the sale of the manuscript by parts, as well as Minuwi’s references to separate pages of manuscripts he viewed personally, indicate the fact that fabrications had no genuine bindings to fit the dates of copying indicated in the text. In other words, these fabrications were apparently either a set of individual sheets or notebooks, 336 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 or had a binding manufactured by the forgers. The absence of genuine bindings can be considered to some extent a distinctive feature of the workshop producing physical forgeries. or had a binding manufactured by the forgers. The absence of genuine bindings can be considered to some extent a distinctive feature of the workshop producing physical forgeries. Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Methods to detect fabrications Modern methods of identifying fabrications have gone far ahead, as compared with those used in the 20th century. Therefore, visual signs of a forgery are no longer the final proof for its identification. An expert assessment by guess-work will satisfy and convince nobody. One expert has more experience, while another has less. As already shown above, expert opinions can be diametrically opposite. Today, in order to eliminate the human factor as much as possible, the questionable artifacts of the written tradition are examined in several directions. 337 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 1. Comprehensive multidisciplinary source study analysis. Some disciplines were separated into independent areas of scientific analysis; others shifted to the digital do- main due to the development of digital humanities (DH). The general principle underlying these sciences is digitizing everything that can be digitized and creating a representative database. And the general algorithm is the comparison of heterogeneous statistics gener- ated by processing a huge data arrays. Preparing a database for automated analysis and retrieving desired statistics is the most time consuming part of operations in any DH area. g g p p y At present, the stage of placement of scanned (PDF) Arabographic texts published in the pre-digital era (20th century) on dedicated websites is actually finalized. The technical issue of their optical recognition (OCR) and full digitization is being successfully solved and the required database is under creation. In other words, the corpus of many thou- sands of medieval Arabographic works gains a new life, this time in digital form. All these processes somewhat remind of a short transitional period from lithographed editions (19th — early 20th centuries) to typesetting. Today, we face another dynamic change of the media with a clear trend to increase its accessibility: memory → manuscript → litho- graphy → typesetting book → digitized text → … g y y g g A good database of digitized texts in a format suitable for search engines (searchable) already provides for all kinds of source research in record time [39], such as comparison of texts and the search for common locations, same persons, toponyms and terms within different texts, etc. This work was performed previously in the analysis of written sources. 5 An overview of problems and methods [see: 41]. Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Methods to detect fabrications This includes a list of books used by the author, citing common locations, text pointers, concordances, various kinds of special-purpose dictionaries, limited by some timeframe, school or even by the author, etc. But the indisputable advantage of automated compari- son is the speed of processing a huge amount of uploaded data. For instance, the Chasto- tnyi slovar’ Unsuri (The ‘Unsuri Frequency Dictionary, 1970) was compiled by its author, M.-N. O. Osmanov (d. 2015), for more than one year. Today, with all the digitized texts by the Ghaznavid court poet Abu’l-Qasim ‘Unsuri (d. 431/1039), the same dictionary can be generated in a couple of hours. The difference is obvious. g Programs developed for digitizing the pre-scanned printed texts can be possibly adapted for optical recognition of the medieval handwritten texts. But so far we can only dream of some breakthroughs in the recognition and digitization of several thousand Arabographic works, yet unpublished and copied in different languages and different handwritings. Single cases of application of computer graphology by 3-5 graphic markers of individual handwriting do not count here. g 2. An independent area of source study, or rather codicology, is a comprehensive tech- nological analysis of the material text carrier, i. e. primarily, the paper: sheet thickness; their color characteristics; surface relief; fiber length, etc. The International Association of Paper Historians (IPH) founded almost 60 years ago [40] has been very successful; its members regularly publish impressive results of recent advances, including new paper dating methods5. 2. An independent area of source study, or rather codicology, is a comprehensive tech- nological analysis of the material text carrier, i. e. primarily, the paper: sheet thickness; their color characteristics; surface relief; fiber length, etc. The International Association of Paper Historians (IPH) founded almost 60 years ago [40] has been very successful; its members regularly publish impressive results of recent advances, including new paper dating methods5. With regard to physical forgeries created in Iran in the mid-20th century, this means that technological analysis will compare the paper of previously identified forgeries, which are still kept in the above listed manuscript repositories, with the paper of copies, whose authen- ticity is questioned. Indeed, in this case, fabrications were apparently made in one workshop, where paper was always produced with the same technology and from the same components. 338 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. Stylometry Stylometry makes one of the directions in the computer text diagnostics. Various types of stylometry software are being developed in different countries, which operate with the authorial signal, or the author’s fingerprint. It is detected by measuring the most frequent words (MFW), combinations of two or more words and other markers appro- priate for automated processing and producing the desired statistics. With regard to the identifying fabrications, this means that the program is first trained to identify foot fea- tures in an authentic text(s). Then it compares the identified foot features with those in the doubtful text. The language, fonts and text direction (from right to left or vice versa) is absolutely unimportant for automated processing. However, the objective limitations of stylometry application are already obvious. Its main disadvantage is a fairly significant number of MFW needed to identify the author’s signal and produce a reliable result. Starting with a certain minimum, their number should be as large as possible, and they should not be stylistically neutral. The results of computer-aided diagnostics of the Roman de la Rose, a French poem of the XIIIth century presented in the article by Maciej Eder [42, pp. 457–469], demonstrate that stylometry can be successfully applied to identifying medieval fabrications in poet- ry, though if only where the MFW number is adequate for the purpose. The poem was written jointly by two authors: Jean de Meun and Guillaume de Lorris. The second author added his part to the verses of the first author and both styles were clearly identified by stylometry. In other words, we can expect that literary fabrications made by the first meth- od, i. e., with the addition of a bulky inauthentic text to the end of the authentic part — a double-layer pie, will also be identified by stylometry. A case study of multi-layered fabrications analysis is shown by the Queen Sophia’s Bible stylometry. According to its results, given in the same article, M. Eder characterizes the book as a “multifaceted collaborative work, in which the translatorial, authorial, and scribal signals are heavily mixed”. Stylometry reflected the work of five translators who had their own stylistic markers. y But there is a catch. All texts taken by M. Eder for computer diagnostics are volumi- nous — a lengthy poem, the Bible translation. In these cases, the authors or translators had enough space to express themselves and leave their individual fingerprints. Methods to detect fabrications 3 Apart from writing material, the handwriting of an uncertain manuscript can also be subjected to technological analysis and digital graphology, if the derived results can be compared with a certain reference standard, i. e., an autograph sample. However, the number of medieval autographs survived until present is far less than the number of man- uscripts copied by thousands by unknown copyists. p p y y py 3. Traditional textology is also being transformed into digital format. Novel DH areas are emerging on the basis of the existing database of digitized and searchable texts, aiming at their multifaceted machine analysis. This is a computer text diagnostics, which some- what resembles the computed tomography of physical organs. It will not only allow, but also require a qualitatively new level of critical attitude to the texts of specified authors, avoiding a blind trust in what came to us under their authorship. At the same time, the limitations of such diagnostic applications are already visible. 3. Traditional textology is also being transformed into digital format. Novel DH areas are emerging on the basis of the existing database of digitized and searchable texts, aiming at their multifaceted machine analysis. This is a computer text diagnostics, which some- what resembles the computed tomography of physical organs. It will not only allow, but also require a qualitatively new level of critical attitude to the texts of specified authors, avoiding a blind trust in what came to us under their authorship. At the same time, the limitations of such diagnostic applications are already visible. Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Stylometry If the machine diagnostics is applied to a multi-layered text even of an innovative tasnif (not to mention a 339 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 compilation-ta’lif), which features a combination of synthesis and analysis, the stylometry of such text would give an erroneous result when searching for the author’s fingerprint. Suppose, two thirds of the tasnif size is made of small quotations taken as illustrative and evidence base from different sources, where each quotation has its intrinsic style attrib- utes: quotations from the Quran, statements of the Prophet Muhammad, a few quotes from his companions, etc. — their styles are different. And only the remaining one-third of the size, or even less, presents the author’s original conclusions with the unique author’s style. The author’s fingerprint in the end of quotation series would make almost no differ- ence from any other quotation for computer-aided analysis, even less so if placed among them. The same difficulties related to the lack of the authorial text will apparently arise with stylometry of short quatrains attributed to Khayyam. Nevertheless, a stylometry method for relatively voluminous non-attributed qasi- das of medieval Persian poets is proposed in the first book of the announced series. This method was tested on an anonymous qasida, which makes an integral part of the Siyar al-muluk at the Institute of Polish language (Instytut Języka Polskiego, PAN, Kraków). The test was kindly made by Joanna Byszuk, a member of the research team of Maciej Eder. General design of the series Each of the texts included in the series is opened by an analytical introduction to the translation, which considers the widest range of related issues, with emphasis on the target audience analysis. Translations are followed by the original Persian texts at the end of each book preceding the indexes and bibliography. For the purpose of design unification in the reference and bibliographic aids and transliteration, all the books in this series follow The Chicago Manual of Style [43], long developed and adopted by most international Oriental publishing houses. The preference given to this manual is explained by the simplicity and clarity of the rules developed, on the one hand, and the attempt to achieve some uniformity throughout the national Cyril- lic publications, in accordance with the principles of Western publications, on the other. Here, these rules are as follows: 1. Arabographic proper names, toponyms and original terms are given: 1. Arabographic proper names, toponyms and original terms are given: a) in a simplified strong transliteration, i. e., using only three vowels of the Arabic alphabet, corresponding in the spelling to three diacritic marks: fatha ‘a’, kasra ‘i’, damma ‘u’, and their diphthongs: ‘ay’, ‘iy’, ‘uy’, as well as two additional signs for rendering the Arab-Persian letter ‘ayn and hamza. In my opinion, translitera- tion with numerous diacritic marks is low-informative for the reader who does not speak Arabic or Persian, except for visualizing a set of obscure words, while an expert can understand their meaning even in a simplified transliteration, especially with the original at hand; b) without assimilation in the Arabic definite article; c) without a soft mark. All this prevents mixing transliteration with transcription. Since the translation is based on Arabographic texts, where vowels are conveyed only by the specified diacritic marks, this transliteration also includes Turkic proper 340 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 names. Their transliteration in this work may sometimes differ from the generally ac- cepted in Russian-language publications (for example, Seljuqs → Saljuqs, Turkan/Terk- en-khatun → Tarkan-khatun, etc.) and is based on the diacritic marks given in the online Persian dictionary — the Lughat-nama by ‘Ali Akbar Dihkhuda (d. 1334/1956) [44, 45]. The logic is simple: medieval authors transliterated the pronunciation of the original Turkic names in Arabic script, which was not fully adapted to represent all the phonetic features of the Turkic language. General design of the series But today it is the Arabographic texts that serve as orig- inals, and not some Turkic runic artifacts, where these names could have been written in some other way. y In my opinion, such an approach to the Russian-language transmission of the Turkic proper names written with the Arabic graphics, even if in a partially distorted form, looks more reasonable and consistent than their transmission in writing through “reconstruct- ed” transcription in Cyrillic, i. e., Russian written record of how they were probably pro- nounced by the Turks some 900 years ago, or how they are pronounced now. To avoid a similar anachronism, all texts provide full formulas of eulogies and good wishes, cited by the authors after mentioning the names of prophets, imams and promi- nent Muslim persons, instead of their obscure abbreviations, as is done in some modern Muslim publications. 2. References to sources and studies in page notes are given at the first mention in ex- panded form, further — in the abbreviated form. References to Arabographic publications are given in Latin transliteration in order to avoid confusing them with published Russian translations of the same-title texts. 2. References to sources and studies in page notes are given at the first mention in ex- panded form, further — in the abbreviated form. References to Arabographic publications are given in Latin transliteration in order to avoid confusing them with published Russian translations of the same-title texts. Preference in the reference and encyclopedic literature is given to web-based pub- lications in the public domain that do not require registration or with minimal formal registration. The same applies to links to dedicated websites where digitized printed pub- lications are posted (marked as ‘available online at’) or online versions of publications only (marked as ‘online edition’). Their accessibility ensures rapid verification and clarification of the information provided, as well as to view a list of sources and studies. Today, the accessibility, rapid verification and critical evaluation have become the main criteria for data providing. To meet these criteria, citations from sources and studies included in the analytical introduction to the texts of this series are given both in transla- tion and in their original form. g 3. All interpolations in texts are taken in square brackets, including references to the Quran. All Arabic sayings, Oriental words and terms, as well as verses and Quranic quota- tions, are italicized. 4. References 1. Daftary Farhad. Hasan Sabbah. Encyclopædia Iranica. Available at: http://www.iranicaonline.org/arti- cles/hasan-sabbah (accessed: 11.11.2019). Henceforth: Encyclopædia Iranica — EIr. 2. ‘Abd al-Jalil Qazwini Razi. Demolishment (Some pieces of defamation by the Shiite haters demolishing “Some Infamies of the Rafidites”). Ed. Mir Jalal al-Din Hussaini Urmawi Muhaddith, Tehran, 1358/1979. (In Persian) ) 3. Torkamany Azar, Parvin. Saljuqs: Religious Dissensions and the Results. Pazhuhish-nama-yi ‘ul- um-i insani, no. 51, Autumn 1385/2006, pp. 53–74. Available at: http://ensani.ir/fa/article/144047/   (accessed: 11.11.2019). (In Persian) 4. Shaked S., Safa Z. Andarz and andarz literature in pre-Islamic Iran; Andarz literature in New Persian. Eir. online edn. Available at: http://www.iranicaonline.org/articles/andarz-precept-instruction-advice (ac- cessed: 31.05.2019).h 5. Khismatulin A. Amir Mu‘izzi Nishapuri. The Siyasat-nama/Siyar al-muluk: A Fabrication Ascribed to Nizam al-Mulk. St. Petersburg-Moscow, Peterburgskoe vostokovedenie, Sadra Publ., 2019. (In Russian and Persian)h 6. Siaset-name. The book on the rule of the XI century wazir Nizam al-Mulk. Transl. by B. N. Zakhod- er. Moscow-Leningrad, Izdatel’stvo AN SSSR, 1949. Available (without proofreading after OCR) at: http:// www.vostlit.info/Texts/rus10/Siaset_name/framepred.htm (accessed: 11.11.2019). (In Russian) 7. Davarpanah Hormoz. Mo‘ezzi Nišāburi. Eir. online edn. Available at: http://www.iranicaonline.org/ articles/moezzi-nisaburi (accessed: 11.11.2019). 8. Durand-Guédy David. Malekšah. Eir. online edn. Available at: http://www.iranicaonline.o maleksah (accessed: 11.11.2019). ( ) 9. Nizam al-Mulk. Siyar al-muluk, 2nd ed. H. Darke, Tehran, Bungah-i tarjuma wa nashr-i kitab, 1347/1968. (In Persian). There are two introductions in the book: in Persian and in English. h 10. Ra’isniya Rahim. One of the sides of biography and works of Hajj Muhammad agha Nakhjawani. Ba qafila-yi shawq: Arj-nama-yi duktur-i Muhammad ‘Ali Muwahhid, Tehran, Sutude, 2014, pp. 537–554. Avail- able at: http://www.mirasmaktoob.ir/sites/default/files/archive/pdf/raeesnia-rahim.pdf (accessed: 11.11.2019). (In Persian)h 11. Khismatulin A. The Writings of Imam al-Ghazali. St. Petersburg-Moscow, Peterburgskoe vostokove- denie, Sadra Publ., 2017. (In Russian and Persian) 12. Likhachev D. S. with collaboration of Alekseev A. A. and Bobrov A. G. Textology (on the material of Russian literature of the 10th–17th centuries). St. Petersburg, Aletheia Publ., 2001. Available at: http://www. lihachev.ru/pic/site/files/fulltext/textologia/0070.pdf (accessed: 11.11.2019). (In Russian)i pi g p 13. Petrov А. Е., Shnirelman V. А. Introduction. Falsification of historical sources and construction of ethnocratic myths. Moscow, The Institute of Archaeology of the RAS Publ., 2011, pp. 5–14. Available at: http://static.iea.ras.ru/books/falysifikatsiya_istorichyeskikh_istochnikov.pdf (accessed: 11.11.2019). (In Russian) 14. Bakonina M. S. A Favorite Poet of the President. Available at: http://www.fontanka.ru/2007/06/19/053/ (accessed: 11.11.2019). (In Russian) 15. Soudavar Abolala. Forgeries i. Introduction; Oscar White Muscarella. Forgeries ii. Of pre-Islamic art; Sheila Blair. Forgeries iii. Of Islamic art; Richard Francis. General design of the series Persian originals are attached to the translations. They give the reader an oppor- tunity to suggest, if desired, his/her own version of the translation, not limited to that published in this series. No matter how good the translation may seem, it can only bring us closer to the original, in no case replacing it, of course, if the original has reached us. At the same time, as shown by the centuries-old practice of oriental studies, everything be- comes outdated with time: scholarly approaches, research findings and even translations, though their life is slightly longer. Only the original texts are never outdated — the sources that remain unchanged in the same form they were created by their authors hundreds of 341 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 References Forgeries iv. Of Islamic manuscripts. Eir. online edn. Available at: http://www.iranicaonline.org/articles/forgeries-i (accessed: 11.11.2019). p g g 16. Tor Deborah. Sanjar, Ahmad b. Malikšah. Eir. online edn. Available at: http://www.iranicaonline. org/articles/sanjar (accessed: 11.11.2019). g j 17. Abu’l-Hasan ‘Ali al-Hujwiri. Kashf al-mahjub. Ed. by V. Zhukovskiy. Leningrad, 1929. (In Persian) 18. Jalal al-Din al-Suyuti. The Differentiator between the Сomposer and Plagiarist. Ed. by Hilal Naji. Beirut, 1998. (In Arabic) 19 Khi li А Th M di l t ’lif T l i l l i d l i f h Ri l i Abd li b 19. Khismatulin А. The Medieval ta’lif: Textological analysis and translation of the Risala-yi Abdaliya by Ya‘qub Charkhi. Ars Islamica. Eds A. Alikberov, M. Piotrovsky. Moscow, Vostochnaya literature Publ., 2016, pp. 392–436. Available at: http://orientalstudies.academia.edu/AlexeyKhismatulin (accessed: 11.11.2019). (In Russian and Persian)h 20. Rosenthal Fr. The Technique and Approach of Muslim Scholarship. Analecta Orientalia, 1947, is- sue 24, pp. 1–74. pp 21. Omidsalar Mahmoud. Minovi, Mojtaba. Eir. online edn. Available at: http://www.iranicaonline.org/ articles/minovi-mojtaba (accessed: 11.11.2019). j 22. Minuwi M. A note by the expert Mujtaba Minuwi on the number of the forged manuscripts. Rahna- ma-yi kitab, issue 2, sal-i 6, 1342/1963, pp. 238–240; idem., ibid. in: Nama-yi Baharistan, issue 5, 1381/2002, 342 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 pp. 191–192. Available at: http://www.ensani.ir/fa/content/259990/default.aspx (accessed: 11.11.2019). (In Per- sian) 23. ‘Omar Khayyam. Ruba‘yyat, tr. and introduction by R. M. Aliev and M.-N. Osmanov, ed. by E. Ber- tel’s. Moscow, Vostocnhaia literature Publ., 1959. Pt. 1 — the MS faximile; Pt. 2 — the Russian translation provided with the Persian text. (In Russian and Persian)h 24. Minuwi M. The Kapus-nama by Frye (an exercise in the skill of identifying the forgeries). Yaghma, issue 9, 1335/1956, pp. 449–465, pp. 481–495. Available at: http://rasekhoon.net/article/show/902513/ (ac- cessed: 11.11.2019). (In Persian)h 25. Bayani Mahdi. The Diwan of Qatran Tabrizi by Anwari Abiwardi’s handwriting. Yaghma, 1329/1950, issue 11, no. 3, pp. 461–474. Available at: http://www.ensani.ir/fa/content/278733/default.aspx (accessed: 11.11.2019). (In Persian) 26. Ibn ‘Abbad Sahib. An Epistle on the Right Guidance and Deviancy. Eds Husayn ‘Ali Mahfuz, Muham- mad Muwaqqar. Tehran, Anjuman-i farhangi-yi mihr, 1374 AH/1955 AD. (In Persian) 27. Omar Khayyam. A New Version Based upon Recent Discoveries. Ed. by A. J. Arberry. London, J. Mur- ray, 1952. y 28. Omar Khayyam. References Les Robaiyyat d’Omar Khayyam de Neyshaboor, pour la première fois, traduits en vers français par Pierre Pascal d’après les plus anciens manuscrits, Roma, Éditions du Coeur Fidèle, 1958.h 29. Ibn Sina, Abu ‘Ali Husayn b. ‘Abd Allah. The Mi‘raj-nama by imam Fakhr Razi’s handwriting. Chap-i ‘aksi ba kushish-i Mahdi Bayani, Tehran, Anjuman-i dustdaran-i kitab, 1331/1952. (In Persian) 29. Ibn Sina, Abu ‘Ali Husayn b. ‘Abd Allah. The Mi‘raj-nama by imam Fakhr Razi’s handwriting. Chap-i ‘aksi ba kushish-i Mahdi Bayani, Tehran, Anjuman-i dustdaran-i kitab, 1331/1952. (In Persian) 30. Rastgar Fasayi Mansur. Khayyam, Farzad and the forged manuscripts of the Ruba‘yyat. Farhang, issue 29–32, 1378/1999, pp. 181–202. Available at: http://www.ensani.ir/fa/content/266314/default.aspx (ac- cessed: 11.11.2019). (In Persian) 30. Rastgar Fasayi Mansur. Khayyam, Farzad and the forged manuscripts of the Ruba‘yyat. Farhang, issue 29–32, 1378/1999, pp. 181–202. Available at: http://www.ensani.ir/fa/content/266314/default.aspx (ac- cessed: 11.11.2019). (In Persian) 31. de Bruijn J. T. P. Kaykavus b. Eskandar. Eir. online edn. Available at: http://www.iranicaonline.org/ articles/kaykavus-onsor-maali (accessed: 11.11.2019).h 31. de Bruijn J. T. P. Kaykavus b. Eskandar. Eir. online edn. Available at: http://www.iranicaonline.org/ articles/kaykavus-onsor-maali (accessed: 11.11.2019).h y 32. ‘Unsur al-Ma‘ali Kaika’us Ibn Iskandar Ibn Qabus. The Nasihat-Nama known as Qabus-Nama. Ed. by Reuben Levy. London, Luzac, 1951 (GMS, N. S. 18). y 32. ‘Unsur al-Ma‘ali Kaika’us Ibn Iskandar Ibn Qabus. The Nasihat-Nama known as Qabus-Nama. Ed. by Reuben Levy. London, Luzac, 1951 (GMS, N. S. 18). 33. Mujarrad Mujtaba. On the forged and illustrated MS of Diwan by Amir Mu‘izzi in Harvard University. Guzarish-i mirath 5–6, sal-I 6, 1391/2012, pp. 87–91. Available at: http://www.mirasmaktoob.ir/fa/system/ files/nashriat/GM-54&55-Mojarad-Noskhe-p87.pdf (accessed: 11.11.2019). (In Persian) i j 34. Sundermann Werner. Henning, Walter Bruno. Eir. online edn. Available at: http://www.iranicaon- line.org/articles/henning-walter-bruno (accessed: 11.11.2019). 35. Frye Richard N. Greater Iran: A 20th-century Odyssey. Costa Mesa, Mazda, 2005. 36. Afšar Iraj. Eqbal Aštiani ‘Abbas. Eir. online edn. Available at: http://www.iranicaonline.org/articles/ eqbal-astiani (accessed: 11.11.2019). ‘ b b ll h h l l f h k d f f d f b 37. ‘Azimi Habibollah, Hashemi-e Asl Helya. Examination of the kinds of forgeries and fabrications in the manuscripts provided with their examples. Ayina-yi Mirath, 1394/2015, issue 56, pp. 265–291; available online at: http://www.am-journal.ir/article_46428.html (accessed: 11.11.2019). (In Persian) 38. Monzawi ‘A. N. Adib Natanzi. Eir. online edn. Available at: http://www.iranicaonline.org/articles/ adib-natanzi-badi-al-zaman-abu-abdallah-hosayn-b (accessed: 11.11.2019). 39. Romanov M. Digital Age, Digital Methods. Ars Islamica. Eds A. Alikberov, M. Piotrovsky. Moscow, Vostochnaia literature Publ., 2016, pp. 129–156.h 39. Romanov M. Digital Age, Digital Methods. Aut hor’s i nfor mat i on: pp. 191–192. Available at: http://www.ensani.ir/fa/content/259990/default.aspx (accessed: 11.11.2019). (In Per- sian) 23. ‘Omar Khayyam. Ruba‘yyat, tr. and introduction by R. M. Aliev and M.-N. Osmanov, ed. by E. Ber- tel’s. Moscow, Vostocnhaia literature Publ., 1959. Pt. 1 — the MS faximile; Pt. 2 — the Russian translation provided with the Persian text. (In Russian and Persian)h References Ars Islamica. Eds A. Alikberov, M. Piotrovsky. Moscow, Vostochnaia literature Publ., 2016, pp. 129–156.h 40. The International Association of Paper Historians. Available at: http://www.paperhistory.org/index.php (accessed: 31.05.2019). 40. The International Association of Paper Historians. Available at: http://www.paperhistory.org/index.php (accessed: 31.05.2019). 41. Balachenkova A. P., Tsypkin D. O. Possibilities of technological analysis of historical papers in source-study investigation of monuments. Vestnik of Saint Petersburg University. History, 2017, vol. 62, is- sue 2, pp. 375–399. https://doi.org/10.21638/11701/spbu02.2017.213. (In Russian) 41. Balachenkova A. P., Tsypkin D. O. Possibilities of technological analysis of historical papers in source-study investigation of monuments. Vestnik of Saint Petersburg University. History, 2017, vol. 62, is- sue 2, pp. 375–399. https://doi.org/10.21638/11701/spbu02.2017.213. (In Russian) source-study investigation of monuments. Vestnik of Saint Petersburg University. History, 2017, vol. 62, is- sue 2, pp. 375–399. https://doi.org/10.21638/11701/spbu02.2017.213. (In Russian) sue 2, pp. 375–399. https://doi.org/10.21638/11701/spbu02.2017.213. (In Russian) d ll l l h l h 42. Eder M. Rolling Stylometry. Digital Scholarship Humanities, isuue 31-3, 2016, pp. 457–469. 43. The Chicago Manual of Style Online. Available at: https://www.chicagomanualofstyle.org/home.html (accessed: 11.11.2019). 43. The Chicago Manual of Style Online. Available at: https://www.chicagomanualofstyle.org/home.html (accessed: 11.11.2019). 43. The Chicago Manual of Style Online. Available at: https://www.chicagomanualofstyle.org/home.html (accessed: 11.11.2019). 44. Sa‘idi Sirjani ‘A.-A. Dehkhoda, Mirza ‘Ali-Akbar Qazvini. Eir. online edn. Available at: http:// www.iranicaonline.org/articles/dehkoda (accessed: 11.11.2019). 44. Sa‘idi Sirjani ‘A.-A. Dehkhoda, Mirza ‘Ali-Akbar Qazvini. Eir. online edn. Available at: http:// www.iranicaonline.org/articles/dehkoda (accessed: 11.11.2019). g 45. Dekhoda, ‘A.-A. Lughat-nama. Available at: http://www.vajehyab.com/dehkhoda/ (accessed: 11.11.2019). g 45. Dekhoda, ‘A.-A. Lughat-nama. Available at: http://www.vajehyab.com/dehkhoda/ (accessed: 11.11.2019). Received: April 10, 2019 Accepted: June 17, 2019 Aut hor’s i nfor mat i on: Alexey A. Khismatulin — PhD (History), Senior Researcher; khism@mail.ru Alexey A. Khismatulin — PhD (History), Senior Researcher; khism@mail.ru 343 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3 Назидательная литература эпохи Салджукидов на персидском языке: анонс книжной серии А. А. Хисматулин Хисматулин Алексей Александрович — канд. ист. наук, старший научный сотрудник; khism@mail.ru Институт восточных рукописей РАН, Институт восточных рукописей РАН, Российская Федерация, 191186, Санкт-Петербург, Дворцовая наб., 18 Для цитирования: Khismatulin A. A. The Persian Mirrors for Princes Written in the Saljuq Period: the Book Series // Вестник Санкт-Петербургского университета. Востоковедение и африканистика. 2019. Т. 11. Вып. 3. С. 321–344. https://doi.org/10.21638/spbu13.2019.306 Дается сжатая характеристика жанра, периода и классических персидских текстов, ко- торые охвачены анонсируемой книжной серией из трех книг. 1. Амир Му‘иззи Ниша- пури. Сийасат-нама/Сийар ал-мулук («Книга о правлении/Жития владык»): подделка, приписанная Низам ал-мулку; этот текст до сих пор издается под авторством Низам ал-мулка, выдающегося премьер-министра Салджукидов. Однако, как показывают результаты исторического, кодикологического и текстологического анализов, текст был скомпилирован Мухаммадом Му‘иззи Нишапури, главой департамента поэтов при салджукидских правителях Малик-шахе и его сыне Санджаре, а затем приписан им перу убитого Низам ал-мулка с совершенно определенными целями. 2. Сочинения имама ал-Газали — это книга, включающая в себя шесть текстов, из них три подлин- ных: руководство-пособие для студентов, озаглавленное автором как Зад-и ахират («Путевой припас для грядущей жизни»); аутентичная часть послания ал-Газали сул- тану Санджару под названием Насихат ал-мулук («Совет владыкам») и сборник писем имама к различным адресатам под заглавием Фаза’ил ал-анам мин раса’ил Худжжат ал-ислам («Достоинства людей из посланий Довода ислама»). Оставшиеся три текста представляют подделки: это письмо ал-Газали, до сих пор считавшееся подлинным и получившее известность в персидском оригинале и арабском переводе под двумя названиями — Эй, фарзанд/Аййуха ал-валад («О дитя»); компилятивное письмо к не- коему правителю под названием Панд-нама («Письмо с советами»); текст, который был адресован салджукидскому султану и стал известен как самостоятельный под названием ал-Фарк байна ал-салих ва гайр ал-салих («Разница между благочестивым и неблагочестивым»), так и в качестве «второй части» Насихат ал-мулук, намеренно или механически присоединенной к аутентичному тексту. 3. Кай Кавус б. Искандар б. Кабус. Кабус-нама («Книга Кабуса») и Низами ‘Арузи Самарканди. Чахар макала / Маджма‘ ал-навадир («Четыре беседы/Собрание редкостей») — это книга, включаю- щая в себя два подлинных текста. Также затрагивается проблема существования под- делок в исламской средневековой литературе, их категории и способы идентификации. Ключевые слова: назидательная литература средневековые исламские подделки лите Ключевые слова: назидательная литература, средневековые исламские подделки, лите- ратурные подделки, Низам ал-мулк, Амир Му‘иззи, Салджукиды, ложная атрибуция, талбис, тазвир, музаввир, китаб мадж‘ул. Статья поступила в редакцию 10 апреля 2019 г., рекомендована к печати 17 июня 2019 г. Кон т а к тная инф о рм а ц и я: Хисматулин Алексей Александрович — канд. ист. наук, старший научный сотрудник; khism@mail.ru 344 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. Институт восточных рукописей РАН, 3 Вестник СПбГУ. Востоковедение и африканистика. 2019. Т. 11. Вып. 3
https://openalex.org/W3185377593	https://media.fupress.com/files/pdf/24/7263/19368	Italian	null	Traiettorie tecno-economiche	Studi e saggi	2,021	cc-by	7,970	Mauro Lombardi, University of Florence, Italy, mauro.lombardi@unifi.it, 0000-0002-3234-7039 FUP Best Practice in Scholarly Publishing (DOI 10.36253/fup_best_practice) Mauro Lombardi, Traiettorie tecno-economiche, pp. 113-132, © 2021 Author(s), CC BY 4.0 International, DOI 10.36253/978-88-5518-310-9.08, in Mauro Lombardi, Transizione ecologica e universo fisico- cibernetico. Soggetti, strategie, lavoro, © 2021 Author(s), content CC BY 4.0 International, metadata CC0 1.0 Universal, published by Firenze University Press (www.fupress.com), ISSN 2704-5919 (online), ISBN 978-88-5518-310-9 (PDF), DOI 10.36253/978-88-5518-310-9 CAPITOLO 6 Traiettorie tecno-economiche In un panorama tecno-economico in profondo cambiamento è difficile, ma non impossibile, individuare traiettorie, che possono essere numerose in ragione dell’intensa dinamica come quella odierna, anche se tendono a prevalere infor- mazioni parziali e ambigue. Nelle pagine successive cercheremo di individuare alcune tendenze di fondo, che definiamo traiettorie in quanto si tratta di model- li concettuali per la messa fuoco di problemi, sulla base dei quali si sviluppano nuove conoscenze, tecnologie e strumenti, la cui elaborazione richiede appun- to intelligenza strategica e adaptive strategic thinking. Ci soffermeremo su quelli che, alla luce dell’analisi svolta, appaiono i trend evolutivi potenzialmente più rilevanti, che collochiamo nei livelli di incertezza 3 e 4 nello schema concettua- le di MGI (Courtney, Kirkland e Viguerie 2000). Peraltro lo stesso MGI (2019) riprende inconsapevolmente la metafora della navigazione (navigating the world of disruption) per descrivere un quadro evolutivo sul piano geo-politico, caratterizzato dall’erosione delle tradizionali fondamenta del contratto sociale post-bellico, in seguito alla diffusione delle tecnologie digitali e all’emergere di potenti forze globali. t In questo volume ci limitiamo ad indicare alcune direttrici tecno-econo- miche, tali da poter essere assunte come punti di riferimento per disegni di in- novation policy. Avanziamo a tale scopo una proposta metodologica, una serie di strumenti operativi e architetture funzionali d’intervento mirati per ambiti tecnico-scientifici e specifici processi decisionali. TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO Introduzione: Proposta metodologica, proprietà, attori, architettura funzionale, strumenti operativi-indicatori Lo scenario odierno di computazione ubiquitaria e iperconnettività globale genera continuamente flussi informativi; pertanto nel produrre un bene o un servizio occorre perseguire simultaneamente molteplici linee di attività:i t • monitorare costantemente la frontiera delle conoscenze tecnico-scientifiche; tt b f i bl i i lti lli d i l f i i d l t • monitorare costantemente la frontiera delle conoscenze tecnico-scientifiche; • mettere bene a fuoco i problemi risolti e quelli da risolvere, funzioni da svol- gere e performance da ottenere; i ; • mettere bene a fuoco i problemi risolti e quelli da risolvere, funzioni da svol- gere e performance da ottenere; t • analizzare le interdipendenze sistemiche tra le molteplici componenti delle varie sequenze di attività e funzioni da scegliere;t t g • selezionare attentamente le tecnologie appropriate per i temi emersi nei pre- cedenti passaggi. CAPITOLO 6 Traiettorie tecno-economiche Non un secondo Leonardo da Vinci, forse non riproducibile nel contesto tecnico-scientifico attuale, bensì una leadership con precise caratteristiche psicologiche: fermezza nella visione, apertura mentale, visione sistemica e adattativa, discovery-oriented nell’individuare opportunità di mercato e potenzialità tecnologiche, confronto incessante con la frontiera.i g Per misurarsi con le sfide globali odierne l’EDP ha un banco di prova im- mediato, cioè delineare tendenze rispetto alle quali definire strategie flessibi- li, adattative e strumenti per valutare ipotesi di sperimentazione per ciascuna traiettoria. CAPITOLO 6 Traiettorie tecno-economiche Dai quattro punti si deduce logicamente una peculiare concezione dell’im- presa, quindi dell’Entrepreneurial Discovery Process (EDP), come unità d’a- nalisi e punto di congruenza tra insiemi di flussi fisici, informativi ed energetici, tra i quali realizzare un matching dinamico in relazione ad un ambiente in con- tinua evoluzione. In sostanza è necessario perseguire la congruenza dinamica tra comportamenti/variabili tecno-economiche del prodotto/processo e varia- bili ambientali (evoluzione e cambiamenti dei paradigmi tecno-economici).i g A questo fine è importante che l’impresa così concepita acquisisca alcuni requisiti fondamentali: q • capacità di percepire e interpretare i segnali provenienti dall’ambiente;lt g • flessibilità operativa e capacità di adattarsi ad un ambiente evolutivo; lt • adozione di modelli cognitivi idonei ad acquisire e generare nuove conoscenze. La funzione imprenditoriale nell’orizzonte odierno richiede, pertanto, capa- cità di coordinamento strategico di competenze da attivare per la risoluzione di problemi connessi alle traiettorie definite. ti La funzione di coordinamento strategico a sua volta dipende da alcune caratteristiche: t • propensione ad estrarre input da processi inferenziali in domini conosciti- vi non controllati direttamente, mediante obiettivi di performance multidi- mensionali e business da definire sulla base di strutture interattive interne ed esterne;ttt • attitudine a promuovere e favorire strutture interattive multi-scala in molti campi tecnico-economici;t • tendenza ad adottare una modello sistemico aperto, incentrato su una plu- ralità di sotto-sistemi, in parte indipendenti ma connessi su temi progettua- li e strategici;t g • Open minded, cultura manageriale, pubblica e privata, con elevata attitudine a catalizzare energie materiali e immateriali di matrice eterogenea, in vista di obiettivi non ben definiti a priori, ma talvolta la funzione catalizzatrice è 114 TRAIETTORIE TECNO-ECONOMICHE un’opera -per così dire- di ingegneria inversa: il punto di partenza è una vi- sione quasi irrealizzabile, che induce particolari individualità a ricercare le competenze idonee per avviare processi di ricerca esplorativa verso l’ignoto (Lombardi e Macchi 2016). In definitiva, quindi, si tratta di un’attività imprenditoriale svolta da un at- tore (individuale o micro-collettivo) in possesso di informazioni/intuizioni rilevanti, come base per l’organizzazione di strutture interattive a vari livelli. Tale attore deve al tempo stesso essere dotato di capabilities per l’esercizio di le- adership strategico-progettuale. 1. I traiettoria: verso la smart specialisation Nello scenario descritto, in cui i cambiamenti possono essere introdotti dall’infinitamente piccolo alla scala astronomica, le attività umane sono desti- nate a cambiare profondamente. Uno degli elementi fondamentali è la perva- sività delle tecnologie dell’informazione, che costituiscono una ‘tecnologia di portata generale’ (General Purpose Technology, GPT). Il concetto di GPT è stato introdotto da Bresnahan e Trajtenberg (1995, 84): «The central notion is that, at any point of time, there are a handful of ‘general purpose technologies’ (GPT’s) characterized by the potential for pervasive use in a wide range of sec- tors and by their technological dynamism». Successivamente è stato ripreso, tra gli altri, da Lipsey et al. (2005, cap. IV) e da Jovanovic e Rousseau (2005). Ai nostri fini adottiamo la definizione degli ultimi due, che individuano tre ca- ratteristiche della GPT: 1) pervasività; 2) incentivo al continuo miglioramen- to, nel senso che evolve incessantemente con diminuzione dei costi per i suoi utilizzatori); 3) generatrici di innovazione, in quanto facilitano l’invenzione di nuovi prodotti e processi. t Il connubio di hardware sempre più miniaturizzato e di sistemi algoritmici, presenti ovunque nell’ambiente (ambient intelligence), dà origine a dispositivi in grado di indurre mutamenti dappertutto, proprio perché producono ed elabora- no informazioni senza sosta. I sistemi economico-produttivi si trasformano in sistemi socio-tecnici organizzati come reti di reti attraverso dinamiche di auto- organizzazione a livello globale. g g In questo orizzonte l’analisi delle organizzazioni e delle loro attività, dalla progettazione alla realizzazione, cambia sostanzialmente, perché il focus non più essere un’entità compatta, che raggruppa funzioni, operazioni, task, ben- 115 TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO sì la sequenza di operazioni economico-produttive, le quali frequentemente si intersecano, sovrappongono, comunque interagiscono. La discontinuità è profonda, perché l’approccio sequenziale comporta una ridefinizione strate- gico-operativa: per ottenere un output, di cui all’inizio si ha solo un’idea, suc- cessivamente elaborata in forma di progetto, si sviluppa un processo ad elevata dimensionalità, come sostiene Hausmann (2008, 15) a proposito della smart specialisation, mettendo in luce l’alto numero di variabili che influenzano l’evo- luzione delle conoscenze su cui si basa il processo economico-produttivo, dove le interdipendenze tra le variabili da considerare rende lo spazio delle decisioni complesso, con la conseguenza dell’emergere di problemi di coordinamento (pp. 17-18) specialmente in un processo di sviluppo che si configura «as a co- evolution of products and capabilities». 1. I traiettoria: verso la smart specialisation È a questo punto che Hausman propo- ne l’interrogativo al centro della nostra riflessione: qual è il ruolo della «policy in a high-dimensional world?». La riduzione dell’elevata dimensionalità per le imprese e le organizzazioni private in generale tradizionalmente avviene mediante il contenimento dello spazio delle decisioni a tre variabili: prezzo, profittabilità, mercato dei capita- li, attraverso cui arrivano i segnali generati dalla dinamica tecno-economica. Questo modello mentale tridimensionale ha gravi limiti soprattutto in fasi di trasformazione strutturale, quando le dimensioni dello spazio decisionale sono molte e in gran parte incognite. g g Quale dunque lo spazio decisionale per le politiche pubbliche? Il punto da cui partire oggi pensiamo sia l’idea che la smart specialization è un processo (Foray 2018, 818) e la Transformative activity is a key concept. It is neither an individual project nor a sector as a whole but rather a collection of innovation capacities and actions that have been “extracted” as it were from an existing structure or several structures, to which can be added extra-regional capacities and that is oriented toward a certain structural change. Entro questa prospettiva generale la questione decisiva è quella di identifi- care le priorità (Kuznetsov e Sabel 2011), tenendo presente che siamo di fronte ad innovazioni generate da molteplici fonti e che si diffondono trasversalmente in molti settori. Un compito fondamentale quindi, aggiungiamo noi, consiste nel contribuire a determinare la direzionalità dei processi innovativi facendo le- va sulle attività ‘trasformative’ con maggiore capacità di generare effetti diffusi (leverage e spillover effect). f Occorre enfatizzare l’importanza a questo riguardo delle complementarità da sviluppare tra meccanismi propulsivi e attività di coordinamento dei pro- cessi innovativi, il che richiede funzioni di monitoraggio per la correzione degli errori, la rimozione degli ostacoli, la fluidificazione delle scelte e della loro at- tuazione, consapevoli che non si tratta di effettuare scelte ottime in condizioni statiche e/o stazionarie. Lo spazio decisionale complesso, che evolve incessan- temente grazie all’infrastruttura fisico-informativa globale, induce mutamenti nelle variabili decisionali, per cui risulta arduo – spesso addirittura impossibile 116 TRAIETTORIE TECNO-ECONOMICHE – individuare regolarità. Se inquadriamo i processi decisionali come processi bottom-up basati sull’EDP)1 (Gómez Prieto et al. 2019), per i quali occorre de- finire appropriati strumenti di misurazione, si comprende agevolmente come il problema del coordinamento, la direzionalità e la verifica puntuale delle priori- tà siano fattori fondamentali. 1. I traiettoria: verso la smart specialisation Occorre peraltro sottolineare che l’EDP si svolge in un contesto globale di flussi e interdipendenze, che rendono lo spazio con- cettuale per la risoluzione dei problemi un insieme topologico (Olsson 2000, 2005) aperto e in espansione, con ritmi imprevedibili di accelerazione e rallen- tamenti/stasi, su cui si devono misurare una serie di soggetti pubblici e privati, investitori e stakeholder sociali. – individuare regolarità. Se inquadriamo i processi decisionali come processi bottom-up basati sull’EDP)1 (Gómez Prieto et al. 2019), per i quali occorre de- finire appropriati strumenti di misurazione, si comprende agevolmente come il problema del coordinamento, la direzionalità e la verifica puntuale delle priori- tà siano fattori fondamentali. Occorre peraltro sottolineare che l’EDP si svolge in un contesto globale di flussi e interdipendenze, che rendono lo spazio con- cettuale per la risoluzione dei problemi un insieme topologico (Olsson 2000, 2005) aperto e in espansione, con ritmi imprevedibili di accelerazione e rallen- tamenti/stasi, su cui si devono misurare una serie di soggetti pubblici e privati, investitori e stakeholder sociali. ft In un siffatto contesto è logico che, in assenza di input ‘direzionali’, diventi elevato il rischio di profonde divaricazioni decisionali, dovute alle differenze nel possesso e nelle capacità di acquisizione delle informazioni, con la con- seguenza di rendere problematica l’evoluzione delle società, fino a produrre eventi ‘catastrofici’. i Questo discorso va tenuto presente in modo particolare per quanto riguarda le aree regionali e i Paesi dove prevalgono culture tecnico-produttive consolida- te per i successi del passato, quindi restie ad effettuare cambiamenti per vari e comprensibili ragioni: difesa di patrimoni culturali, diffidenza rispetto all’igno- to, timori connessi all’apparente perdita di prestigio e di potere ecc. La dinamica innovativa verso la smart specialisation richiede, invece, una profonda ed estesa trasformazione intersettoriale di attività, incentrate sull’eser- cizio di veri e propri EDP, a loro volta basati su una nuova knowledge-base, com- pletamente diversa dal passato e estremamente diversificata. La sua evoluzione avviene inevitabilmente nell’ambito di strutture interattive che danno origine a output multi-technology e multi-knowledge domain. Focus Area: Industria 4.0 Mission: Rappresentazione digitale di processi e prodotti Focus Area: Industria 4.0 Focus Area: Industria 4.0 Mission: Rappresentazione digitale di processi e prodotti Mission: Mission: Attori: t Imprese medio-grandi Reti di imprese Centri di Ricerca Entità erogatrici di KIBS 1 Così definita da Marinelli e Perianez-Forte (2017, 5): EDP «as a continuum process […]». E ancora: «bottom up identification of investment-priorities on research and innovation, within the design of a Smart Specialisation Strategy»; sono ingredienti essenziali della tra- iettoria verso la smart specialization. 117 TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO Indicatori: 1. Impiego di AI. 2. % stimata di riduzione degli errori di processo e di o 3. % stimata riduzione bottlenecks di processo. 4. % stimata riduzione dei costi per controllo di qualità 5. Utilizzo di modellazione con IA e predictive mainten Focus Area: Industria 4.0 Mission: Nuovi materiali per riduzione carbon footprint Attori: Startup Innovative Reti di Imprese Centri di Ricerca Indicatori: 1. Utilizzo genomica dei materiali. 2. Riduzione carbon footprint dei prodotti e dei process 3. Standard di sicurezza nel processo di lavoro. 4. Riduzione consumi di input. Focus Area: Economia Circolare Mission: EDP e piattaforma intersettoriale multi-settoriale e tran Attori: Reti di imprese Centri di Ricerca Startup innovative Indicatori: 1. Riduzione scarti e rifiuti (micro). 2. % reimpiego input materiali. 3. % impiego di materiali innovativi. 4. Riduzione consumi energetici. 5. Utilizzo di modellazione con IA. Indicatori: 1. Impiego di AI. 2. % stimata di riduzione degli errori di processo e di output. 3. % stimata riduzione bottlenecks di processo. 4. % stimata riduzione dei costi per controllo di qualità continuo. 5. Utilizzo di modellazione con IA e predictive maintenance. Indicatori: 1. Impiego di AI. 2. % stimata di riduzione degli errori di processo e di output. 3. % stimata riduzione bottlenecks di processo. 4. % stimata riduzione dei costi per controllo di qualità continuo. 5. Utilizzo di modellazione con IA e predictive maintenance. 2. Riduzione carbon footprint dei prodotti e dei processi. t 3. Standard di sicurezza nel processo di lavoro. 4. Riduzione consumi di input. Focus Area: Economia Circolare Mission: EDP e piattaforma intersettoriale multi-settoriale e trans-locale Focus Area: Economia Circolare 1. Riduzione scarti e rifiuti (micro). 5. Utilizzo di modellazione con IA. 118 TRAIETTORIE TECNO-ECONOMICHE 2. II traiettoria: digitalizzazione dei processi produttivi di beni e servizi. Living in a networked world 2. II traiettoria: digitalizzazione dei processi produttivi di beni e servizi. Living in a networked world Il nucleo propulsivo, ormai acquisito a livello teorico e operativo, è la per- vasività di dispositivi di information processing in grado di cooperare ed agire in rete (Geisberger e Broy 2015, cap. 1). I nessi dinamici tra potenza computa- zionale e strumenti di Intelligenza Artificiale costituiscono la trama di fondo dell’universo fisico-cibernetico, precedentemente indicato. 3 Per ulteriori approfondimenti dei temi trattati in questo paragrafo si rinvia a Lombardi (2017) e ai contributi apparsi su Agenda Digitale.eu nel 2018-2019-2020, tutti scaricabili. 2 «The term Cyber-Physical Systems refers to embedded systems, i.e. devices, buildings, ve- hicles and medical equipment, as well as logistics, coordination and management processes and Internet services that – use sensors – interpret and store data […] are connected to each other via digital networks […] – use data and services […] – possess a range of multimodal human-machine interfaces» (Geisberger e Broy 2015, 25-26). TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO Valutazione della riduzione di traffico urbano e della qualità dell’aria pe micro-aree oggi a rischio di congestione. Focus Area: Living cities Mission: Valorizzazione su nuove basi negozi di vicinato e micro-filiere di qualità Attori: Enti pubblici Piccoli Esercizi commerciali Reti di negozi Reti di aziende a livello di città metropolitane Associazioni di cittadini Indicatori: 1. Stabilizzazione e possibile aumento dei negozi di prossimità. 2. Incremento delle micro-funzioni di servizio alla popolazione. 3. Riduzione traffico veicolare su micro-scala. 4. Miglioramento qualità dell’aria. 5. Organizzazione di nuovi flussi di approvvigionamento su basi ‘intelligenti Micro-piattaforme per l’incontro di domanda e offerta, impiego di mezzi d trasporto con minore consumo energetico e da fonti rinnovabili. Focus Area: Living cities Mission: Riassetto funzionale di aree urbane Attori: Enti pubblici Associazioni imprenditoriali Grandi Imprese Associazioni di cittadini Indicatori: 1. Effetti di flusso della redistribuzione di funzioni al fine di ridurre i flussi di per sone e veicoli, oltre che per rispondere ad esigenze di imprese e popolazione 2 Stime dei flussi e della riorganizzazione logistica con modellazion 4. Valutazione della riduzione di traffico urbano e della qualità dell’aria per micro-aree oggi a rischio di congestione. Focus Area: Living cities Mission: Valorizzazione su nuove basi negozi di vicinato e micro-filiere di qualità Attori: Enti pubblici Piccoli Esercizi commerciali Reti di negozi Reti di aziende a livello di città metropolitane Associazioni di cittadini Indicatori: 1. Stabilizzazione e possibile aumento dei negozi di prossimità. 2. Incremento delle micro-funzioni di servizio alla popolazione. 3. Riduzione traffico veicolare su micro-scala. 4. Miglioramento qualità dell’aria. 5. Organizzazione di nuovi flussi di approvvigionamento su basi ‘intelligenti’. Micro-piattaforme per l’incontro di domanda e offerta, impiego di mezzi di trasporto con minore consumo energetico e da fonti rinnovabili. Focus Area: Living cities Mission: Riassetto funzionale di aree urbane Attori: Enti pubblici Associazioni imprenditoriali Grandi Imprese Associazioni di cittadini d Living cities 1. Stabilizzazione e possibile aumento dei negozi di prossimità. p g p Incremento delle micro-funzioni di servizio alla popolazione.fi TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO Siamo in condizioni di realizzare un’intelligenza distribuita che auto-organizza scambi informativi, indipendentemente dall’intervento continuo degli umani, anzi i dispositivi pro- grammati sono in grado di scambiare in formazioni, auto-diagnosticarsi e trarre conseguenze in termini di pattern evolutivi su cui intervenire.tfit g pt Una traiettoria che può essere delineata con sufficiente attendibilità sul piano tecnico-scientifico e logico è la creazione di sistemi fisico-cibernetici gerarchici e annidati2, che connettono vari livelli delle sequenze economico-produttive, dei sistemi urbani, degli apparati logistici multi-scala. È di grande interesse la tra- sversalità del loro impiego e la interoperabilità multi-modale, che apre un grade spazio esplorativo per la progettazione di processi di produzione e l’erogazione di un enorme potenziale di servizi3. Focus Area: Living cities Mission: Ristrutturazione Logistica urbana Mission: Ristrutturazione Logistica urbana Mission:t Attori: Enti Pubblici Imprese manifatturiere Imprese Commerciali e KIBS Istituzioni Indicatori: 1. Rappresentazione computazionale ‘reti di flussi’.t pp pl 2. Stima dei sotto-sistemi urbani e del carico energetico. t g 3. Stime della riduzione di consumi energetici e di carbon footprint per progetti coordinati di riorganizzazione dei flussi di approvvigionamento. t g 3. Stime della riduzione di consumi energetici e di carbon footprint per progetti coordinati di riorganizzazione dei flussi di approvvigionamento. 2 «The term Cyber-Physical Systems refers to embedded systems, i.e. devices, buildings, ve- hicles and medical equipment, as well as logistics, coordination and management processes and Internet services that – use sensors – interpret and store data […] are connected to each other via digital networks […] – use data and services […] – possess a range of multimodal human-machine interfaces» (Geisberger e Broy 2015, 25-26). 2 «The term Cyber-Physical Systems refers to embedded systems, i.e. devices, buildings, ve- hicles and medical equipment, as well as logistics, coordination and management processes and Internet services that – use sensors – interpret and store data […] are connected to each other via digital networks […] – use data and services […] – possess a range of multimodal human-machine interfaces» (Geisberger e Broy 2015, 25-26). 3 Per ulteriori approfondimenti dei temi trattati in questo paragrafo si rinvia a Lombardi (2017) e ai contributi apparsi su Agenda Digitale.eu nel 2018-2019-2020, tutti scaricabili. 3 Per ulteriori approfondimenti dei temi trattati in questo paragrafo si rinvia a Lombardi (2017) e ai contributi apparsi su Agenda Digitale.eu nel 2018-2019-2020, tutti scaricabili. 119 TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO 4. Indicatori: ftl 1. Effetti di flusso della redistribuzione di funzioni al fine di ridurre i flussi di per- sone e veicoli, oltre che per rispondere ad esigenze di imprese e popolazione. g 2. Stime dei flussi e della riorganizzazione logistica con modellazione computazionale. p 3. Stime della riduzione di carbon footprint a livello urbano e di micro-area. 120 TRAIETTORIE TECNO-ECONOMICHE 3. III Traiettoria: sostenibilità ambientale e resilienza sistemica Ciò che attualmente sta vivendo l’Umanità in conseguenza della pandemia da Covid-19 enfatizza al massimo livello di allarme due questioni di importanza essenziale per l’evoluzione del Pianeta Terra: la sostenibilità ambientale delle at- tività economico-produttive umane e la resilienza dei sistemi, così definita dalle National Academies USA: «the ability to prepare and plan for, absorb, recover from, and more successfully adapt to adverse events» (National Academies 2012, 16). Questa definizione è fatta esplicitamente propria da Acatech (Thoma 2014, 89): «A society can be described as resilient if it has the ability to defend itself against actual or potential adverse events, prepare and plan for them, cope with and recover from them and continuously improve its ability to adapt to them». y p y p Lo sviluppo sostenibile richiede la trasformazione dell’economia a livello globale: «Sustainable development is not a destination, but a dynamic process of adaptation, learning and action. It is about recognizing, understanding and acting on interconnections – above all those between the economy, society and the natural environment» (UN 2012, 6) e «Achieving sustainability requires us to transform the global economy. Tinkering on the margins will not do the job» (UN 2012, 7). j Joan Copper dell’Università di Warwick, uno degli estensori del Rapporto Acatech (Thoma 2014) chiarisce che resilienza e sostenibilità non sono sinoni- mi, perché la seconda incorpora la prima, come risulta dall’ampia analisi svolta delle iniziative progettuali di resilienza a livello internazionale (Thoma 2014). Il concetto di sviluppo sostenibile è sempre quello definito dal famoso Rapporto Bruntland (1987) per le Nazioni Unite4, nel quale sono anche indicati principi, oltre che direttive e soluzioni a livello globale, che siano giuste, sostenibili dal punto di vista economico e ambientale. La resilienza ha d’altro canto tre proprie- tà fondamentali: persistenza, adattabilità e trasformabilità (Miller et al. «Sustainable development is development that meets the needs of the present without compro- mising the ability of future generations to meet their own needs» (cap. 2.1, unformatted Report). Indicatori: ftl Calcolo della riduzione del consumo di energia. 3. Stime della riduzione di carbon footprint. 4. Creazione e incremento di reti progettuali per ridurre l’impatto ambient e aumentare la resilienza urbana. 5. Startup innovative multidisciplinari. Focus Area: Risparmio energetico e riduzione delle emissioni di CO2 Mission: Sviluppo di Entrepreneurial Discovery Process in ambito energetico Attori: Enti Pubblici Famiglie Grandi Imprese Associazioni di cittadini Organismi di quartiere Società di progettazione Indicatori: 1. Quantità di energia da rinnovabili prodotta autonomamente. 2. Numero di microgrid create in sotto-sistemi urbani e in Comunità Local varia scala territoriale. 3. Stime della riduzione di carbon footprint. 4. Startup innovative multidisciplinari per la creazione, gestione e manute zione tramite piattaforme aperte. Focus Area: Risparmio energetico e riduzione delle emissioni di CO2 Mission: Rigenerazione aree industriali Attori: Enti Pubblici Imprese Partnership pubblico-privato Reti di Imprese a varia scala Indicatori: 1. Stime energetiche del retrofitting di edifici in aree industriali per il risparm e la produzione di energia da innovabili e/o impiego della cogenerazione 2. Stima della diminuzione dei costi per le imprese. 2. Calcolo della rid 3. Stime della ridu 4. Creazione e incr e aumentare la r 5. Startup innovat Focus Area: Risparmio energeti Mission: Sviluppo di Entrepr Attori: Enti Pubblici Famiglie Grandi Imprese Associazioni di citt Organismi di quart Società di progettaz Indicatori: 1. Quantità di ene 2. Numero di micr varia scala territ 3. Stime della ridu 4. Startup innovat zione tramite pi Focus Area: Risparmio energeti Mission: Rigenerazione aree Attori: Enti Pubblici Imprese Partnership pubblic Reti di Imprese a va Indicatori: 1. Stime energetich e la produzione 2. Stima della dim 2. Calcolo della riduzione del cons 3. Stime della riduzione di carbon f 4. Creazione e incremento di reti p e aumentare la resilienza urbana 5. Startup innovative multidiscipli Focus Area: Risparmio energetico e riduzione d Mission: Sviluppo di Entrepreneurial Discov Attori: Enti Pubblici Famiglie Grandi Imprese Associazioni di cittadini Organismi di quartiere Società di progettazione Indicatori: 1. Quantità di energia da rinnovab 2. Numero di microgrid create in s varia scala territoriale. 3. Stime della riduzione di carbon f 4. Startup innovative multidiscipl zione tramite piattaforme aperte Focus Area: Risparmio energetico e riduzione d Mission: Rigenerazione aree industriali Attori: Enti Pubblici Imprese Partnership pubblico-privato Reti di Imprese a varia scala Indicatori: 1. Stime energetiche del retrofitting e la produzione di energia da inn 2. Stima della diminuzione dei cos 2. Indicatori: ftl 2010, 4).l pt ( ) Dagli spunti di analisi e riflessione esposti si evince che in un mondo iper- connesso è necessario integrare in un solo sistema simbiotico gli aspetti tecno- logici, legali, economici, politici e sociali (Thoma 2014, 51). In estrema sintesi, la prospettiva sistemica è essenziale, l’attenzione alla complessità derivante dalle interdipendenze è decisiva, insieme alla capacità adattativa come sostiene anche Edwards (2009, 18): «The capacity of an individual, community or system to adapt in order to sustain an acceptable level of function, structure and identity».tt p p y Un altro punto da mettere in rilievo è quello delle infrastrutture, alcune fai- lures delle quali (Italia compresa) sono analizzate da Kröger (2011), a cui si deve una definizione più tecnica di resilienza, del tutto coerente con quella più gene- rale prima riportata: «the ability of a system or a system-of-systems to resist/ absorb initial adverse effects of a disruptive (shocking or creeping) internal or external event/force (stressor) and the time/speed at which it is able to return to an appropriate functionality/equilibrium» (in Thoma 2014, 68-69). Lo stesso 121 TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO Kröger ha elaborato una rappresentazione grafica molto interessante del grado di resilienza di un Sistema rispetto ad uno shock (Fig. 1).tt t Alla luce della sintesi dei concetti basilari appena esposti, del tutto congruen- ti con quelli introdotti nei paragrafi iniziali, è da ritenere che la traiettoria della resilienza sistemica, declinata ad ogni livello, sia assunta come una delle priori- tà fondamentali delle politiche per l’innovazione, anche perché riassume in sé quasi tutti gli aspetti considerati finora. Figura 1 – Come rispondono a uno shock sistemi resilienti e non resilienti. [Fonte: Thoma 2014, 70] Figura 1 – Come rispondono a uno shock sistemi resilienti e non resilienti. [Fonte: Thoma 2014, 70] Figura 1 – Come rispondono a uno shock sistemi resilienti e non resilienti. [Fonte: Thoma 2014, 70] Figura 1 – Come rispondono a uno shock sistemi resilienti e non resilienti. [Fonte: Thoma 2014, 70] Focus Area: Risparmio energetico e riduzione delle emissioni di CO2 Mission: Risparmio energetico e riduzione di emissioni di CO2 Attori: Enti Pubblici Famiglie Grandi Imprese Associazioni di cittadini Organismi di quartiere Società di progettazione Indicatori: 1. Retrofitting energetico e bio-climatico di edifici pubblici e privati, aree residenziali. 1. Retrofitting energetico e bio-climatico di edifici pubblici e privati, aree residenziali. 122 TRAIETTORIE TECNO-ECONOMICHE 1 TRAIETTORIE TECNO-ECONOMIC 2. Indicatori: ftl Calcolo della riduzione del consumo di energia. 3. Stime della riduzione di carbon footprint. 4. Creazione e incremento di reti progettuali per ridurre l’impatto ambientale e aumentare la resilienza urbana. 5. Startup innovative multidisciplinari. Focus Area: Risparmio energetico e riduzione delle emissioni di CO2 Mission: Sviluppo di Entrepreneurial Discovery Process in ambito energetico Attori: Enti Pubblici Famiglie Grandi Imprese Associazioni di cittadini Organismi di quartiere Società di progettazione 2. Calcolo della riduzione del consumo di energia. 3. Stime della riduzione di carbon footprint.t f p 4. Creazione e incremento di reti progettuali per ridurre l’impatto ambientale e aumentare la resilienza urbana. 4. Creazione e incremento di reti progettuali per ridurre l’impatto ambientale e aumentare la resilienza urbana. 5. Startup innovative multidisciplinari. Focus Area: Risparmio energetico e riduzione delle emissioni di CO2 Mission: Sviluppo di Entrepreneurial Discovery Process in ambito energetico Indicatori: 1. Quantità di energia da rinnovabili prodotta autonomamente.t t 2. Numero di microgrid create in sotto-sistemi urbani e in Comunità Locali a varia scala territoriale. 3. Stime della riduzione di carbon footprint. 4. Startup innovative multidisciplinari per la creazione, gestione e manuten- zione tramite piattaforme aperte. 4. Startup innovative multidisciplinari per la creazione, gestione e manuten- zione tramite piattaforme aperte. Mission: Integrazione di flussi informativi, Management della transizione sistemica Attori: Integrazione di flussi informativi, Management della transizione sistemica Indicatori: 1. Creazione di modelli integrati di sistemi a varia scala: sotto-sistemi urbani, sistemi urbani, livello regionale.t 2. Modelli computazionali delle reti di reti: idrauliche, elettriche, energetiche, informative.t 3. Elaborazione di scenari evolutivi con simulazioni di bottlenecks, failures mi- nori e sistemiche.i 4. Previsione di implicazioni e costi di eventi ‘catastrofici’. i 5. Ipotesi di meccanismi per consentire assorbimenti degli shocks. 4. IV Traiettoria: Intelligence Analysis. Servizi ad alta intensità di conoscenza Focus Area: Focus Area: Risparmio energetico e riduzione delle emissioni di CO2 Indicatori: 1. Stime energetiche del retrofitting di edifici in aree industriali per il risparmio e la produzione di energia da innovabili e/o impiego della cogenerazione. 2. Stima della diminuzione dei costi per le imprese. 123 TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO 3. Calcolo degli investimenti in nuovi materiali (isolamento termico, assorbi- mento da solare ecc.). 3. Calcolo degli investimenti in nuovi materiali (isolamento termico, assorbi- mento da solare ecc.). ) 4. Stime della riduzione di carbon footprint di area. ) 4. Stime della riduzione di carbon footprint di area. Focus Area: Focus Area: Resilienza sistemica Resilienza sistemica Mission: Integrazione di flussi informativi, Management della transizione sistemica Attori: Enti Pubblici Istituzioni Grandi Imprese Centri di Ricerca Enti Formativi di Alto Livello 4. IV Traiettoria: Intelligence Analysis. Servizi ad alta intensità di conoscenza Un’implicazione logica dell’analisi dell’evoluzione sistemica e complessa dello spazio informativo globale è la seguente: le attività non direttamente manipola- tive dei processi fisici assumono un’importanza decisiva, pur con una profonda asimmetria al loro interno. Da un lato avranno preminenza quelle a più alto con- tenuto di conoscenza, in quanto capaci di influenzare e controllare –entro cer- ti limiti – i processi fisici, e dall’altro vi saranno attività a contenuti conoscitivi di natura prevalentemente operativa, cioè servizi tradizionali, che assumeranno un ruolo residuale, nonostante la loro grande estensione quantitativa (assisten- za alla persona, mansioni esecutive in molti ambiti della vita socio-economica). Non è peraltro da escludere la permanenza di attività ‘di nicchia’ di significativa importanza funzionale (manutenzione e controllo di dispositivi meccanici, elet- tromeccanici, idraulici in molti settori produttivi, attività pratico manipolative). Non essendo questa la sede per un approfondimento della questione relativa alle asimmetrie, ci concentriamo sulla necessità, a livello individuale e collettivo, di input ad alta intensità di conoscenza. Un’attenzione precipua va assegnata allo sviluppo dell’Intelligence Analysis (Deloitte Insights 2019) e quindi delle attività 124 TRAIETTORIE TECNO-ECONOMICHE che richiedono input tecnico-scientifici interdisciplinari e trans-disciplinari per esplorare soluzioni innovative in merito ai problemi e alle sfide generate dalle tra- iettorie già indicate , cioè I4.0, intesa come digitalizzazione di processi e prodotti multi-technology e multi knowledge-domain, nonché lo sviluppo di tecnologie di IA da impiegare per la realizzazione di cooperazione tra umani e macchine, median- te Human-Computer-Interaction e amplified intelligence. In sostanza, la traiettoria ipotizzata consiste nella tendenza verso l’impiego di agenti artificiali che siano in accordo con finalità a beneficio dell’Umanità, potenziando le capacità inter- pretative degli esseri umani, senza sostituirsi ad essi. A tale scopo un’attenzione particolare deve essere prestata agli sviluppi e alle applicazioni delle reti neurali di ultima generazione (cfr. cap. 4, par. 5), il cui fine dovrebbe consistere nel raf- forzare l’Umanità nello svolgere alcune funzioni: 1) estrarre e analizzare le in- formazioni da mezzi audiovisivi distribuiti a varia scala territoriale; 2) sviluppare l’automazione e il controllo dei processi lungo le sequenze economico-produttive e logistiche; 3) sostenere e rendere più affidabili processi decisionali di grande ri- lievo, per es. nel rispondere ad eventi catastrofici e quindi potenziare la resilienza sistemica; 4) incrementare la capacità previsiva circa l’evoluzione dei sistemi mul- ti-scala tramite lo sviluppo al tempo stesso della cultura manageriale appropriata e l’impiego diffuso di predictive analytics per ogni tipologia di sistema. 5. V° Traiettoria: Sperimentazione clinica. Predictive Analytics e trattamenti sanitari. Outbreak Analytics (Polonsky et al. 2019) 5. V° Traiettoria: Sperimentazione clinica. Predictive Analytics e trattamenti sanitari. Outbreak Analytics (Polonsky et al. 2019) 5. V° Traiettoria: Sperimentazione clinica. Predictive Analytics e trattamenti sanitari. Outbreak Analytics (Polonsky et al. 2019) Gli sviluppi dell’IA hanno generato impulsi notevoli in molti campi discipli- nari delle Life Sciences e dei trattamenti clinici, per cui si aprono grandi poten- zialità ai fini della prevenzione e la cura delle malattie, soprattutto grazie a nuovi strumenti di analisi predittiva di andamenti individuali e dinamiche collettive (epidemie, eventi atmosferici, episodi catastrofici).it i Utilizziamo a fini puramente euristici la rappresentazione dei trend attuali e di quel- li potenziali in campo sanitario, secondo proiezioni di Deloitte Insights (Figg. 2-3-4). 126 li potenziali in campo sanitario, secondo proiezioni di Deloitte Insights (Figg. 2 3 4). Figura 2 – L’approccio tradizionale allo sviluppo clinico è lungo e ha successo solo nel 10% dei casi. [Fonte: Deloitte Insights 2020, fig. 1] 4. IV Traiettoria: Intelligence Analysis. Servizi ad alta intensità di conoscenza Nella tra- iettoria in questione diventa essenziale, infatti, la diffusione di cultura e tecniche di management dei sistemi complessi in modo pervasivo nelle organizzazioni di ogni tipo; 5) realizzare sistemi di Augmented Reality e Virtual Reality sia in tutti i processi di lavoro (ad es. per problemi di sicurezza e di controllo ambientale), sia nelle attività di fruizione dei beni artistici e culturali (mostre, Musei, sistemi urbani di pregio); 6) realizzare piattaforme e progetti per l’Economia Circolare seguendo gli approcci più consolidati: Circular Economy della Fondazione Ellen MacArthur Foundation, Cradle to Cradle, Blue Economy, Industrial Symbiosis. Sviluppi dell’Intelligenza Artificiale e della Human Computer Interaction 1. Numero di robot introdotti dalla ‘robotica avanzata’ in ambito industriale. Stima dei risultati qualitativi e quantitativi (riduzione pezzi difettosi, minori consumi energetici, controlli pervasivi di qualità, minori costi). 125 TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO 2. Augmented reality e Virtual Reality nella fruizione di beni artistici (musei) e paesaggistici (visite di aree urbane di pregio). 2. Augmented reality e Virtual Reality nella fruizione di beni artistici (musei) e paesaggistici (visite di aree urbane di pregio).li gg g 3. Riorganizzazione dei flussi turistici con finalità programmatorie e redistri- buzione dei flussi con il risultato di una fruizione di più alto livello.i gg g 3. Riorganizzazione dei flussi turistici con finalità programmatorie e redistri- buzione dei flussi con il risultato di una fruizione di più alto livello.i l p 4. Previsione di implicazioni e costi di eventi ‘catastrofici’. i 5. Ipotesi di meccanismi per consentire assorbimenti degli shocks. Figura 2 – L’approccio tradizionale allo sviluppo clinico è lungo e ha successo solo nel 10% dei casi. [Fonte: Deloitte Insights 2020, fig. 1] Focus Area: Sviluppi dell’Intelligenza Artificiale e della Human Computer Interaction Mission: 1. Rete di Centri di Integrazione di flussi informativi da database. 3. Modelli previsivi dei trend sanitari emergenti nella popolazione umana e nel mondo animalet gt 6. Analisi sistematiche di sperimentazioni cliniche, specie se di frontiera. 8. Team di competenze multiple per elaborare visioni e strategiche di evolu- zione sistemica. Figura 2 – L’approccio tradizionale allo sviluppo clinico è lungo e ha successo solo nel 10% dei casi. [Fonte: Deloitte Insights 2020, fig. 1] Figura 2 – L’approccio tradizionale allo sviluppo clinico è lungo e ha successo solo nel 10% dei casi. [Fonte: Deloitte Insights 2020, fig. 1] 126 TRAIETTORIE TECNO-ECONOMICHE Figura 3 – Applicazioni dell’AI in campo sanitario. [Fonte: Deloitte Insights 2020, fig. 2] Figura 3 – Applicazioni dell’AI in campo sanitario. [Fonte: Deloitte Insights 2020, fig. 2] Figura 3 – Applicazioni dell’AI in campo sanitario. [Fonte: Deloitte Insights 2020, fig. 2] Figura 4 – Previsione dei tempi di adozione delle tecnologie AI. [Fonte: Deloitte Insights 2020, fig. 5] Figura 4 – Previsione dei tempi di adozione delle tecnologie AI. [Fonte: Deloitte Insights 2020, fig. 5] Emerge uno scenario futuro ricco di input innovativi multi-disciplinari (bio-chimica, chimica, fisica, computer science, sociologia, strategic thinking), i quali risultano pienamente coerenti con gli spunti di riflessione che abbiamo cercato di sviluppare. 127 TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO Focus Area: Sviluppi dell’Intelligenza Artificiale e della Human Computer Interaction Mission: Applicazione dell’AI in ambito sanitario Focus Area: Sviluppi dell’Intelligenza Artificiale e della Human Computer Interaction Mission: Applicazione dell’AI in ambito sanitario Focus Area: Sviluppi dell’Intelligenza Artificiale e della Human Computer Interaction Mission: Applicazione dell’AI in ambito sanitario Bioeconomia Nell’era della rarefazione delle risorse del Pianeta, dei cambiamenti climatici e di problemi nell’accesso al cibo per ampie fasce della popolazione mondiale, vi è un interesse sempre più forte degli studiosi per la Bioeconomia ranging from the usage of mostly untapped feedstock (CO2, waste, algae), optimized microorganisms, digitalization in farming, social innovations (urban gardening, collective agriculture, etc.). Bio-based innovations are not only relevant in high-tech sectors, but also in traditional segments, such as the provision of new materials in textiles (e.g. spider silk) or developing new protein alternatives in the food sectors (Wydra 2020, 1). ranging from the usage of mostly untapped feedstock (CO2, waste, algae), optimized microorganisms, digitalization in farming, social innovations (urban gardening, collective agriculture, etc.). Bio-based innovations are not only relevant in high-tech sectors, but also in traditional segments, such as the provision of new materials in textiles (e.g. spider silk) or developing new protein alternatives in the food sectors (Wydra 2020, 1). Naturalmente ai fini delle politiche per l’innovazione è importante compren- dere bene quali attività sono incluse, al fine di poterle sottoporre a monitoraggio e misurazione abbastanza precisi. Wydra (2020) discute molti studi ed esperien- ze di politiche a livello internazionale, indicando il fatto che sono finora inserite nel concetto di bioeconomia solo attività che impiegano integralmente risorse derivanti da fonti biogeniche (es. biomassa), mentre sono escluse gli sviluppi di plastiche bio-based e biochimiche, con il risultato di non rendere gli indicatori del tutto precisi. Vi sono poi settori difficili da classificare come la carta e la cel- 128 TRAIETTORIE TECNO-ECONOMICHE lulosa. La varietà di definizioni porta ad includere una nutrita serie di comparti: agricoltura, alimentari, carta e cellulosa, input basati su risorse biogeniche nella chimica, plastiche. Gli indicatori impiegati sono quelli consueti: R&D, ricerca bibliometrica e analisi dei brevetti, risorse umane nella commercializzazione delle innovazioni (ovvero adozione e diffusione). Sulla base delle informazioni raccolte anche in altri studi sono individuate alcune traiettorie innovative: 1) sostituzione di combustibili fossili con materiali bio-based; 2) innovazioni che aumentano la produttività del settore primario; 3) nuovi e più efficienti impie- ghi di biomassa; 4) applicazioni di alto valore e minore impatto, ad esempio che riducano l’uso di sostanze tossiche. La Bioeconomia è un insieme ad alto potenziale innovativo e può incidere in misura sostanziale all’interno dei processi finalizzati alala sostenibilità ambien- tale e alla resilienza sistemica. Bioeconomia Per questi motivi è altamente auspicabile l’ado- zione di una definizione ampia e l’impiego di indicatori multipli, come avviene nella vasta letteratura analizzata in Wydra (2020). 5 «A food can be regarded as “functional” if it is satisfactorily demonstrated to affect benefi- cially one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either an improved state of health and well-being and/or reduction of risk of disease» (Ashwell 2002, 5). 6 «the recently born “foodomics” is to be intended as a global perspective of knowledge about foods, which covers the assessment of their composition, the effects of (bio)technological processes for their production, their modifications over time and the impact that food con- sumption has on human health. Food proteomics and metabolomics, along with their de- rived “omic” branches such as peptidomics, lipidomics and glycomics, are still evolving technologies capable of tackling the nature and the transformations of foods» (Picariello et al. 2012, 286). 6 «the recently born “foodomics” is to be intended as a global perspective of knowledge about foods, which covers the assessment of their composition, the effects of (bio)technological processes for their production, their modifications over time and the impact that food con- sumption has on human health. Food proteomics and metabolomics, along with their de- rived “omic” branches such as peptidomics, lipidomics and glycomics, are still evolving technologies capable of tackling the nature and the transformations of foods» (Picariello et al. 2012, 286). 5 «A food can be regarded as “functional” if it is satisfactorily demonstrated to affect benefi- cially one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either an improved state of health and well-being and/or reduction of risk of disease» (Ashwell 2002, 5). Agro-alimentare Nell’attuale contesto globale, interessato dalla pandemia da Covid-19, l’agro- alimentare diviene un ambito di rilevanza strategica e viene anch’esso a confi- gurarsi come un insieme di sequenze di operazioni e task che interagiscono, si intersecano e sovrappongono. Particolare rilievo dunque assume la progettazione innovativa in molteplici forme (Galanakis et al. 2016): open innovation anche e soprattutto per le piccole e medie imprese, creazione di network per la sicurezza dei dati, la produzione e l’utilizzo di energia, l’introduzione di nuove emergenti tecnologie, l’impiego di un’ampia gamma di tecnologie che favoriscono la so- stenibilità ambientale, sociale ed economica, lo sviluppo di functional food5, la foodomics6 (Picariello et al. 2012), che comprende una serie di discipline impie- gate per valutare la composizione, gli effetti dell’applicazione di biotecnologie, l’impatto sui modelli di consumo e sulla popolazione, come si vede in figura 5. ti Questa gamma di potenzialità tecnologiche potrebbe ulteriormente con- tribuire alla valorizzazione dell’enorme varietà e alto livello qualitativo delle produzioni regionali, esaltandone le proprietà e rafforzandone la proiezione sui mercati con strumenti di certificazione e controllo delle filiere non solo di mar- 129 TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO chi noti, ma anche e soprattutto di microfiliere di piccole e medie imprese locali mediante nuove tecnologie (Blockchain, piattaforme dedicate) e input manage- riali innovativi (startup, unità di comunicazione specializzate nei nuovi media). Ciò potrebbe avvenire mediante strategie congiunte pubblico-privato. Figura 5 – Integrazione tra campi disciplinari (‘omics’) in grado di rappresentare gusto, olfatto e altre proprietà essenziali del cibo. [Fonte: Picariello et al. 2012, fig. 1] Figura 5 – Integrazione tra campi disciplinari (‘omics’) in grado di rappresentare gusto, olfatto e altre proprietà essenziali del cibo. [Fonte: Picariello et al. 2012, fig. 1] Figura 5 – Integrazione tra campi disciplinari (‘omics’) in grado di rappresentare gusto, olfatto e altre proprietà essenziali del cibo. [Fonte: Picariello et al. 2012, fig. 1] È superfluo, ma doveroso, sottolineare la stretta correlazione tra questo ti- po di iniziative strategiche e il water-energy-food nexus, a cui si aggiunge anche la razionalizzazione in termini di efficienza e sostenibilità dell’infrastruttura logistica, sulla quale va sviluppata una progettazione innovativa di alto livel- lo qualitativo. Focus Area: Bioeconomia, Nuove tecnologie in agricoltura Sviluppo e diffusione, di attività con risorse da fonti biogeniche t Piccole imprese private Società cooperative e Associazioni tra Comuni rurali Indicatori: 1. Sostituzione di combustibili fossili con input bio-based. 2. Incrementi nella produzione nel consumo di functional foods. 3. Agro-alimentare Incrementi degli investimenti in foodomics. g f 4. Riduzione dell’uso di sostanze nocive per la salute umana e diffusione di tecniche di coltivazione nature-oriented. 4. Riduzione dell’uso di sostanze nocive per la salute umana e diffusione di tecniche di coltivazione nature-oriented. 130 TRAIETTORIE TECNO-ECONOMICHE Focus Area: Agro-alimentare Mission: Nuove tecnologie nella produzione e diffusione di prodotti agricoli Focus Area: Agro-alimentare Mission: Nuove tecnologie nella produzione e diffusione di prodotti agricoli Attori: Enti pubblici Istituzioni Piccole imprese e cooperative agricole Negozi di prossimità Centri di Ricerca Imprese agri-turistiche e turistiche Unità Terziarie Attori: Enti pubblici Istituzioni Piccole imprese e cooperative agricole Negozi di prossimità Centri di Ricerca Imprese agri-turistiche e turistiche Unità Terziarie Attori: Enti pubblici Istituzioni Piccole imprese e cooperative agricole Negozi di prossimità Centri di Ricerca Imprese agri-turistiche e turistiche Unità Terziarie Attori: Piccole imprese e cooperative agricole d Imprese agri-turistiche e turistiche Indicatori: 1. Riduzione nell’uso combustibili fossili.t 1. Riduzione nell’uso combustibili fossili.t 2. Incrementi di produttività dopo l’introduzione di strumenti e metodi per l’agricoltura di precisione.f 3. Riduzione dell’uso di sostanze nocive per la salute umana e diffusione di tecniche di coltivazione nature-oriented.tii 4. Piattaforme per la valorizzazione di output da micro-filiere e la certificazio- ne dei prodotti (per es. mediante blockchain).t 4. Piattaforme per la valorizzazione di output da micro-filiere e la certificazio- ne dei prodotti (per es. mediante blockchain). t 5. Variazioni di fatturato. t 6. Numerosità delle micro-filiere aderenti. Bibliografia Ashwell, M. 2002. Concepts of functional food. ILSI (International Life Science Institute) Europe Concise Monograph Series 1-40. Bresnahan, T., e M. Trajtenberg. 1995. “General purpose technologies ‘Engines of growth?’.” Journal of Econometrics 65 (1): 83-108. Bruntland, G.H. 1987. “Report of the World Commission on Environment and Development: our Common Future.” <https://sustainabledevelopment.un.org/ content/documents/5987our-common-future.pdf> (2021-10-03). p ( ) Courtney, H.G., Kirkland, J., e S.P. Viguerie. 2000. Strategy under uncertainty. MGI.ttt Deloitte Insights. 2019. Future-of-intelligence analysis <https://www2.deloitte.com/ content/dam/insights/us/articles/6306_future-of-intel-analysis/DI_Future-of- intel-analysis.pdf> (2021-10-03). y p Deloitte Insights. 2020. “Intelligent clinical trials Transforming through AI-enabled engagement.” <https://www2.deloitte.com/content/dam/insights/us/articles/22934_ intelligent-clinical-trials/DI_Intelligent-clinical-trials.pdf> (2021-10-03). g _ g Edwards, C. 2009. Resilient Nation. London: Demos. g _ g p ( ) Edwards, C. 2009. Resilient Nation. London: Demos. 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https://openalex.org/W2171092515	https://europepmc.org/articles/pmc1590026?pdf=render	English	null	Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway	BMC genomics	2,006	cc-by	15,401	This article is available from: http://www.biomedcentral.com/1471-2164/7/205 This article is available from: http://www.biomedcentral.com/1471-2164/7/205 This article is available from: http://www.biomedcentral.com/1471-2164/7/205 © 2006 Mormann 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. BioMed Central BioMed Central BioMed Central BMC Genomics Open Access Open Acc Research article Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway Sascha Mormann1,2, Alexander Lömker1,2, Christian Rückert1,2, Lars Gaigalat1,2, Andreas Tauch1, Alfred Pühler2 and Jörn Kalinowski1 Address: 1Institut für Genomforschung, Universität Bielefeld, D-33594 Bielefeld, Germany and 2Lehrstuhl für Genetik, Universität B 33594 Bielefeld, Germany Email: Sascha Mormann - smormann@genetik.uni-bielefeld.de; Alexander Lömker - aloemker@genetik.uni-bielefeld.de; Christian Rückert - christian.rueckert@genetik.uni-bielefeld.de; Lars Gaigalat - lars.gaigalat@genetik.uni-bielefeld.de; Andreas Tauch - tauch@genetik.uni-bielefeld.de; Alfred Pühler - puehler@genetik.uni-bielefeld.de; Jörn Kalinowski - joern.kalinowski@genetik.uni-bielefeld.de * Corresponding author Received: 19 May 2006 Accepted: 10 August 2006 Received: 19 May 2006 Accepted: 10 August 2006 Published: 10 August 2006 BMC Genomics 2006, 7:205 doi:10.1186/1471-2164-7-205 Background Insertion sequences (IS) are the simplest form of mobile genetic elements. These elements generally possess one or two open reading frames encoding the protein that causes its mobility, the transposase. The majority of IS elements is flanked by terminal inverted-repeat sequences (IR). As a result of the integration mechanism IS generate directly repeated sequences (DR) of the target DNA flanking the element of a fixed length, which are characteristic for a given element. g Corynebacterium glutamicum is a non-sporulating soil bac- terium, belonging to the high G+C Gram-positive Actino- bacteria. The bacterium, originally isolated as a natural glutamic acid excreter, is nowadays widely used as an industrial relevant producer of several L-amino acids and vitamins [1,2]. The common method to isolate amino acid-producing strains of C. glutamicum uses the iterative approach of general mutagenesis and subsequent screen- ing for mutants with a high capacity to secrete the amino acid of interest [3]. With the development of genetic engi- neering methods, like gene disruption and replacement [4,5] or over-expression and deregulation of single genes [6,7], tools for more rational strain improvement are pro- vided and successfully applied [8,9]. The availability of the complete genome sequence of C. glutamicum ATCC 13032 in combination with automatic annotation tools [10] enhanced the potential of corynebacterial research greatly. However, despite single gene manipulation tech- niques and the acquired knowledge about genes or the function and regulation of their corresponding enzymes in the metabolic context of the cell, there are still gaps in some metabolic pathways and a large amount of genes for which functions could not yet be assigned. A practicable strategy to analyse genes of unknown function is to mutate them, e.g. by generating a genomic insertion mutant library, and subsequently characterize the pheno- typic properties of individual clones within the library. An ideal resource for this purpose would be an ordered or random collection of insertion mutants, each containing a defined insertion in a particular non-essential gene, with which a large number of clones could be analysed simul- taneously [11,12]. A large number of native insertion sequences and their isoforms as part of transposons could be isolated from chromosomes and plasmids of coryneform bacteria and assigned to different members of mobile element families [17]. Furthermore, insertion sequences can be used to construct artificial transposons [18] and artificial transpo- son vectors [19] to circumvent, for instance, the natural limitation of missing antibiotic resistance markers. Background Sev- eral active mobile IS have been published for C. glutami- cum. Most of these appear to transpose randomly, but are of limited usability for random mutagenesis due to a more or less distinctive target site preference. The IS ele- ments IS1249 and IS1513 as part of the transposons Tn5432 and Tn5564, prefer triple A/T or a central palin- dromic tetranucleotide (CTAG) as target sequences [20,21]. Recent studies with IS31831, as part of Tn14751 or IS14999 revealed A/T-rich regions [22] or an 8-bp pal- indromic sequence as preferred target sites [23]. In 2003, Bonamy et al. [24] reported about an IS1207- based transposon Tn5531 with an apparent low target site-specificity in the C. glutamicum strain ATCC 14752. However, the endogenous occurrence of seven copies of the highly similar insertion sequences (ISCg1a – ISCg1d, ISCg7, ISCg10 and ISCg17) in the strain ATCC 13032 impede the application of this transposon there since inte- grations would mainly be a result of RecA-mediated homologous recombination events. In response to this demand, transposon mutagenesis sys- tems became of increasing interest for genome-scale manipulation, analyses and single-gene studies in bacte- ria. Transposon systems are efficient mutagenesis tools in a wide variety of bacterial species [13]. The advantages of a mutagenesis system employing antibiotic resistance conferring transposons are the ability of a random inte- gration into the chromosome, a positive selection for transposon mutants and the physical marker introduced at the site of mutation [14]. The insertion element IS6100 was initially isolated as part of the composite transposon Tn610 of Mycobacterium for- tuitum [25]. Later studies revealed its presence in a wide spectrum of host organisms from different bacterial line- ages, e.g. Arthrobacter sp. [26], Pseudomonas aeruginosa [27], Xanthomonas campestris [28], or Aeromonas salmonic- ida [29]. It is 880-bp in size and is bordered by 14-bp per- fect terminal inverted repeats. In common with other members of the IS6 family it translocates by replicative transposition which results in both the formation of a cointegration complex with an additional copy of the ele- ment itself as an end-product and the occurrence of an 8- bp direct repeat at the target site [17,30]. Tauch et al. [31] isolated IS6100 from the C. glutamicum antibiotic resist- ance-plasmid pTET3 present in the strain C. http://www.biomedcentral.com/1471-2164/7/205 http://www.biomedcentral.com/1471-2164/7/205 http://www.biomedcentral.com/1471-2164/7/205 Page 2 of 20 (page number not for citation purposes) Abstract Background: Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. Results: A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100- containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in C. glutamicum. The cg0910 gene, renamed hisN, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes. Conclusion: The absence of regional and sequence preferences of IS6100-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain C.glutamicum ATCC 13032. The identification of the hisN gene encoding histidinol-phosphate phosphatase in C. glutamicum closed the last gap in histidine synthesis in the Actinobacteria. The system might be a valuable genetic tool also in other bacteria due to the broad host- spectrum of IS6100. Page 1 of 20 (page number not for citation purposes) BMC Genomics 2006, 7:205 http://www.biomedcentral.com/1471-2164/7/205 BMC Genomics 2006, 7:205 active in the strain ATCC 13032. Subsequent Southern hybridization analyses of a small number of clones sug- gested that IS6100 inserts in a random manner into the C. glutamicum chromosome. was tested. Therefore, 26 clones were randomly selected and plasmid rescue cloning was carried out by digesting chromosomal DNA of the selected clones with EcoRI or XbaI, respectively, religating of the rescue constructs, transferring to E. coli and subsequent sequencing of the genomic insert with IS6100-specific primers. The sequence data obtained was analysed for sequence homologies with BLASTN searches against the C. glutami- cum ATCC 13032 whole genome sequence [10]. The BLAST results revealed the common 14-bp inverted repeat of either left and right flank of the cointegrate followed by the individual 8-bp direct repeat target site for each clone, indicating the position of the transposon integration (Table 1). As the target sites of the clones were each differ- ent, with no duplicates with the same or similar sequences, it can be assumed that these 26 transposition events occurred independently. The position of the trans- poson in every mutant, as well those selected randomly (Fig. 1, red bars) and those investigated in auxanographic analyses (Fig. 1, black bars) were plotted on a circular map representing the C. glutamicum ATCC 13032 chromo- some. The picture indicates a random distribution throughout the genome without regions of significant accumulation of insertions. Both, intragenic and inter- genic insertions were detected. The G+C content was cal- culated from the eight nucleotides of the target site and ten flanking nucleotides upstream and downstream. It showed a high variance ranging from 39.3% to 78.5%, whereas the medium G+C content for the chromosome of the strain ATCC 13032 is 53.8%. The two possible orien- tations of the cointegrate with respect to the directions of replication or transcription are present in nearly equal proportions indicating that neither orientation is favoured. In this study a transposon library of the completely sequenced type strain ATCC 13032 with a statistically rep- resentative size was constructed using an transposon vec- tor based on the IS6100 element. By auxanography analyses several auxotrophic mutants could be obtained. Plasmid rescue and sequencing of the insertion sites revealed a number of known biosynthesis genes as well hitherto unknowns. The additional development of a PCR-based screening strategy permitted the rapid detec- tion of insertion mutants in any chromosomal region of interest. http://www.biomedcentral.com/1471-2164/7/205 Taken together, the transposon used and the library created represent novel tools with high impact on functional genome analyses in C. glutamicum ATCC 13032. Transposon mutagenesis of Corynebacterium glutamicum ATCC 13032 with an IS6100-based transposon vector In vivo mutagenesis of the C. glutamicum ATCC 13032 genome was performed with the IS6100-based artificial transposon vector named pAT6100. The vector is a con- struct resulting from cloning IS6100 into the vector pK18mob2 [31]. The pAT6100 plasmid DNA extracted from Escherichia coli DH5αMCR was transformed into electrocompetent cells of C. glutamicum RES167, a restric- tion-deficient strain derived from the wild-type, by elec- troporation. Since the vector shares no homologous sequences with the host genome and it is not able to rep- licate autonomously in C. glutamicum the kanamycin resistance phenotype is the result of a transposition event into the chromosome. The transposition efficiencies ranged from five to ten c.f.u. per µg of desalted plasmid DNA. An additional test for target site preferences was carried out by performing sequence pattern searches with the TEIRESIAS algorithm [33]. For this purpose a total of 172 target sites acquired from the random selected clones and from mutants of the auxanographic analyses (see below) were used. No sequence pattern, palindromic sequences or regions of nucleotide symmetry could be detected with this bioinformatic approach (data not shown). Addition- ally, the occurrence of the bases at a distinct position of the 8-bp target sequence was calculated (data not shown). The values and therefore the appearance of bases appear to be distributed evenly. Neither the pattern searches nor the nucleotide distributions in the TSD indicate a signifi- cant target site preference for insertion. An ordered clone library was constructed comprising 10,080 independent mutant clones. With a coverage more than three-fold with respect to the 3,002 coding regions determined for C. glutamicum ATCC 13032 [10] we expect that nearly all non-essential genes contain insertions. In order to estimate the theoretical quality of the library, sta- tistical analyses were made: Given the number of clones in the transposon library (10,080), the estimated number of non-essential genes (2,400) [32], and the average gene length (952 bp), the average probability for any given gene to be disrupted by at least one transposon insertion is 97%. The cointegration of the transposon vector forms long direct repeats in the form of IS6100 copies at the site of insertion. Background glutamicum LP-6 and showed that the element is transpositionally Transposons and insertion sequences (IS) are defined seg- ments of DNA that can relocate as a unit between genomic regions [15]. Transposons range from class I composite transposons consisting of a pair of IS elements that enclose additional genetic information for antibiotic resistance or other properties, to complex class II trans- posons, to conjugative transposons that combine hybrid features of transposons, plasmids and bacteriophages [16]. Page 2 of 20 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/205 cum tr ene pr BC-2 t ermeas onserv BC-typ ranspo ldo/ket BC-typ utative onserv aleylac omose ypothe utative ranslat pstream robable TPase ranche utative BC-typ pstream rans rodu type se ved pe c osase to r pe c me ved cetat erine ene p10b F rB p I nQ aK rans rodu type se ved pe c osase to r pe c me ved cetat erine etica pro tion m pu e ph com ed-ch dith pe M m ac utami G A P C A b T A A P C M H H P T u P A B P A u H Transposon mutagenesis of Corynebacterium glutamicum ATCC 13032 with an IS6100-based transposon vector Such direct repeats might cause instability by replication slippage or by homologous recombination, thereby losing the integrated vector and one or both of the In order to perform experimental analyses of the library, first a potential target site preference of the transposon Page 3 of 20 (page number not for citation purposes) + + + + + + + + + uct Target site duplication (TSD) d G+C content [%] e Transpo orientati e transporter GGCGCGCG 78.5 - AGTGAACC 53.5 + hypothetical protein CCCAACGG 60.7 + cobalamin/Fe3+-siderophores transport system periplasmic components CTCAACGG 53.6 + CTCTTTTT 39.3 + e – fragment ISCg10a AAAAATAC 57.1 + reductase CCCTAGCG 46.5 + cobalt transport system, ATPase component CCTACGTT 42.9 + embrane protein CCTTTGAG 42.9 + hypothetical protein TAAGGAAG 39.2 - ate reductase CGATTACG 50.0 - e Kinase CCTATTAC 46.4 - al protein GCGATATC 46.4 - otein kinase ArgK or related GTPase of G3E family AGGTGAAG 60.7 + elongation factor P CGGGTGTC 60.7 + utative membrane protein (cg2054) CTTGATTC 42.9 + hosphoribosyl-AMP cyclohydrolase CGCCAAAG 57.1 - mponents of ABC transporters with duplicated ATPase domains TTCTGGAA 64.3 - hain amino acid uptake carrier GTTTCATT 42.8 + hiol-disulfide isomerase involved in polyketide biosynthesis ACTGGACT 53.6 + Mn2+/Zn2+ transport system, permease component TTGCTGAT 57.2 + cyltransferase (cg3050) CTCGACTG 57.1 + k protein Hsp70 TTCTCAGC 53.5 + GTCAGCCG 60.7 - ncharacterized enzyme related to sulfurtransferases (cg3319) CTTCAGTC 46.4 + egulatory protein, ArsR family CTTCCGCG 60.8 + nome [GenBank:BX927147]. coring) and 10 nt down-/upstream). ockwise (-) and counterclockwise (+) direction BMC Genomics 2006, 7:205 http://www.biomedcentral.com/1471-2164/7/205 Distribution of transposon insertions on a circular plot of the C. glutamicum ATCC 13032 genome [GenBank:BX927147] Figure 1 Distribution of transposon insertions on a circular plot of the C. glutamicum ATCC 13032 genome [GenBank:BX927147]. Col- oured bars of the outer circle pointing inward and outward show the orientation of the cointegrates with respect to the genome in clockwise and counterclockwise direction, respectively. Red bars indicate the integration positions mapped for ran- domly selected clones and black bars map those investigated by auxotrophy analysis. Additional circles (from inward to out- ward) represent relative G+C content and coding regions transcribed in clockwise and counterclockwise direction, respectively. A positive deviation in G+C content from the average (53.8%) is shown by bars pointing outward and a negative deviation by bars pointing inward. Transposon mutagenesis of Corynebacterium glutamicum ATCC 13032 with an IS6100-based transposon vector Annotated genes are coloured according to the colour scheme of the functional classes sys- tem COG (cluster of orthologous groups) [89]. The plot was generated with GenDB version 2.2 [90]. Auxanographic screening and targeted gene identification using PCR techniques In the majority of clones the observed auxotrophic pheno- type is well explained by the existing annotation of the disrupted gene. For example amino acid biosynthesis genes like argG (arginine), thrB (threonine), proC (pro- line), serA (serine), leuD (leucine) or lysA (lysine) were subject to intensive studies in C. glutamicum before, and the transposon insertions in these genes resulted in the expected auxotrophic phenotypes. This was also the case for insertions into genes that show high similarities to genes with a known function in closely related species, e.g. guaA and guaB2, known to be involved in the guanosine monophosphate biosynthesis in Corynebacterium ammoni- agenes [37], or to genes in more remote organisms, e.g. purA and purB which are involved in adenosine mono- phosphate biosynthesis in E. coli [38]. Another example are the carAB genes: even though these genes are not char- acterized in C. glutamicum they are well known to cause a double requirement for both arginine and uracil in E. coli since they encode for the subunits of a protein that pro- vides the essential carbamoylphosphate for arginine and pyrimidine biosynthesis [39]. The ilvC gene is well studied in C. glutamicum and is known to cause a double auxotro- phy for isoleucine and valine when disrupted [40]. The successful supplementation of a metE transposon mutant strain with either methionine or cobalamin (vitamin B12) is explained by the fact that a cobalamin-dependent (MetH) and a cobalamin-independent enzyme (MetE) perform the same step in methionine biosynthesis in C. glutamicum [41]. The loss of the MetE enzyme, the pre- ferred route during aerobic growth, can be compensated by supporting the MetH function with appropriate amounts of cobalamin. The initial step of performing genome-scale auxano- graphic analyses was the plating of the entire transposon library on minimal medium plates. After 32 h of incuba- tion this screening approach delivered 342 mutants which exhibit a clear growth-deficient phenotype. The appear- ance of potential auxotrophic mutants which are unable to form colonies under such growth conditions was used as an initial criterion for efficient mutagenesis. These mutants were further characterized by the rapid method for identification of bacterial biochemical requirements according to Holliday [34]. Therefore, 12 plates of mini- mal medium were each supplemented with different com- binations of six growth factors. Mutants with a single biochemical requirement would grow on two plates. http://www.biomedcentral.com/1471-2164/7/205 ing plates. No loss of the resistance phenotype and therefore the integrated transposon could be observed. It has to be concluded that the integrations are stably main- tained in C. glutamicum even in the absence of selective pressure. Additionally, isolated DNA from randomly selected clones which had not been grown under selective growth conditions was used to perform PCR experiments. In all cases the presence of the cointegrate was verified (data not shown). vitamins. Additionally, we have found twelve clones that show a double and six with an alternative requirement. Altogether, the auxotrophic requirements of nearly two thirds of the mutants could be identified. From all of the auxotrophy categories a number of clones was selected and the transposon integration sites were determined using the plasmid rescue technique described above. Table 2 summarizes the phenotypic and genotypic data obtained from these clones. Distributio Figure 1 Distribution of transposon insertions on a circular plot of the C. glutamicum ATCC 13032 genome [GenBank:BX927147] Figure 1 Distribution of transposon insertions on a circular plot of the C. glutamicum ATCC 13032 genome [GenBank:BX927147]. Col- oured bars of the outer circle pointing inward and outward show the orientation of the cointegrates with respect to the genome in clockwise and counterclockwise direction, respectively. Red bars indicate the integration positions mapped for ran- domly selected clones and black bars map those investigated by auxotrophy analysis. Additional circles (from inward to out- ward) represent relative G+C content and coding regions transcribed in clockwise and counterclockwise direction, respectively. A positive deviation in G+C content from the average (53.8%) is shown by bars pointing outward and a negative deviation by bars pointing inward. Annotated genes are coloured according to the colour scheme of the functional classes sys- tem COG (cluster of orthologous groups) [89]. The plot was generated with GenDB version 2.2 [90]. IS6100 elements in the absence of antibiotic selection. To assess this, five randomly selected individual clones were subcultured in antibiotic-free liquid complex medium in twelve rounds of 48 h each. The last culture was plated on solid complex medium. From this culture, 900 single col- onies from each clone were tested on kanamycin-contain- Page 5 of 20 (page number not for citation purposes) Page 5 of 20 (page number not for citation purposes) Page 5 of 20 (page number not for citation purposes) BMC Genomics 2006, 7:205 http://www.biomedcentral.com/1471-2164/7/205 Page 6 of 20 (page number not for citation purposes) Auxanographic screening and targeted gene identification using PCR techniques The double auxotrophic mutants could be determined by growth on only a single plate and an alternative require- ment by growth on more than two plates. For the latter two cases the exact nutritional requirements have to be determined by further tests combining individual growth factors. With this method, a total of 36 different growth factors, which are adapted to corynebacterial needs by omitting biotin, choline, and thiosulfate, and adding cobalamine, glutamine, as well as asparagine, could be tested simulta- neously. These growth factors comprise 21 amino acids, six nucleotides and nine vitamins, including some of their precursors. Each candidate auxotrophic clone was inocu- lated on all twelve plates and incubated for 32 h. Forty- seven out of the 342 clones tested formed slow growing colonies on every plate, suggesting that these are generally growth-deficient but not auxotrophic mutants. With the redefined number of 295 clones circa 2.9% of the transpo- son library are genuine auxotrophic mutants. This roughly correlates to prior studies with different transposons in which auxotrophic mutants were obtained with frequen- cies of 1.3% in C. glutamicum [24], 2.5% in Rhodococcus spp. [35] and 2% in Rhodococcus fascians [36]. In some cases the correlation of observed phenotype and the gene targeted by transposon integration are not so obvious. An example is the guanine auxotrophy of the nadA insertion. The nadA gene is located together with nadB and nadC and encodes a protein which is apparently involved in quinolinate and nicotinamide adenine dinu- cleotide biosynthesis. It is currently unclear why guanine supplements growth of this mutant. Also in the case of the integration in gene thiO the connection between gene function and auxotrophy is not clear yet. The gene as part of the thiEOSGF cluster encodes a putative D-amino acid Out of these 295 clones 167 display a requirement for a single growth factor (57%). Auxanographic screening and targeted gene identification using PCR techniques Among these, 141 exhibit an auxotrophy for one of eleven different amino acids, 24 for one of four different nucleotides and two for different Page 6 of 20 (page number not for citation purposes) Page 6 of 20 (page number not for citation purposes) 0699 guaA (2) guaB2 (2) nadA (1) putative GMP synthase, Inositol-monophosphate dehydrogenase, Quinolate synthase GMP biosynthesis Quinolinate biosynthesis [37]d [91] 0617 (1) pyrB (1) - Aspartate carbamoyltransferase catalytic chain, Putative Molybdopterin- guanine dinucleotide biosynthesis protein Pyridmidine biosynthesis [92]e purF (1) b Amidophosphoribosyltransferase Purine biosynthesis [93]f argG (1) Argininosuccinate synthase Arginine biosynthesis [94] 3207 aroG (2) pheA (4) Phospho-2-dehydro-3deoxyheptonate aldolase, Prephenate dehydratase Phenylalanine biosynthesis [95] [96] g2303, g1699, g1698, g0910 hisA (1), hisB (4),hisD (3), hisE (1), hisF (2), hisG (2), hisI (1), hisN (1) Phosphoribosylformimin-5-aminoimidazole carboxamide ribotideisomerase, Imidazoleglycerol-phosphate dehydratase, Histidinol dehydrogenase, Anthranilate synthase component I, probabale Cyclase (imidazole glycerol phosphate synthase-subunit), Phosphoribosyltransferase, prob. Phosphoribosyl-AMP cyclohydrolase, Histidinol-phosphate phosphatase Histidine biosynthesis [97] [49] thiO (1) Putative D-amino acid oxidase flavoprotein oxidoreductase Thiamine biosynthesis [42]g proC (1) Pyrroline-5-carboxylate reductase Proline biosynthesis [98] nd c serA (1) Phosphoglycerate dehydrogenase Serine biosynthesis [99] cysI (2) Ferredoxin-sulfite reductase Assimilatory sulfate reduction [65] leuD (1) 3-Isopropylmalate dehydratase, small chain Leucine biosynthesis [100] thrB (1) Homoserine kinase Threonine biosynthesis [101] Refe [3 [37]d [9 [9 [9 [9 [9 [9 [4 h an auxotrophic phenotype ocus a Integrated in gene a Encoded protein Funct 6 purA (5) purB (3) Adenylosuccinate synthase Adenylosuccinate lyase AMP 9 guaA (2) guaB2 (2) nadA (1) putative GMP synthase, Inositol-monophosphate dehydrogenase, Quinolate synthase GMP Quin 7 (1) pyrB (1) - Aspartate carbamoyltransferase catalytic chain, Putative Molybdopterin- guanine dinucleotide biosynthesis protein Pyrid purF (1) b Amidophosphoribosyltransferase Purin argG (1) Argininosuccinate synthase Argin 7 aroG (2) pheA (4) Phospho-2-dehydro-3deoxyheptonate aldolase, Prephenate dehydratase Phen 3, 9, 8, 0 hisA (1), hisB (4),hisD (3), hisE (1), hisF (2), hisG (2), hisI (1), hisN (1) Phosphoribosylformimin-5-aminoimidazole carboxamide ribotideisomerase, Imidazoleglycerol-phosphate dehydratase, Histidinol dehydrogenase, Anthranilate synthase component I, probabale Cyclase (imidazole glycerol phosphate synthase-subunit), Phosphoribosyltransferase, prob. http://www.biomedcentral.com/1471-2164/7/205 BMC Genomics 2006, 7:205 oxidase flavoprotein oxidoreductase that is known to be involved in thiamine biosynthesis [42]. Although a polar effect on the downstream genes thiSGF is not experimen- tally verified, such an effect would furthermore affect a more extensive part of the thiamine synthesis. The mutant is supplementable by histidine and not, as expected, by thiamine. from the trp operon. For this, a PCR-based screening strat- egy was chosen and the workflow by Hobom et al. [44] was adapted so that the entire library could be screened for insertion events by iterative rounds of three-step PCR experiments simultaneously. This strategy delivered another transposon insertion which was mapped 35 bp upstream from the predicted trpP translation start. This mutant did not exhibit a tryptophan-auxotrophic pheno- type in subsequent growth tests which coincides with the assumption that the trpP gene encodes a permease neces- sary for tryptophan- and 5-methyltryptophan uptake in C. glutamicum [45]. Since the de novo purine and pyrimidine nucleotide syn- thesis pathways are poorly characterized in C. glutamicum especially the functions of genes like purF or pyrB require further studies as well. Mutants with transposon inser- tions in the C. glutamicum genes purC, purE, and purK, known as purine biosynthesis genes in E. coli [38], were found among those which grew on more than three plates (data not shown). These mutants appear interesting to be investigated in further studies. Identification of a novel C. glutamicum gene closing the last gap in histidine biosynthesis The whole genome sequence of C. glutamicum ATCC 13032 is known and the genes are annotated based on sequence similarity and experimental data [10]. Due to the fact that a large number of amino acid biosynthesis genes have been characterized before, only few pathways remained incomplete. With the help of sequence similar- ities and subsequent genetic analyses further genes have been identified [46] leaving only few gaps. These "miss- ing" genes mostly encode transaminases which are noto- rious for their involvement in more then one pathway and were also partly characterized by bioinformatic and bio- chemical studies [47,48]. Beside this, only a single step in histidine biosynthesis remained, where no gene was known or a candidate gene could be identified by sequence similarity. The genes of most biosynthetic pathways in C. glutamicum are scattered in different genetic loci. A special case where the distribution of transposon insertions in a limited genomic region could be tested is the trp operon contain- ing six genes which encode all functions of tryptophan biosynthesis in C. glutamicum [43]. For this, the transpo- son insertion sites of 43 tryptophan-auxotrophic clones were determined. The insertion sites were all different and located within every gene from trpE to trpA (Fig. 2). Although the trp operon contains more insertions than statistically expected, this example gives additional evi- dence that the transposon library is random and that the transposon system can be used to mutagenise the C. glutamicum chromosome up to a high density. The metabolic pathway of the histidine biosynthesis, reac- tions, enzymes, as well as the organization of the corre- sponding genes display a high degree of conservation in bacteria [49]. According to the KEGG PATHWAY database [50] the histidinol-phosphate phosphatase (HolPase) is To find a gene of interest that is mutated by a transposon is sometimes not possible by phenotypic screening. We were interested to map additional insertions upstream Physical map of the tryptophan gene cluster in C. glutamicum ATCC 13032. Within the trpPEGDCFBA genes, indicated by blue arrows, the coloured bars denote the mapped integrations of 43 independent tryptophan auxotrophic mutants Figure 2 Physical map of the tryptophan gene cluster in C. glutamicum ATCC 13032. Within the trpPEGDCFBA genes, indicated by blue arrows, the coloured bars denote the mapped integrations of 43 independent tryptophan auxotrophic mutants. The two orien- tations with respect to the genome are indicated in black (clockwise) and green (counterclockwise direction). Identification of a novel C. glutamicum gene closing the last gap in histidine biosynthesis The transposon integration identified by PCR screening is also marked (orange bar). The left stem-loop symbol denotes the transcriptional attenuator involved in the regulation of the tryptophan biosynthesis [43] and the right loop a Rho-independent transcriptional terminator structure. Physical map of the tryptophan gene cluster in C glutamicum ATCC 13032 Within the trpPEGDCFBA genes, indicated by blue arrows, the coloured bars denote the mapped integrations of 43 independent tryptophan auxotrophic mutants Figure 2 Physical m arrows, th Figure 2 Auxanographic screening and targeted gene identification using PCR techniques Phosphoribosyl-AMP cyclohydrolase, Histidinol-phosphate phosphatase Histid thiO (1) Putative D-amino acid oxidase flavoprotein oxidoreductase Thiam proC (1) Pyrroline-5-carboxylate reductase Prolin nd c serA (1) Phosphoglycerate dehydrogenase Serin cysI (2) Ferredoxin-sulfite reductase Assim reduc leuD (1) 3-Isopropylmalate dehydratase, small chain Leuc thrB (1) Homoserine kinase Thre [102 [43] [47] [103] [40] (2) Diaminopimelate decarboxylase Lysine biosynthesis [102 (5), trpB (11), F (10), trpD rpE (6), trpG Tryptophan synthase alpha chain, Tryptophan synthase beta chain, Indole-3- glycerol-phosphate synthase/phosphoribosylanthranilate isomerase, Anthranilate phosphoribosyltransferase, Anthranilate synthase component I, Anthranilate synthase component II Tryptophan biosynthesis [43 BioY family membrane protein Biotin transporter (1) Pyridoxine biosynthesis transcriptional regulator, Aminotransferase Pyridoxine/valine biosynthesis [47 (1) carA (1) Carbamoyl phosphate synthase subunit, Carbamoyl phosphate synthase small subunit Arginine/pyrimidine biosynthesis [103 2) ilvC (2) Acetolactate synthase, Acetohydroxy acid isomeroreductase Isoleucine/valine biosynthesis [40 ROK-type transcriptional regulator Cysteine/pyridoxine biosynthesis putative membrane protein E (2) Homocysteine methyltransferase Methionine biosynthesis [41 espective gene. type strain phic phenotype (Continued) rpB ), trp 6), tr arA C (2) phe http://www.biomedcentral.com/1471-2164/7/205 http://www.biomedcentral.com/1471-2164/7/205 http://www.biomedcentral.com/1471-2164/7/205 BMC Genomics 2006, 7:205 an exception which was not characterized at a genetic level in C. glutamicum and closely related Actinobacteria. This enzyme (L-histidinol-phosphate phosphohydrolase; EC 3.1.3.15) catalyses the penultimate step of the biosynthe- sis, the dephosphorylation of histidinol phosphate to his- tidinol, the direct precursor of histidine. In the reference organisms E. coli and Salmonella typhimurium the HolPase activity is associated with the N-terminal domain of the HisB bifunctional enzyme. The C-terminal domain exhibit similarity to an imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19), which catalyses the sixth step of the histidine synthesis. However, amino acid sequence homology searches revealed that in C. glutamicum, like in most bacteria, the imidazoleglycerol-phosphate dehy- dratase is encoded as a monofunctional enzyme. Addi- tional similarity searches for the E. coli homologs of the HolPase in C. glutamicum and other organisms, including the entire class of Actinobacteria, does neither provide any significant sequence homologies to ORFs in the C. glutamicum genome nor to well-conserved domains in other Actinobacteria. transposon mutants. Determination of the transposon integration sites in these mutants revealed insertions in seven of the nine histidine synthesis genes known so far, hisGE, hisHAFI and hisDCB, and another one in the gene cg0910 (Table 2). The integration in this gene, which com- prises 783 bp, is located 125 bp downstream from the pre- dicted cg0910 translation start, suggesting that the gene product is non-functional. The cg0910 gene is preceded by the gene cg0911 and both might be transcribed together (Fig. 3). The cg0911cg0910 locus is located far from known histidine biosynthesis genes. BLASTP searches with the deduced protein sequence of both genes revealed significant similarities to each other and to inositol- 1(or4)-monophosphatases (IMP; EC 3.1.3.25) which hydrolyse the ester bond of inositol phosphate to generate inositol. Schematic representation of the 3-k histidine-auxotrophic or -prototrop Figure 3 Validation of cg0910 gene function by deletion and homologous complementation In order to confirm the phenotype of the transposon mutation, a cg0910 deletion mutant was constructed using the "gene splicing by overlap extension" (gene SOE- ing) technique [51]. Therefore, a fusion product of chro- mosomal DNA regions of 622 bp and 541 bp directly up- and downstream, respectively, of the cg0910 target locus was created, which was subsequently cloned into the Since the loss of the HolPase activity is supposed to exhibit a histidine-auxotrophic phenotype the corre- sponding unidentified gene in C. glutamicum was pre- sumed to be among the 16 histidine-auxotrophic Schematic representation of the 3-kb chromosomal region around cg0910 (blue arrow) in C. glutamicum. His- and his+ denote a histidine-auxotrophic or -prototrophic phenotype of the corresponding mutant or strain, respectively Figure 3 Schematic representation of the 3-kb chromosomal region around cg0910 (blue arrow) in C. glutamicum. His- and his+ denote a histidine-auxotrophic or -prototrophic phenotype of the corresponding mutant or strain, respectively. The transposon integra- tion (A) is marked with a green flag. Red arrows indicate the binding positions of the primers used to amplify the deletion frag- ments (B; yellow boxes). The features of the deletion- (B) and complementation (C) constructs are also designated. The ruler indicates the absolute position in the genome. ation of the 3-kb chromosomal region around cg0910 (blue arrow) in C. glutamicum. His- and his+ denote a c or -prototrophic phenotype of the corresponding mutant or strain, respectively Physical m arrows, th Figure 2 y p yp p g g p g y pp g p yp p p g Physical map of the tryptophan gene cluster in C. glutamicum ATCC 13032. Within the trpPEGDCFBA genes, indicated by blue arrows, the coloured bars denote the mapped integrations of 43 independent tryptophan auxotrophic mutants. The two orien- tations with respect to the genome are indicated in black (clockwise) and green (counterclockwise direction). The transposon integration identified by PCR screening is also marked (orange bar). The left stem-loop symbol denotes the transcriptional attenuator involved in the regulation of the tryptophan biosynthesis [43] and the right loop a Rho-independent transcriptional terminator structure. Page 9 of 20 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/205 Schematic histidine-au Figure 3 Schematic representation of the 3 kb chromosomal region around cg0910 (blue arrow) in C. glutamicum. His and his denote a histidine auxotrophic or prototrophic phenotype of the corresponding mutant or strain, respectively Figure 3 Schematic representation of the 3-kb chromosomal region around cg0910 (blue arrow) in C. glutamicum. His- and his+ denote a histidine-auxotrophic or -prototrophic phenotype of the corresponding mutant or strain, respectively. The transposon integra- tion (A) is marked with a green flag. Red arrows indicate the binding positions of the primers used to amplify the deletion frag- ments (B; yellow boxes). The features of the deletion- (B) and complementation (C) constructs are also designated. The ruler indicates the absolute position in the genome. Page 10 of 20 (page number not for citation purposes) Page 10 of 20 (page number not for citation purposes) Page 10 of 20 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/205 http://www.biomedcentral.com/1471-2164/7/205 BMC Genomics 2006, 7:205 method and, subsequently, the tree was evaluated by bootstrap analysis (Fig. 4). pK18mobsacB vector system [5]. The resulting vector pK18mobsacB-∆cg0910 was finally used for targeted gene deletion. The deleted internal cg0910 fragment was 702 bp of size (Fig. 3). The phylogenetic tree separates the five C. glutamicum IMP paralogs (Fig. 4, bold) into distinct classes which all include apparent orthologs from other Actinobacteria. The denotation of the respective class was derived from members for which a physical function has been described (Fig. 4, box): CysQ in E. coli [53], ImpA in Myco- bacterium smegmatis [54], SuhB in M. tuberculosis [55] and HisN (Cg0910) in C. glutamicum. The initially observed phenotype of the transposon mutant with the integration in cg0910 (clone 99G01) could be reproduced with the resulting deletion mutant, termed CG281, which was tested negative for its ability to grow on minimal medium without and positive with his- tidine supplementation. This indicated that possible sec- ondary effects in the transposon mutant could be ruled out and that the loss of the cg0910 gene product is respon- sible for histidine auxotrophy. Both the mutants 99G01 and CG281 also grew on minimal medium supplemented with histidinol, the end product of the histidinol phos- phatase reaction, comparable to the wild-type strain. These results suggest that cg0910, assigned with the gene name hisN, encodes the hitherto unknown but essential HolPase activity in C. glutamicum, and is the functional homolog of the E. coli N-terminal HisB protein domain (Table 2). Discussion In this study we describe a transposon system applicable for efficient random mutagenesis in C. glutamicum ATCC 13032. The use of an IS6100-based transposon vector gave rise to a transposon library of statistically representative size comprising independent mutant clones. To provide further evidence that the cg0910 gene encodes the HisN function, homologous complementation was carried out in both mutants 99G01 and CG281. For this, the complete cg0910 coding sequence was amplified by PCR with primers providing an optimized ribosome bind- ing site and additional restriction enzyme recognition sites. The amplificate was subsequently cloned into the IPTG-inducible shuttle expression vector pEC-XT99A [52]. The resulting plasmid pEC-XT99A-cg0910ex (Fig. 3) was subsequently transferred to the corresponding mutant strains which were then tested for histidine pro- totrophy. In both cases the extrachromosomally located cg0910 gene product complemented the loss of the corre- sponding chromosomal region in trans (data not shown). It was shown earlier that IS6100 is capable of transposing in vivo in C. glutamicum with unique transposition events by forming a cointegrate with the chromosome [31]. The excision frequency and therefore the stability of a replica- tive transposon integration is usually controlled by a resolvase protein, which is not encoded by the pAT6100 transposon vector. Nevertheless, cointegrate resolution involving either the two identical IS6100 copies or the 8- bp direct repeats might be caused by homologous recom- bination or by replication slippage resulting in the loss of the vector part and one or both copies of the IS6100 ele- ment. In this study an antibiotic sensitivity could not be observed after prolonged growth in the absence of antibi- otic pressure during cultivation, indicating that IS6100 generates cointegrations that are stably maintained in C. glutamicum. This finding conforms with the study of Weaden and Dyson [56] in which the stable cointegration of IS6100 was observed in Streptomyces avermitilis. Schematic histidine-au Figure 3 The putative orthologs of Cg0911 form a separate class. None of its members, comprising only corynebacterial and one Nocardioides species, was functionally character- ized up to now. Page 11 of 20 (page number not for citation purposes) Phylogenetic analysis of Cg0910 and related inositol monophosphatases (IMP) BLAST searches revealed four IMP paralogs in the C. glutamicum ATCC 13032 genome with significant similar- ity to cg0910, by name cg0911, cg0967 (cysQ), cg2090 (suhB), and cg2298 (impA). For a functional classification of the inositol monophosphatase family-like proteins, phylogenetic analysis in comparison to sequence-related proteins from other Actinobacteria and the model organ- ism E. coli was conducted. For this, similarity searches with the deduced amino acid sequences of the C. glutami- cum IMPs in non-redundant databases were carried out, comprising, beside E. coli K-12, the genomes of Actinobac- teria including completed as well as draft status genomes. Proteins with reliable similarity scores were used to per- form a multiple alignment. Based on this alignment a phylogenetic tree was created by the neighbour-joining The already developed transposon systems are not well applicable for random mutagenesis in the sequenced C. glutamicum type strain ATCC 13032 because of similar endogenous insertion sequences, pronounced target site preferences, or low transposition frequencies. For instance, seven copies of ISL3 family-like sequences in the ATCC 13032 genome prevent the usage of IS31831 [18] and related elements (e.g. IS1207) [24] as well as their derived transposons (e.g. Tn5531) [24]. Very recently a mutagenesis system was described that used IS31831- and Tn5-based minitransposons to generate a comprehensive library of the C. glutamicum strain R which covers nearly Page 11 of 20 (page number not for citation purposes) Page 11 of 20 (page number not for citation purposes) BMC Genomics 2006, 7:205 http://www.biomedcentral.com/1471-2164/7/205 Dendrogram showing the relationship of inositol monophosphatase family proteins (IMP) in Actinobacteria and E. coli K-12 Figure 4 Dendrogram showing the relationship of inositol monophosphatase family proteins (IMP) in Actinobacteria and E. coli K-12. A multiple alignment with amino acid sequences of proteins with high similarity to the C. glutamicum IMPs was generated with the use of the DIALIGN2 software. Based on this alignment an unrooted phylogenetic tree was constructed using the neighbour- joining algorithm integrated in the CLUSTALX package and visualized as a radial tree by the TreeTool software. The branches were combined to classes that delivered within the bootstrapping analyses in at least two thirds of the cases the same subtree. These classes, marked by different colours, were named according to the designations in the boxed leaves. The locus tags (leaves) were obtained from the GenBank genome entries. C. glutamicum proteins are printed in bold letters. Dendrogra Figure 4 Dendrogram showing the relationship of inositol monophosphatase family proteins (IMP) in Actinobacteria and E. coli K-12 Figure 4 Dendrogram showing the relationship of inositol monophosphatase family proteins (IMP) in Actinobacteria and E. coli K-12. A multiple alignment with amino acid sequences of proteins with high similarity to the C. glutamicum IMPs was generated with the use of the DIALIGN2 software. Based on this alignment an unrooted phylogenetic tree was constructed using the neighbour- joining algorithm integrated in the CLUSTALX package and visualized as a radial tree by the TreeTool software. The branches were combined to classes that delivered within the bootstrapping analyses in at least two thirds of the cases the same subtree. These classes, marked by different colours, were named according to the designations in the boxed leaves. The locus tags (leaves) were obtained from the GenBank genome entries. C. glutamicum proteins are printed in bold letters. Locus tag prefixes denote following organisms (c, complete genome sequence; da, draft assembly): Arth (Arthrobacter sp. FB24; da), BL (Bifidobac- terium longum NCC2705; c), BLinB01 (Brevibacterium linens BL2; da), DIP (Corynebacterium diphtheriae NCTC13129; c), CE (C. efficiens YS-314; c), cg (C. glutamicum ATCC 13032; c), jk (C. jeikeium K411; c), Ecoli (Escherichia coli K-12; c), Francci3 (Frankia sp. CcI3; c), Franean1 (Frankia sp. EAN1pec; da), JNB (Janibacter sp. HTCC2649; da), Krad (Kineococcus radiotolerans SRS30216; da), Lxx (Leifsonia xyli subsp. xyli str. CTCB07; c), Micol (Micromonospora olivasterospora; da), MAP (Mycobacterium avium subsp. paratuberculosis K-10; c), ML (M. leprae TN; c), Rv (M. tuberculosis H37Rv; c), Mycsm (Mycobacterium smegmatis str. MC2 155; da), nfa (Nocardia farcinica IFM 10152; c), Noca (Nocardioides sp. JS614; da), PPA (Propionibacterium acnes KPA171202; c), RhoDS7 (Rhodococcus sp. DS7; da), Rxyl (Rubrobacter xylanophilus DSM 9941; da), SAV (Streptomyces avermitilis MA-4680; c), SCO (S. coelicolor A3(2); c) and Tfu (Thermobifida fusca YX; c). Dendrogram showing the relationship of inositol monophosphatase family proteins (IMP) in Actinobacteria and E. coli K-12 Figure 4 Dendrogram showing the relationship of inositol monophosphatase family proteins (IMP) in Actinobacteria and E. coli K-12. A multiple alignment with amino acid sequences of proteins with high similarity to the C. glutamicum IMPs was generated with the use of the DIALIGN2 software. Based on this alignment an unrooted phylogenetic tree was constructed using the neighbour- joining algorithm integrated in the CLUSTALX package and visualized as a radial tree by the TreeTool software. Phylogenetic analysis of Cg0910 and related inositol monophosphatases (IMP) Locus tag prefixes denote following organisms (c, complete genome sequence; da, draft assembly): Arth (Arthrobacter sp. FB24; da), BL (Bifidobac- terium longum NCC2705; c), BLinB01 (Brevibacterium linens BL2; da), DIP (Corynebacterium diphtheriae NCTC13129; c), CE (C. efficiens YS-314; c), cg (C. glutamicum ATCC 13032; c), jk (C. jeikeium K411; c), Ecoli (Escherichia coli K-12; c), Francci3 (Frankia sp. CcI3; c), Franean1 (Frankia sp. EAN1pec; da), JNB (Janibacter sp. HTCC2649; da), Krad (Kineococcus radiotolerans SRS30216; da), Lxx (Leifsonia xyli subsp. xyli str. CTCB07; c), Micol (Micromonospora olivasterospora; da), MAP (Mycobacterium avium subsp. paratuberculosis K-10; c), ML (M. leprae TN; c), Rv (M. tuberculosis H37Rv; c), Mycsm (Mycobacterium smegmatis str. MC2 155; da), nfa (Nocardia farcinica IFM 10152; c), Noca (Nocardioides sp. JS614; da), PPA (Propionibacterium acnes KPA171202; c), RhoDS7 (Rhodococcus sp. DS7; da), Rxyl (Rubrobacter xylanophilus DSM 9941; da), SAV (Streptomyces avermitilis MA-4680; c), SCO (S. coelicolor A3(2); c) and Tfu (Thermobifida fusca YX; c). http://www.biomedcentral.com/1471-2164/7/205 glutamicum [31]. 80% of the presently unpublished genome [57]. The C. glutamicum strain R has the advantage of not possessing insertion elements of the IS31831 family. In contrast to this, IS6100 is absent from the strain ATCC 13032 chro- mosome, ruling out integration by homologous recombi- nation and thus it is suitable to perform transposon mutagenesis in this strain. Beside IS6100 quite a few active mobile elements have been published for C. glutamicum which are of restricted usability of generating a compre- hensive random transposon library since they prefer spe- cific sequences, e.g. a triple A or T (IS1249) [20] or palindromic sequences (e.g. IS14999) [23], as a target for integration. On the other hand, prior studies with IS6100- based transposons suggested independent transposition and absence of a target site preference. This was first shown in Streptomyces lividans and S. coelicolor by nucle- otide sequence comparison for a small number of mutants [58] and, on the basis of Southern hybridization studies in S. lividans [59] and C. glutamicum [31]. In this study, the usability of IS6100 for mutagenesis in the type strain ATCC 13032 was shown by analysis of a larger number of clones. The sequence data determined from 172 insertions delivered definite position informa- tions and, consequently, allowed comprehensive analyses on the transposon target sites. Each clone investigated car- ried the transposon in a different chromosomal location. The absence of regional preferences together with the absence of sequence preferences (sequence pattern, nucle- otide-usage for the TSD positions or G+C content) as a tar- get demonstrated the randomness of the library constructed and applied in auxanography analyses. Fur- thermore, this system might be a practicable genetic tool in other organisms as well because of the broad host-spec- trum of the IS6100 element. It is known that transposon insertions near the beginning or within an operon can attenuate or interrupt the expres- sion of downstream genes preventing RNA polymerase readthrough [63,64]. A polar effect on downstream genes was experimentally shown for an IS6100 integration in the gene cysI (cg3118) with transcriptional analysis using Real Time RT-PCR [65]. This example points out that the transposon system is not only applicable for high density mutagenesis of chromosomal regions (e.g. tryptophan operon, Fig. 2) but might be utilised for the determination of operons. This study integrated genomic sequence data with genome-scale auxotrophy analyses. http://www.biomedcentral.com/1471-2164/7/205 BMC Genomics 2006, 7:205 Transposon integrations were found in a variety of known amino acid, nucleotide and vitamin pathway genes as well in genes encoding hypothetical proteins or such of pres- ently unknown function. The vast majority of the observed auxotrophic phenotypes could be correlated and explained with the knowledge of the mutated genomic region since most genes of amino acid, nucleotide and vitamin biosyntheses are annotated in C. glutamicum. In contrast, for some observed phenotypes the connection between gene and auxotrophy is not yet clear, delivering interesting targets for further studies. The loss of gene products associated with de novo synthesis or recycling pathways may be compensated by influx of necessary metabolite entries from other pathways that share com- mon intermediates or precursors to a given intermediate. For instance, the PurF protein, an amidophosphoribosyl- transferase, is known to be involved in more than one pathway. In Salmonella typhimurium PurF is the first of five enzymes shared by the de novo purine and HMP (hydroxymethylpyrimidine) synthesis, an essential com- pound of thiamine biosynthesis. Thus, PurF is expected to be required for both purine and thiamine biosynthesis [60]. It is not obvious why a purF transposon mutant is supplementable solely by hypoxanthine. Alternative path- ways were discovered which could bypass the requirement for all pur genes in thiamine synthesis [61,62]. Therefore, hypoxanthine, a purine derivative, might be sufficient to compensate the growth-deficiency of this mutant. 80% of the presently unpublished genome [57]. The C. glutamicum strain R has the advantage of not possessing insertion elements of the IS31831 family. In contrast to this, IS6100 is absent from the strain ATCC 13032 chro- mosome, ruling out integration by homologous recombi- nation and thus it is suitable to perform transposon mutagenesis in this strain. Beside IS6100 quite a few active mobile elements have been published for C. glutamicum which are of restricted usability of generating a compre- hensive random transposon library since they prefer spe- cific sequences, e.g. a triple A or T (IS1249) [20] or palindromic sequences (e.g. IS14999) [23], as a target for integration. On the other hand, prior studies with IS6100- based transposons suggested independent transposition and absence of a target site preference. This was first shown in Streptomyces lividans and S. coelicolor by nucle- otide sequence comparison for a small number of mutants [58] and, on the basis of Southern hybridization studies in S. lividans [59] and C. Dendrogra Figure 4 The branches were combined to classes that delivered within the bootstrapping analyses in at least two thirds of the cases the same subtree. These classes, marked by different colours, were named according to the designations in the boxed leaves. The locus tags (leaves) were obtained from the GenBank genome entries. C. glutamicum proteins are printed in bold letters. Locus tag prefixes denote following organisms (c, complete genome sequence; da, draft assembly): Arth (Arthrobacter sp. FB24; da), BL (Bifidobac- terium longum NCC2705; c), BLinB01 (Brevibacterium linens BL2; da), DIP (Corynebacterium diphtheriae NCTC13129; c), CE (C. efficiens YS-314; c), cg (C. glutamicum ATCC 13032; c), jk (C. jeikeium K411; c), Ecoli (Escherichia coli K-12; c), Francci3 (Frankia sp. CcI3; c), Franean1 (Frankia sp. EAN1pec; da), JNB (Janibacter sp. HTCC2649; da), Krad (Kineococcus radiotolerans SRS30216; da), Lxx (Leifsonia xyli subsp. xyli str. CTCB07; c), Micol (Micromonospora olivasterospora; da), MAP (Mycobacterium avium subsp. paratuberculosis K-10; c), ML (M. leprae TN; c), Rv (M. tuberculosis H37Rv; c), Mycsm (Mycobacterium smegmatis str. MC2 155; da), nfa (Nocardia farcinica IFM 10152; c), Noca (Nocardioides sp. JS614; da), PPA (Propionibacterium acnes KPA171202; c), RhoDS7 (Rhodococcus sp. DS7; da), Rxyl (Rubrobacter xylanophilus DSM 9941; da), SAV (Streptomyces avermitilis MA-4680; c), SCO (S. coelicolor A3(2); c) and Tfu (Thermobifida fusca YX; c). Page 12 of 20 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/205 http://www.biomedcentral.com/1471-2164/7/205 This theory has been falsified for a M. smegmatis impA mutant. This mutant is not auxotrophic for histidine, but exhibits altered cell envelope permeability properties, with a notable reduc- tion in the synthesis of phosphatidylinositol dimanno- side, the precursor of LAM [54]. The SuhB inositol monophosphatase activity has been characterized biochemically in M. tuberculosis. Inositol-1- phosphate was shown to be the preferred target of SuhB for dephosphorylation in order to provide the PI synthase with inositol [75]. The specificity of Cg0910 (HisN) for histidinol phosphate was experimentally proven for C. glutamicum. For all other members of the phylogenetic IMP class labelled Cg0910 the intrinsic HolPase function could be proposed because of the close relationship and the high sequence conserva- tion. Furthermore, in the respective Actinobacteria a pro- tein having the enzymatic function of histidinol phosphate dephosphorylation was not identified. Although the IMP-like proteins analysed in this study appear to be sequence homologs phylogenetic analysis revealed that they are significantly different as they branch into distinct classes. Despite their amino acid conserva- tion, they seem to form a protein family of diverse func- tions and/or with diverse substrates. The assumption of different substrate specificities corresponds to previously published studies performed either in closely related mycobacteria or in E. coli. The Cg0911 class comprises only corynebacterial mem- bers and one Nocardioides subspecies. The fact that in all these organisms cg0911 and cg0910 orthologs are located next to each other leads to the assumption of a recent gene duplication that exclusively occurred in corynebacteria or in a common ancestor. However, the finding that the respective proteins of cg0910 and cg0911 are members of different IMP classes indicate that they accomplish differ- ent functions and thus solely the cg0910 gene encodes the HolPase activity in C. glutamicum. Additional evidence that the unknown Cg0911 function, in contrast to Cg0910, does not play an important role in the cell was obtained by the inactivation of the respective gene (data not shown). The mutant showed no obvious phenotype under the applied growth conditions. The E. coli CysQ homolog is required for cysteine synthe- sis during aerobic growth. The sulfate assimilation branch of the cysteine pathway comprises sulfate uptake, its acti- vation by formation of adenosine 5'-phosphosulfate (APS) and conversion to 3'-phosphoadenosine 5'-phos- phosulfate (PAPS) by APS kinase, and its reduction to sulfite. It has been suggested that CysQ acts on PAPS as a target. http://www.biomedcentral.com/1471-2164/7/205 BMC Genomics 2006, 7:205 BMC Genomics 2006, 7:205 BMC Genomics 2006, 7:205 For detailed studies on gene functions, transposon mutants have to be confirmed experimentally in order to rule out secondary mutations, polar effects or leaky phe- notypes. In this study, this was carried out by a concrete example in which, by the means of supplementation, genetic deletion and complementation assays, the last gap in the histidine biosynthesis pathway of C. glutamicum could be closed. homolog is missing and thus PAPS as an intermediate is not formed. Sulfite is released from APS by direct reduc- tion through APS reductase [65]. Therefore, the target sub- strate for a PAPS CysQ protein would be missing as well. It might be considered that CysQ in C. glutamicum and the other actinobacterial members of this phylogenetic class indeed does not possess a PAPS phosphatase activity, but most likely an inositol-phosphate phosphatase activity. homolog is missing and thus PAPS as an intermediate is not formed. Sulfite is released from APS by direct reduc- tion through APS reductase [65]. Therefore, the target sub- strate for a PAPS CysQ protein would be missing as well. It might be considered that CysQ in C. glutamicum and the other actinobacterial members of this phylogenetic class indeed does not possess a PAPS phosphatase activity, but most likely an inositol-phosphate phosphatase activity. Initial similarity searches revealed that the Cg0910 pro- tein (HisN), together with another four paralogs in the C. glutamicum genome, apparently belongs to the mono- phosphatase-family proteins that usually hydrolyse the ester bond of myo-inositol-1(or4) phosphate. Inositol monophosphatases (IMP) play a crucial role in the bio- synthesis of inositol and inositol phospholipids [66]. The role of IMP in bacteria is not completely clear yet. Bacteria of the genus Mycobacterium contain a number of inositol- derived cell wall constituents, like phosphatidylinositol (PI), phosphatidylinositol mannosides (PIM), lipoarabi- nomannan (LAM) and lipomannan (LM) [67-69]. LAM- like molecules and other inositol-containing phospholip- ids are not only present in mycobacteria but also in other Actinobacteria, including the genus Corynebacterium [70- 72]. Actually, little is known about inositol synthesis in bacteria. The known de novo pathway in mycobacteria basically comprises the cyclization of glucose-6-phos- phate to inositol-1-phosphate (I-1-P) and, subsequently, the dephosphorylation of I-1-P by IMP producing inositol [73]. Initially, impA has been proposed to encode the missing HolPase, as it is clustered with the histidine biosynthesis genes in the Actinobacteridae [74]. http://www.biomedcentral.com/1471-2164/7/205 It is proposed to help controlling the levels of PAPS, which may be toxic to the cell in high concentra- tions, or the generation of sulfite [53]. The actinobacterial CysQ IMP homologs in the phylogenetic tree branch later as compared to CysQ of E. coli, thus functional differences between the E. coli and the actinobacterial CysQ proteins might be possible. In C. glutamicum an APS kinase http://www.biomedcentral.com/1471-2164/7/205 By means of auxano- graphic assays, for 63% of the 295 isolated auxotrophic mutants with distinct phenotypes various nutritional requirements could be identified. Additionally, out of this contingent the different integration loci of 101 clones were determined. The value of 2.9% auxotrophic clones only slightly differs from those in other transposon muta- genesis studies (1.3% [24], 2.5% [35] and 2% [36]) but is remarkably higher compared to studies in which auxo- trophic mutants were obtained with frequencies of 0.2% in "Brevibacterium flavum" with IS31831 [18], 0.2% in C. glutamicum with IS1249 (Tn5432) [20] and 0.5% in Strep- tomyces avermitilis with IS6100 [56]. This might be explained by the fact that latter systems use mobile ele- ments for which an insertion sequence specificity was identified (IS31831 and IS1249). The screening approach for characterization of auxo- trophic mutants used in this study shows a limitation in resolving complex growth phenotypes, exhibited by one thirds of these mutants. However, determination of trans- poson positions in randomly selected members of this auxotrophy category revealed for example mutations within purC, purE, and purK, genes known to be involved in purine biosynthesis. Such genes, as mentioned above, might be of interest to be investigated in further experi- mental studies. These findings indicate that also the com- plex phenotypes are not a result of additional spontaneously occurring mutations, but caused by disrup- tion of the corresponding gene or by the accompanying polar effect. Page 13 of 20 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/205 Transformation of C. glutamicum f f g The artificial transposon vector pAT6100 was electrotrans- ferred into the restriction-deficient strain C. glutamicum RES167 by means of a method previously described [79]. To force transposition, the cells were additionally incu- bated by shaking at 200 rpm for 50 min at 37°C directly after electroporation procedure before pelleting and spreading them on solid complex media plates containing 25 µg ml-1 kanamycin to select transformants. Kanamycin- resistant colonies were picked with sterile sticks, trans- ferred to separate wells of 96-well microtiter plates (Greiner, Solingen, Germany) containing 200 µl selective complex medium and inoculated for 48 h at 30°C. Long- term storage was carried out in 96-well microtiter plates containing 10% Hogness modified freezing medium (HMFM), composed of 87% (v/v) glycerol, 1.7 mM tri- sodium citrate, 6.8 mM (NH4)2SO4, 0.4 mM MgSO4 · 4 H2O, 36 mM Na2HPO4 · 2 H2O, 13.2 mM KH2PO4, and 90% TSB at -80°C. The closure of the last gap in the histidine biosynthesis pathway by identification of the hisN gene encoding a his- tidinol-phosphate phosphatase not only complements the knowledge of the pathway in C. glutamicum but in the entire class of Actinobacteria as well as in several Proteo- bacteria and Chlorobia [76]. Furthermore, with the classi- fication of the inositol monophosphate proteins based on sequence similarities, it could be proposed that the IMP homologs in Actinobacteria, although generally acting on phosphorylated metabolites, appear to have diverse sub- strate specificities. Bacterial strains, plasmids, oligonucleotides and culture conditions Relevant bacterial strains, plasmids, the transposon vector and oligonucleotides constructed and used in this study are listed in Table 3. E. coli and C. glutamicum strains were routinely grown on Tryptic Soy Broth (TSB) complex media (Merck, Darmstadt, Germany) supplemented with 1.5% (w/v) agar (Invitrogen, Karlsruhe, Germany) at 37°C and 30°C, respectively. Antibiotics used for selec- tion were kanamycin (50 µg ml-1 for E. coli and 25 µg ml- 1 for C. glutamicum), tetracycline (5 µg ml-1) and nalidixic acid (50 µg ml-1). The oligonucleotides used as primers were designed with the Clone Manager software (Scien- tific & Educational Software, Cary, USA) and purchased from Operon (Cologne, Germany). The optical density of a culture in liquid medium was determined at a wave- length of 600 nm with a BioPhotometer (Eppendorf, Hamburg, Germany) spectralphotometer. Determination of transposon integration sites f p g Genomic DNA from C. glutamicum transposon mutants was extracted using the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, Taufkirchen, Germany). Cloning and sequencing of insertion sites were carried out by plasmid rescue techniques as described before [20] with the excep- tion that the chromosomal DNA was digested with EcoRI and XbaI to clone either side of the insertion. Sequencing of the resulting plasmids was performed with the syn- thetic oligonucleotide primers s6100e and s6100x for the EcoRI and the XbaI sites, respectively, by IIT Biotech (Bielefeld, Germany). Sequence data/accession numbers Nucleotide homology searches were applied against the annotated C. glutamicum ATCC 13032 genome sequence [GenBank:BX927147] [10,80] obtained from GenBank [81]. Amino acid sequences of predicted inositol mono- phosphatase proteins were obtained from the Swissprot/ TrEMBL databases [82]: Cg0910 [Swiss-Prot:Q8NS80], Cg0911 [Swiss-Prot:Q6M6Y2], CysQ [Swiss-Prot: Q8NS37], SuhB [Swiss-Prot:Q6M4B2] and ImpA [Swiss- Prot:Q8NNT8]. Further protein sequence information used for comparative analyses were retrieved from the fol- lowing GenBank genome entries: [GenBank:AE014295] Bifidobacterium longum NCC2705, [GenBank:BA000035] C. efficiens YS-314, [GenBank:BX248353] C. diphtheriae NCTC13129, [GenBank:CR931997] C. jeikeium K411, [GenBank:U00096] Escherichia coli K-12, [Gen- Bank:CP000249] Frankia sp. CcI3, [GenBank:AE016822] Leifsonia xyli subsp. xyli str. CTCB07, [GenBank:AE016958] Mycobacterium avium subsp. paratuberculosis K-10, [Gen- Bank:AL450380] M. leprae TN, [GenBank:AL123456] M. tuberculosis H37Rv, [GenBank:AP006618] Nocardia farci- nica IFM 10152, [GenBank:AE017283] Propionibacterium Conclusion In conclusion, the random transposon system described in this study together with the availability of the complete genome sequence of C. glutamicum ATCC 13032 [10] rep- resents a powerful tool for genome-scale functional anal- yses in this type strain. Moreover, the transposon library is Page 14 of 20 (page number not for citation purposes) Page 14 of 20 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/205 http://www.biomedcentral.com/1471-2164/7/205 http://www.biomedcentral.com/1471-2164/7/205 http://www.biomedcentral.com/1471-2164/7/205 BMC Genomics 2006, 7:205 BMC Genomics 2006, 7:205 a resource of specific mutants which contribute to the genetic understanding of this organism in the future. Auxotrophy screening and supplementation of auxotrophic phenotypes The identification of auxotrophic mutants was performed by parallel plating on minimal media MM1 (MMYE with- out yeast extract [88]) and TSB complex media. The auxa- nographic characterizations were carried out by picking the colonies on MM1 plates supplemented with growth factors which were added as sterile filtered stock solutions by means of the purchaser's instructions, following a plat- ing pattern described by Holliday [34], and 25 µg ml-1 DNA isolation, manipulation and transfer Plasmid DNA was extracted from E. coli and C. glutamicum by an alkaline lysis technique using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) with a prelimi- nary incubation for 2 h at 37°C in resuspension buffer P1 containing 50 mg ml-1 lysozyme (185,000 U mg-1; Serva, Heidelberg, Germany) for C. glutamicum. DNA modifica- tion, analyses by gel-electrophoresis and ligation were car- ried out by standard procedures [77]. Restriction endonucleases and T4 DNA ligase, together with enzyme buffers, were purchased from Fermentas (St. Leon-Rot, Germany) and used according to the manufacturer's instructions. DNA restriction fragments required for clon- ing were recovered from agarose gels by using QIAEX II gel extraction kit (Qiagen). E. coli and corynebacterial cells were transformed by electroporation [78] using the Bio- Rad Gene Pulser system (Bio-Rad, Munich, Germany). Page 15 of 20 (page number not for citation purposes) Page 15 of 20 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/205 BMC Genomics 2006, 7:205 Table 3: Bacterial strains, plasmids and oligonucleotides used in this study Strain, plasmid, oligonucleotide Relevant genotype/characteristics/information or sequence a Source/reference E. coli DH5αMCR F- endA1 supE44 mcrA thi-1 λ-recA1 gyrA96 relA1 deoR ∆(lacZYA-argF)U169 (Φ80dlacZ∆M15) ∆(mrr-hsdRMS-mcrBC) [104] C. glutamicum ATCC 13032 Nxr, wild-type ATCC b RES167 Nxr, ∆(cglIMR, cglIIR) Restriction deficient mutant of C. glutamicum ATCC 13032 [79] CG281 ∆cg0910 This study Transposon vector pAT6100 RP4mob, oriVE.c., Kmr Mobilizable cloning vector pK18mob2 carrying the IS6100 insertion element [31] Plasmids pK18mobsacB sacB, lacZα, mcs, Kmr/mobilizable E. coli cloning vector, allows selection for double- crossover in C. glutamicum [5] pK18mobsacB-∆cg0910 pK18mobsacB with defined deletion derivative of cg0910 This study pEC-XT99A Tcr, lacIq, mcs, Ptrc inducible E. coli – C. glutamicum shuttle expression vector [52] pEC-XT99A-cg0910ex pEC-XT99A with internal cg0910 exc fragment This study Oligonucleotides 5'-3' IS6100x GGTACAGGTAGGTCCACTTG This study IS6100y CGGCAGGTGAAGTATCTCAA This study gs_trpP CACCTGCATCAAGGTCGATT This study s6100e GCGCCTTGTGGAGAGAGCTT This study s6100x CGGATAGCGACAATACCAGC This study cg0910_del1 GATCTAGGATCCGATGGCACCTACAACTTCAC This study cg0910_del2 AACGATCCAGCGTCTCATCGTCGGCAAGTTCGGCAAGTTC This study cg0910_del3 CGATGAGACGCTGGATCGTT This study cg0910_del4 GATCTACTCGAGAGCTCTTCCAGCGTTCCATT This study cg0910_ex1 GATCTACAATTGAAAGGAGGACAACCATGAGCAAATATGCAGACGA This study cg0910_ex2 GATCTAGGATCCCTATTTTAAACGATCCAGCG This study a r superscript indicates resistance. Nx, nalidixic acid; Km, kanamycin; Tc, tetracycline. b ATCC; American Type Culture Collection Rockville MD USA Strain, plasmid, oligonucleotide Relevant genotype/characteristics/information or sequence a performed with the DIALIGN2 software [84]. DNA isolation, manipulation and transfer The phylo- genic tree was calculated using the neighbour-joining method [85] integrated in the CLUSTALX software pack- age [86] and visualized as a radial tree with the interactive phylogenetic tree plotting program TreeTool [87]. Sequence pattern searches were performed with the TEIR- ESIAS algorithm [33] provided by the IBM Bioinformatics Group. acnes KPA171202, [GenBank:BA000030] Streptomyces avermitilis MA-4680, [GenBank:AL645882] S. coelicolor A3(2), [GenBank:CP000088] Thermobifida fusca YX, [Gen- Bank:AAHG00000000] Arthrobacter sp. FB24, [Gen- Bank:AAGP00000000] Brevibacterium linens BL2, [GenBank:AAII00000000] Frankia sp. EAN1pec, [Gen- Bank:AAMN00000000] Janibacter sp. HTCC2649, [Gen- Bank:AAEF00000000] Kineococcus radiotolerans SRS30216, [GenBank:AAJB00000000] Nocardioides sp. JS614, and [GenBank:AAEB00000000] Rubrobacter xylanophilus DSM 9941. Deletion and genetic complementation of cg0910 kanamycin. The list of growth factors comprises 20 bio- genic L-amino acids (alanine, arginine, asparagine, aspar- tic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, pheny- lalanine, proline, serine, threonine, tryptophan, tyrosine, valine; final concentrations 1 mM each), nucleic acid bases (adenine, cytosine, guanine, thymine, uracil; 1 mM final cc. each), vitamins and precursors (thiamine (vita- min B1; 500 µg l-1), riboflavin (B2; 200 µg l-1), nicotinic acid (B3; 400 µg l-1), pantothenic acid (B5; 400 µg l-1), pyridoxine (B6; 400 µg l-1), cobalamin (B12; 10 µg l-1), folic acid (20 µg l-1), p-aminobenzoic acid (200 µg l-1), inositol (20 µg l-1), ornithine (1 mM), and hypoxanthine (1 mM)). The chemicals were purchased from VWR (Darmstadt, Germany) and Merck. g p f g Defined chromosomal deletions within the cg0910 gene were constructed with the pK18mobsacB vector system which allows to identify an allelic exchange by homolo- gous recombination [5]. The deletion was introduced into the target gene by "gene SOEing" [51]. Therefore, upstream and downstream regions of the gene to be deleted were amplified by two different PCR reactions using primer pairs cg0910_del1+2 and cg0910_del3+4 (Table 3), respectively. After purification the resulting amplificates were used as templates for the second round of PCR. The final products were digested with the restric- tion enzymes BamHI and XhoI, corresponding to the cleavage sites introduced via the PCR primers, and subse- quently cloned into appropriately digested pK18mobsacB vector. The ligation mixture was used to transform E. coli DH5αMCR and the transformants were selected on TSB plates containing 50 µg ml-1 kanamycin and 40 mg l-1 X- Gal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside). The resulting deletion plasmid pK18mobsacB-∆cg0910 was extracted and transformed into C. glutamicum ATCC 13032. Integration of the introduced plasmid into the chromosome by single-crossover was selected on TSB plates supplemented with 25 µg ml-1 kanamycin. The kan- amycin-resistant clones were grown overnight in liquid media and spread on TSB plates containing 10% (w/v) sucrose. Colonies from this plates were tested for the desired kanamycin-sensitive and sucrose-resistant pheno- type by parallel picking. PCR experiments were used to verify the deletion in the C. glutamicum chromosome. Construction of plasmid pEC-XT99A-cg0910ex for in trans-expression of the Cg0910 protein was initiated with the amplification of the corresponding gene by PCR using the primer pair cg0910_ex1 and cg0910_ex2 (Table 3). Deletion and genetic complementation of cg0910 Primer cg0910_ex1 was provided with a sequence of an optimal ribosome binding site upstream from the cg0910 start codon. The resulting 821-bp DNA fragment was puri- fied and cleaved using MunI and BamHI. Appropriate restriction sites were added with the 5'-extension of the PCR primers. The fragment was subsequently ligated into the pEC-XT99A vector, which was prior cut with the corre- sponding enzymes. The ligation mixture was used for transformation of E. coli DH5αMCR, the transformants were selected on TSB plates containing 5 µg ml-1 tetracy- cline. PCR techniques and conditions DNA fragments for deletion and complementation exper- iments were amplified from chromosomal DNA of C. glutamicum ATCC 13032 using Pwo DNA polymerase (Inv- itrogen), and Taq DNA polymerase (Peqlab, Erlangen, Germany) for the verification of chromosomal deletions. PCR reaction conditions were as follows: initial denatura- tion at 94°C for 2 min followed by 35 cycles of denatura- tion for 30 s, annealing for 1 min at a primer-dependent temperature, extension at 72°C for 2 min and a final extension for 4 min at 72°C. PCR products were purified using the QIAquick PCR purification kit (Qiagen). PCR mutant screening was carried out with Eppendorf Taq DNA polymerase (Eppendorf) with pooled transposon mutants as templates, IS6100-specific primers IS6100x and IS6100y for both possible orientations of the transpo- son and a position-specific search primer gs_trpP (Table 3). The basic procedure was adapted from a method pub- lished by Hobom et al. [44]. Iterative rounds of PCR experiments were performed in order to identify the clone of interest in the library. In the first round clone-pools were examined containing all mutants in one microtiter plate. Sequential PCR rounds were carried out with the mutant-pools representing the clones of those individual plates that were tested positively in prior rounds until the exact position of the clone in the library could be deter- mined. The bacterial cells were directly set in the PCR reac- tion and lysed with an initial PCR step of 94°C for 10 min to release the chromosomal DNA. PCR reaction condi- tions were as follows: initial denaturation at 94°C for 10 min followed by 40 cycles of denaturation for 30 s, annealing for 45 s at a primer-dependent temperature, extension at 72°C for 2 min and a final extension for 4 min at 72°C. PCR experiments were performed with the delivered buffers according to the manufacturer's instruc- tions and with the use of a DNA Engine DYAD thermocy- cler from MJ Research (Watertown, MA, USA). Bioinformatic analyses and tools for interpretation of C. glutamicum sequences Database searches and sequence similarity-based searches with nucleotide and amino acid sequences were per- formed with the BLASTN- and BLASTP- algorithms [83], respectively. 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Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge ( ) 98. Ankri S, Serebrijski I, Reyes O, Leblon G: Mutations in the Corynebacterium glutamicum proline biosynthetic path- way: a natural bypass of th proA step. J Bacteriol 1996, 178(15 ):4412-4419. ) 99. Peters-Wendisch P, Netzer R, Eggeling L, Sahm H: 3-Phosphoglyc- erate dehydrogenase from Corynebacterium glutamicum: the C-terminal domain is not essential for activity but is required for inhibition by L-serine. Appl Microbiol Biotechnol 2002, 60(4):437-441. ( ) 100. 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https://openalex.org/W4390663688	https://www.mdpi.com/2223-7747/13/2/156/pdf?version=1704467322	English	null	Identification of Genes Responsible for the Synthesis of Glycitein Isoflavones in Soybean Seeds	Plants	2,024	cc-by	15,496	plants plants Citation: Horitani, M.; Yamada, R.; Taroura, K.; Maeda, A.; Anai, T.; Watanabe, S. Identification of Genes Responsible for the Synthesis of Glycitein Isoflavones in Soybean Seeds. Plants 2024, 13, 156. https://doi.org/10.3390/ plants13020156 Citation: Horitani, M.; Yamada, R.; Taroura, K.; Maeda, A.; Anai, T.; Watanabe, S. Identification of Genes Responsible for the Synthesis of Glycitein Isoflavones in Soybean Seeds. Plants 2024, 13, 156. https://doi.org/10.3390/ plants13020156 Keywords: Glycine max; isoflavone; QTL; glycitein; 6-hydroxydaidzein; flavonoid 6-hydroxylase; O-methyltransferase Article , Risa Yamada 1, Kanami Taroura 1, Akari Maeda 1, Toyoaki Anai 2 and Satoshi Watanabe 1,* Masaki Horitani 1 , Risa Yamada 1, Kanami Taroura 1, Akari Maeda 1, Toyoaki Anai 2 and Sat 1 Faculty of Agriculture, Saga University, 1 Honjo-machi, Saga 840-8502, Japan; horitani@cc.saga-u.ac.jp (M.H.) 2 Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; anai.toyoaki.494@m.kyushu-u.ac.jp * Correspondence: nabemame@cc saga u ac jp; Tel /Fax: +81 952 28 8741 y y jp * Correspondence: nabemame@cc.saga-u.ac.jp; Tel./Fax: +81-952-28-8741 Abstract: Soybean (Glycine max (L.) Merrill) isoflavones are among the most important secondary metabolites, with functional benefits for human health. Soybeans accumulate three aglycone forms of isoflavones: genistein, daidzein, and glycitein. Soybean landrace Kumachi-1 does not accumulate malonylglycitin at all. Gene structure analysis indicated that Glyma.11G108300 (F6H4) of Kumachi-1 has a 3.8-kbp insertion, resulting in a truncated flavonoid 6-hydroxylase (F6H) sequence compared to the wild-type sequence in Fukuyutaka. Mapping experiments using a mutant line (MUT1246) with a phenotype similar to that of Kumachi-1, with a single-nucleotide polymorphism (SNP) in F6H4, revealed co-segregation of this mutation and the absence of glycitein isoflavones. We also identified a mutant line (K01) that exhibited a change in the HPLC retention time of glycitein isoflavones, accumulating glycoside and malonylglycoside forms of 6-hydroxydaidzein. K01 contains an SNP that produces a premature stop codon in Glyma.01G004200 (IOMT3), a novel soybean isoflavone O-methyltransferase (IOMT) gene. We further analyzed transgenic hairy roots of soybeans expressing Glyma.11G108300 (F6H4) and Glyma.01G004200 (IOMT3). Those overexpressing F6H4 accumulated malonylglycoside forms of 6-hydroxydaidzein (M_6HD), and co-expression of F6H4 and IOMT3 increased the level of malonylglycitin but not of M_6HD. These results indicate that F6H4 and IOMT3 are responsible for glycitein biosynthesis in soybean seed hypocotyl. plants plants plants 1. Introduction Soybean (Glycine max (L.) Merrill) is used as an oil and food source for humans and livestock. Soybean secondary metabolites have functional benefits to human health and have received much attention worldwide. These metabolites include isoflavones, which function in plant–microbe interactions [1,2] and have positive nutritional effects in the human diet. Three aglycone forms of isoflavones, genistein, daidzein, and glycitein, which are glycosylated and then malonylated, are synthesized in soybean mature seeds. These aglycones have health benefits, such as reducing the risk of cardiovascular disease and osteoporosis, mitigating menopausal symptoms [3], and preventing the proliferation of certain cancers [4]. Received: 9 November 2023 Revised: 23 December 2023 Accepted: 27 December 2023 Published: 5 January 2024 Three types of alleles—activated type (Aokimame (AOKI) allele), normal type (Fukuyutaka (FUKU) allele), and null type (Kumachi-1 allele)—were characterized at this locus [31]. This QTL regulates the accumulation of glycitein isoflavones in the hypocotyl of soybean seeds. We performed the QTL analysis to find qMgly-11 with two different segregated populations. One population was obtained from a cross between FUKU and AOKI and another was obtained from a cross between FUKU and Kumachi-1. The closest DNA markers positions for qMgly-11 were in the 8.18 to 8.21 Mbp region of Gm11 (Figure S2). Complete co-segregation between marker genotypes and the null allele or accu- mulation of glyciten isoflavones phenotypes was observed [31]. We also performed a GWAS analysis of 158 soybean landrace lines. The strongest association was detected with a DNA marker located at 8.16 Mbp. These results showed that the same chromosomal region (qMgly-11 locus: 8.15 to 8.25 Mbp on Gm11) has significant association with the malonylglycitein content in soybean seeds [31]. Several genes and enzymes involved in glycitein isoflavone biosynthesis have been characterized [12,32], including three F6H genes (F6H1 to F6H3) and two IOMT genes (GmIOMT1 and GmIOMT2), but the relationship between qMgly-11 and these genes had not been elucidated until now. The qMgly-11 locus (8.15 to 8.25 Mbp) include 17 genes (Glyma.11G107200 to Glyma.11G109000) in the soybean genome database (Wm82.a4.v1). We examined the annotation information of these genes and Glyma.11G108300 (8.25 Mbp on Gm11), annotated as p450, showed homology with other F6H proteins. We considered this gene a reasonable a candidate of qMgly-11. In this study, we identified a mutant line (MUT1246) showing the same phenotype as Kumachi-1, with a null allele for glycitein isoflavones. We identified Glyma.11G108300 as a novel F6H gene (F6H4) underlying this QTL via gene structure analysis and mapping experiments. We further identified a novel mutant line, K01, show- ing peak shifts in high-pressure liquid chromatography (HPLC) corresponding to decreases in glycitin and malonylglycitin and increases in glycoside and malonylglycoside forms of 6-hydroxydaidzein. Mapping experiments revealed that a nonsense mutation in a novel IOMT (IOMT3) i i t d ith thi k hift h t The enzymes related to the isoflavones in legumes have been well elucidated (Figure S1), and their physical interactions among key enzymes have been studied [5]. Chalcone synthase (CHS) synthesizes naringenin chalcone from 4-coumaroyl-CoA and three units of malonyl-CoA. CHS, and chalcone reductase (CHR) also catalyze isoliquiritigenin synthesis [6,7]. Chalcone isomerase converts both chalcones (naringenin chalcone and isoliquiritigenin) into isoflavone aglycones (genistein and daidzein) with chalcone isomerase [5], isoflavone synthase (IFS [8,9]), and 2-hydroxyisoflavanone dehydratase (HID) sequentially [10]. Glycitein aglycone is syn- thesized from liquiritigenin by flavonoid 6-hydroxylase (F6H, a member of the cytochrome Copyright: © 2024 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/plants Plants 2024, 13, 156. https://doi.org/10.3390/plants13020156 Plants 2024, 13, 156 2 of 19 P450 family [11]), IFS, HID, and O-methyltransferase (OMT [12]). These aglycones are syn- thesized on the surface of the endoplasmic reticulum, and glycosyltransferases and malonyl- transferases then catalyze the synthesis of glycosides (daidzin, glycitin, and genistin) and malonylglycosides (malonyldaidzin, malonylglycitin, and malonylgenistin) [13,14]. Many QTL studies on isoflavone content have been conducted [15–27], and the soybase database (https://www.soybase.org/, accessed on 1 October 2023) lists at least 289 QTLs related to isoflavones. However, knowledge of the genes responsible for the genetic diversity of isoflavone content in soybean genetic diversity is still limited. Several genome-wide association study (GWAS) analyses of isoflavone content have been performed [28,29]. Although an association has been found between isoflavone content and single-nucleotide polymorphisms (SNPs) in IFS genes, detailed effects of these SNPs have not been elucidated [30]. GWAS analysis has been performed with over 6 million SNP data points and 1500 germplasm accessions mainly obtained from China to identify the association between SNPs and total isoflavone content or malonylglycitin content, resulting in the identification of several SNPs significantly associated with malonylglycitin content and one major QTL on chromosome 11 (Gm11) [28]. They found GWAS peaks in the region 8.25 to 8.26 on Gm11 under four environmental conditions and proposed one candidate gene (Glyma.11G108100, 8.23 Mbp of Gm11). However, there have been no additional experiments, such as transformation and screening of mutant lines, to reveal the function of their candidate genes. We have also identified a major QTL for mal- onylglycitein content, qMgly-11, in the same region [31]. 2.1. Gene Structure of Soybean Landrace Kumachi-1 F6H4 Gene 2.1. Gene Structure of Soybean Landrace Kumachi-1 F6H4 Gene To determine the gene structure of Glyma.11G108300 (F6H4) of Kumachi-1, PCR ampli- fication was attempted from the start codon to the stop codon. We could amplify the start of the second exon from FUKU but not from Kumachi-1 (Figure 1); therefore, we expected an insertion of DNA in F6H4 from Kumachi-1. The combination of long-read sequences from Nanopore and short reads of Illumina sequencing data revealed that a 3.8 kbp insertion Plants 2024, 13, 156 3 of 19 re, we g-read 3 of 19 re, we g-read was present in Kumachi-1 F6H4. The inserted sequence was highly similar to the sequence of the retrotransposon Tnt1 and had 670 bp terminal repeat sequences, which we could detect with PCR (Figure 1). The predicted amino acid sequence of Kumachi-1 F6H4 was truncated (80 aa) compared to the FUKU wild-type amino acid sequence (507 aa). This result indicates that Kumachi-1 has a null allele of F6H4 and cannot synthesize glycitein isoflavones in its seeds. kbp insertion was present in Kumachi-1 F6H4. The inserted sequence was highly similar to the sequence of the retrotransposon Tnt1 and had 670 bp terminal repeat sequences, which we could detect with PCR (Figure 1). The predicted amino acid sequence of Kuma- chi-1 F6H4 was truncated (80 aa) compared to the FUKU wild-type amino acid sequence (507 aa). This result indicates that Kumachi-1 has a null allele of F6H4 and cannot synthe- size glycitein isoﬂavones in its seeds. was present in Kumachi-1 F6H4. The inserted sequence was highly similar to the sequence of the retrotransposon Tnt1 and had 670 bp terminal repeat sequences, which we could detect with PCR (Figure 1). The predicted amino acid sequence of Kumachi-1 F6H4 was truncated (80 aa) compared to the FUKU wild-type amino acid sequence (507 aa). This result indicates that Kumachi-1 has a null allele of F6H4 and cannot synthesize glycitein isoflavones in its seeds. kbp insertion was present in Kumachi-1 F6H4. The inserted sequence was highly similar to the sequence of the retrotransposon Tnt1 and had 670 bp terminal repeat sequences, which we could detect with PCR (Figure 1). The predicted amino acid sequence of Kuma- chi-1 F6H4 was truncated (80 aa) compared to the FUKU wild-type amino acid sequence (507 aa). This result indicates that Kumachi-1 has a null allele of F6H4 and cannot synthe- size glycitein isoﬂavones in its seeds. 2.1. Gene Structure of Soybean Landrace Kumachi-1 F6H4 Gene Figure 1. Gene structure comparison between Fukuyutaka (wild-type allele) and Kumachi-1 (null allele: Mut type). (A) The gene structure of Glyma.11G108300 is shown with exons (black boxes), introns (lines), and 5′ and 3′ untranslated regions (gray boxes). The Kumachi-1 sequence contains an insertion of a Tnt1-like sequence with a long terminal repeat (LTR: narrow gray boxes). The phys- ical locations of the primer pairs (M1 and M2) are shown. The truncated Mut-type amino acid se- quence in Kumachi-1 allele with the insertion of Tnt1-like sequence is shown in a one-letter code with stop codon (asterisk). (B) Agarose gel electrophoresis of PCR products ampliﬁed with the M1 and M2 primer pairs. The far-left lanes contain a 100 bp size marker. 2 2 N l M t t Li f F6H4 G Figure 1. Gene structure comparison between Fukuyutaka (wild-type allele) and Kumachi-1 (null allele: Mut type). (A) The gene structure of Glyma.11G108300 is shown with exons (black boxes), introns (lines), and 5′ and 3′ untranslated regions (gray boxes). The Kumachi-1 sequence contains an insertion of a Tnt1-like sequence with a long terminal repeat (LTR: narrow gray boxes). The physical locations of the primer pairs (M1 and M2) are shown. The truncated Mut-type amino acid sequence in Kumachi-1 allele with the insertion of Tnt1-like sequence is shown in a one-letter code with stop codon (asterisk). (B) Agarose gel electrophoresis of PCR products amplified with the M1 and M2 primer pairs. The far-left lanes contain a 100 bp size marker. Figure 1. Gene structure comparison between Fukuyutaka (wild-type allele) and Kumachi-1 (null allele: Mut type). (A) The gene structure of Glyma.11G108300 is shown with exons (black boxes), introns (lines), and 5′ and 3′ untranslated regions (gray boxes). The Kumachi-1 sequence contains an insertion of a Tnt1-like sequence with a long terminal repeat (LTR: narrow gray boxes). The phys- ical locations of the primer pairs (M1 and M2) are shown. The truncated Mut-type amino acid se- quence in Kumachi-1 allele with the insertion of Tnt1-like sequence is shown in a one-letter code with stop codon (asterisk). (B) Agarose gel electrophoresis of PCR products ampliﬁed with the M1 and M2 primer pairs. The far-left lanes contain a 100 bp size marker. 2 2 No el Muta t Li e for F6H4 Ge e Figure 1. Gene structure comparison between Fukuyutaka (wild-type allele) and Kumachi-1 (null allele: Mut type). 2.1. Gene Structure of Soybean Landrace Kumachi-1 F6H4 Gene (A) The gene structure of Glyma.11G108300 is shown with exons (black boxes), introns (lines), and 5′ and 3′ untranslated regions (gray boxes). The Kumachi-1 sequence contains an insertion of a Tnt1-like sequence with a long terminal repeat (LTR: narrow gray boxes). The physical locations of the primer pairs (M1 and M2) are shown. The truncated Mut-type amino acid sequence in Kumachi-1 allele with the insertion of Tnt1-like sequence is shown in a one-letter code with stop codon (asterisk). (B) Agarose gel electrophoresis of PCR products amplified with the M1 and M2 primer pairs. The far-left lanes contain a 100 bp size marker. 2.2. Novel Mutant Line for F6H4 Gene We then looked for additional m 2.2. Novel Mutant Line for F6H4 Gene y y no glycitein isoﬂavones and identiﬁed a mutant line (MUT1246) with a phenotype similar to that of Kumachi-1 (Figure 2A). Direct sequencing identiﬁed an SNP (G407A) in F6H4 of MUT1246 (Figure 2B), which caused a non-synonymous substitution in the amino acid sequence (Cys136Tyr). We conﬁrmed the co-segregation of this mutation and the absence of glycitein isoﬂavones (glycitin and malonylglycitin) in the hypocotyls of a segregating population obtained from a cross between MUT1246 and TOYO (Figure 2C, Table S2). Co- segregation in two mapping populations, one obtained from a cross between Kumachi-1 and FUKU in a previous study [31] and the other obtained from a cross between MUT1246 and TOYO in this study, indicated that these mutations in F6H4 resulted in the absence of glycitein isoﬂavone in soybean seed hypocotyl parts. We then looked for additional mutant lines in the soybean mutant library that had no glycitein isoflavones and identified a mutant line (MUT1246) with a phenotype similar to that of Kumachi-1 (Figure 2A). Direct sequencing identified an SNP (G407A) in F6H4 of MUT1246 (Figure 2B), which caused a non-synonymous substitution in the amino acid sequence (Cys136Tyr). We confirmed the co-segregation of this mutation and the absence of glycitein isoflavones (glycitin and malonylglycitin) in the hypocotyls of a segregating population obtained from a cross between MUT1246 and TOYO (Figure 2C, Table S2). Co-segregation in two mapping populations, one obtained from a cross between Kumachi-1 and FUKU in a previous study [31] and the other obtained from a cross between MUT1246 and TOYO in this study, indicated that these mutations in F6H4 resulted in the absence of glycitein isoflavone in soybean seed hypocotyl parts. Plants 2024, 13, 156 Plants 2024, 13, x FOR 4 of 19 4 of 19 Figure 2. Identiﬁcation of novel glycitein isoﬂavone-deﬁcient mutants and the responsible genes. (A) Representative chromatograms of wild-type and MUT1246 mutant lines, with the latter showing a null phenotype for glycitein isoﬂavones (peak 2 and 5), as in Kumachi-1. The number of peaks are as follow, 1: daidzein, 2: glycitin, 3: genistin, 4: malonyldaidzin, 5: malonylglycition, 6: malonylgen- istin. (B) Predicted C136Y amino acid substitution in Glyma.11G108300 (F6H4) of MUT1246 and the causal SNP with its position from the start codon of the gene. 2.2. Novel Mutant Line for F6H4 Gene We then looked for additional m 2.2. Novel Mutant Line for F6H4 Gene (B) Predicted C136Y amino acid substitution in Glyma.11G108300 (F6H4) of MUT1246 and the causal SNP with its position from the start codon of the gene. (C) Mapping experiment using a cross between MUT1246 and TOYO showing co-segregation between the SNP genotype of G407A (resulting in amino acid substitution by C136Y) and the glycitein isoflavone phenotype. The mutant homozygous genotype (MUT_homo) showed complete absence of malonylglycitin in the hypocotyls. 2.2. Novel Mutant Line for F6H4 Gene We then looked for additional m 2.2. Novel Mutant Line for F6H4 Gene (C) Mapping experiment using a cross between MUT1246 and TOYO showing co-segregation between the SNP genotype of G407A (result- ing in amino acid substitution by C136Y) and the glycitein isoﬂavone phenotype. The mutant ho- mozygous genotype (MUT_homo) showed complete absence of malonylglycitin in the hypocotyls. Figure 2. Identification of novel glycitein isoflavone-deficient mutants and the responsible genes. (A) Representative chromatograms of wild-type and MUT1246 mutant lines, with the latter showing a null phenotype for glycitein isoflavones (peak 2 and 5), as in Kumachi-1. The number of peaks are as follow, 1: daidzein, 2: glycitin, 3: genistin, 4: malonyldaidzin, 5: malonylglycition, 6: malonylgenistin. (B) Predicted C136Y amino acid substitution in Glyma.11G108300 (F6H4) of MUT1246 and the causal SNP with its position from the start codon of the gene. (C) Mapping experiment using a cross between MUT1246 and TOYO showing co-segregation between the SNP genotype of G407A (resulting in amino acid substitution by C136Y) and the glycitein isoflavone phenotype. The mutant homozygous genotype (MUT_homo) showed complete absence of malonylglycitin in the hypocotyls. Fi 2 Id tiﬁ ti f l l it i i ﬂ d ﬁi t t t d th ibl Figure 2. Identification of novel glycitein isoflavone-deficient mutants and the responsible genes. Figure 2. Identiﬁcation of novel glycitein isoﬂavone-deﬁcient mutants and the responsible genes. (A) Representative chromatograms of wild-type and MUT1246 mutant lines, with the latter showing a null phenotype for glycitein isoﬂavones (peak 2 and 5), as in Kumachi-1. The number of peaks are as follow, 1: daidzein, 2: glycitin, 3: genistin, 4: malonyldaidzin, 5: malonylglycition, 6: malonylgen- istin. (B) Predicted C136Y amino acid substitution in Glyma.11G108300 (F6H4) of MUT1246 and the causal SNP with its position from the start codon of the gene. (C) Mapping experiment using a cross between MUT1246 and TOYO showing co-segregation between the SNP genotype of G407A (result- ing in amino acid substitution by C136Y) and the glycitein isoﬂavone phenotype. The mutant ho- mozygous genotype (MUT_homo) showed complete absence of malonylglycitin in the hypocotyls. Figure 2. Identification of novel glycitein isoflavone-deficient mutants and the responsible genes. (A) Representative chromatograms of wild-type and MUT1246 mutant lines, with the latter showing a null phenotype for glycitein isoflavones (peak 2 and 5), as in Kumachi-1. The number of peaks are as follow, 1: daidzein, 2: glycitin, 3: genistin, 4: malonyldaidzin, 5: malonylglycition, 6: malonylgenistin. 2.2. Novel Mutant Line for F6H4 Gene We then looked for additional m 2.2. Novel Mutant Line for F6H4 Gene (B) Predicted C136Y amino acid substitution in Glyma.11G108300 (F6H4) of MUT1246 and the causal SNP with its position from the start codon of the gene. (C) Mapping experiment using a cross between MUT1246 and TOYO showing co-segregation between the SNP genotype of G407A (resulting in amino acid substitution by C136Y) and the glycitein isoflavone phenotype. The mutant homozygous genotype (MUT_homo) showed complete absence of malonylglycitin in the hypocotyls. Figure 2. Identiﬁcation of novel glycitein isoﬂavone-deﬁcient mutants and the responsible genes. (A) Representative chromatograms of wild-type and MUT1246 mutant lines, with the latter showing a null phenotype for glycitein isoﬂavones (peak 2 and 5), as in Kumachi-1. The number of peaks are as follow, 1: daidzein, 2: glycitin, 3: genistin, 4: malonyldaidzin, 5: malonylglycition, 6: malonylgen- istin. (B) Predicted C136Y amino acid substitution in Glyma.11G108300 (F6H4) of MUT1246 and the causal SNP with its position from the start codon of the gene. (C) Mapping experiment using a cross between MUT1246 and TOYO showing co-segregation between the SNP genotype of G407A (result- ing in amino acid substitution by C136Y) and the glycitein isoﬂavone phenotype. The mutant ho- mozygous genotype (MUT_homo) showed complete absence of malonylglycitin in the hypocotyls. Figure 2. Identification of novel glycitein isoflavone-deficient mutants and the responsible genes. (A) Representative chromatograms of wild-type and MUT1246 mutant lines, with the latter showing a null phenotype for glycitein isoflavones (peak 2 and 5), as in Kumachi-1. The number of peaks are as follow, 1: daidzein, 2: glycitin, 3: genistin, 4: malonyldaidzin, 5: malonylglycition, 6: malonylgenistin. (B) Predicted C136Y amino acid substitution in Glyma.11G108300 (F6H4) of MUT1246 and the causal SNP with its position from the start codon of the gene. (C) Mapping experiment using a cross between MUT1246 and TOYO showing co-segregation between the SNP genotype of G407A (resulting in amino acid substitution by C136Y) and the glycitein isoflavone phenotype. The mutant homozygous genotype (MUT_homo) showed complete absence of malonylglycitin in the hypocotyls. istin. (B) Predicted C136Y amino acid substitution in Glyma.11G108300 (F6H4) of MUT1246 and the causal SNP with its position from the start codon of the gene. (C) Mapping experiment using a cross between MUT1246 and TOYO showing co-segregation between the SNP genotype of G407A (result- ing in amino acid substitution by C136Y) and the glycitein isoﬂavone phenotype. The mutant ho- mozygous genotype (MUT_homo) showed complete absence of malonylglycitin in the hypocotyls. 4. Model of F6H4 Protein Structure 2.4. Model of F6H4 Protein Structure 4. Model of F6H4 Protein Structure The three-dimensional structure of F6H4 (AOKI type) with heme was modeled as entioned in the materials and methods. The overall predicted structure is shown in Fig- re 4. Most plant cytochrome P450s are expressed on endoplasmic reticulum and are nown as microsomal P450s; they have a transmembrane anchor at the N-terminus at- ched to the endoplasmic reticulum. The predicted structure of F6H4 had a long α-helix the N-terminus, suggesting an anchor to the endoplasmic reticulum. The top view howed a triangular overall structure, which is characteristic of cytochrome P450s. The eme iron was co-ordinated by Cys446, and one propionate of the heme was located at a ydrogen-bond distance from arginine 444, which is conserved in all cytochrome P450s. hese observations conﬁrmed that the proposed structure was plausible. Next, we at- mpted to calculate the substrate-binding structures. Unfortunately, no reasonable struc- res with substrates were established by docking simulations. The structure built by Al- haFill can predict the position of not only heme but also of substrates registered in the rotein data bank. We obtained a model structure of F6H4 with 4-phenyl-1H-imidazole PI), which was used as a substrate analog in some P450 crystallographic studies [34,35]. he distance between the hydroxylated carbon of the substrate and the iron ion in the The three-dimensional structure of F6H4 (AOKI type) with heme was modeled as mentioned in the materials and methods. The overall predicted structure is shown in Figure 4. Most plant cytochrome P450s are expressed on endoplasmic reticulum and are known as microsomal P450s; they have a transmembrane anchor at the N-terminus attached to the endoplasmic reticulum. The predicted structure of F6H4 had a long α-helix at the N-terminus, suggesting an anchor to the endoplasmic reticulum. The top view showed a triangular overall structure, which is characteristic of cytochrome P450s. The heme iron was co-ordinated by Cys446, and one propionate of the heme was located at a hydrogen-bond distance from arginine 444, which is conserved in all cytochrome P450s. These observations confirmed that the proposed structure was plausible. Next, we attempted to calculate the substrate-binding structures. Unfortunately, no reasonable structures with substrates were established by docking simulations. The structure built by AlphaFill can predict the position of not only heme but also of substrates registered in the protein data bank. 2.3. Association Study with Soybean Mini‐Core Collection 2.3. Association Study with Soybean Mini-Core Collection Plants 2024, 13, 156 ants 2024, 13, x FOR P 5 of 19 of 19 igure 3. Interactive eﬀect between SNPs in Glyma.11G108300 identiﬁed in an association study sing the soybean mini-core collection. The positions of the SNPs causing amino acid substitutions re shown above the gene map. Dotted vertical lines show positions of SNPs between AOKI and UKU. Non-synonymous amino acid substitutions are shown below the gene structure. Box plots how the amount of glycitein isoﬂavones produced by each genotype based on the amino acid sub- itutions encoded by Glyma.11G108300. Genotypes AOK-AOK, AOK-FUKU, FUKU-AOK, and UKU-FUKU encode amino acid residues 203A and 248T, 203A and 248A, 203S and 248T, and 203S nd 248A, respectively. The number of individuals with each genotype is shown in parentheses. Figure 3. Interactive effect between SNPs in Glyma.11G108300 identified in an association study using the soybean mini-core collection. The positions of the SNPs causing amino acid substitutions are shown above the gene map. Dotted vertical lines show positions of SNPs between AOKI and FUKU. Non-synonymous amino acid substitutions are shown below the gene structure. Box plots show the amount of glycitein isoflavones produced by each genotype based on the amino acid substitutions encoded by Glyma.11G108300. Genotypes AOK-AOK, AOK-FUKU, FUKU-AOK, and FUKU-FUKU encode amino acid residues 203A and 248T, 203A and 248A, 203S and 248T, and 203S and 248A, respectively. The number of individuals with each genotype is shown in parentheses. 2.3. Association Study with Soybean Mini‐Core Collection 2.3. Association Study with Soybean Mini-Core Collection y y We previously found a QTL for glycitein isoﬂavone content in a population obtained from a cross between AOKI and FUKU [31]. The AOKI allele of qMgly‐11 can increase glycitein content compared to that produced by the FUKU allele. If we assume that the gene underlying qMgly‐11 is F6H4 (Glyma.11G108300), there is an explanation for the func- tional diﬀerence between the AOKI and FUKU alleles. These two lines had amino acid substitutions in F6H4 (A66T, T127I, A203S, and T248A, caused by SNPs G196A, C410T, G607T, and A742G, respectively). We performed association mapping with the soybean mini-core collection [33] to identify the amino acid substitutions responsible for the func- tional diﬀerence between the two alleles and found a signiﬁcant association of A203S and T248A with glycitein isoﬂavone content (Figure S3). Linear regression analysis with both alleles in a single model that considered their interaction revealed that T248A had a sig- niﬁcant association with the phenotype (p < 0.001) but that A203S did not (t-value = 0.18, p = 0.86, Figure 3). Mapping experiments with MUT1246 and association mapping using the soybean mini-core collection revealed that the C136Y and T248A substitutions aﬀected the enzymatic activity of F6H4 We previously found a QTL for glycitein isoflavone content in a population obtained from a cross between AOKI and FUKU [31]. The AOKI allele of qMgly-11 can increase glycitein content compared to that produced by the FUKU allele. If we assume that the gene underlying qMgly-11 is F6H4 (Glyma.11G108300), there is an explanation for the functional difference between the AOKI and FUKU alleles. These two lines had amino acid substitutions in F6H4 (A66T, T127I, A203S, and T248A, caused by SNPs G196A, C410T, G607T, and A742G, respectively). We performed association mapping with the soybean mini-core collection [33] to identify the amino acid substitutions responsible for the functional difference between the two alleles and found a significant association of A203S and T248A with glycitein isoflavone content (Figure S3). Linear regression analysis with both alleles in a single model that considered their interaction revealed that T248A had a significant association with the phenotype (p < 0.001) but that A203S did not (t-value = 0.18, p = 0.86, Figure 3). Mapping experiments with MUT1246 and association mapping using the soybean mini-core collection revealed that the C136Y and T248A substitutions affected the enzymatic activity of F6H4. 4. Model of F6H4 Protein Structure 2.4. Model of F6H4 Protein Structure We obtained a model structure of F6H4 with 4-phenyl-1H-imidazole (4PI), which was used as a substrate analog in some P450 crystallographic studies [34,35]. The distance between the hydroxylated carbon of the substrate and the iron ion in the heme is less Plants 2024, 13, 156 Plants 2024, 13, x FO 6 of 19 of 19 than 4.5 Å, according to reported crystal structures of P450s [36]. Thus, the substrate for F6H4, liquiritigenin, was placed with this constraint based on the model structure with 4PI (Figure 4 and Figure S4). Importantly, the hydroxyl (4′-OH) on the side opposite the hydroxylation in liquiritigenin was located at a hydrogen-bond distance of 3.5 Å from threonine 248 in the Aokimame type, which was mutated to alanine in the FUKU type (Figure 4A). In addition, our predicted model revealed that cysteine 136 was located near the heme and formed a part of the heme pocket. Therefore, the C136Y and T248A mutations may be related to F6H4 enzymatic activity. heme is less than 4.5 Å, according to reported crystal structures of P450s [36]. Thus, the substrate for F6H4, liquiritigenin, was placed with this constraint based on the model structure with 4PI (Figures 4 and S4). Importantly, the hydroxyl (4′-OH) on the side op- posite the hydroxylation in liquiritigenin was located at a hydrogen-bond distance of 3.5 Å from threonine 248 in the Aokimame type, which was mutated to alanine in the FUKU type (Figure 4A). In addition, our predicted model revealed that cysteine 136 was located near the heme and formed a part of the heme pocket. Therefore, the C136Y and T248A mutations may be related to F6H4 enzymatic activity. Figure 4. Modeled protein structure of F6H4 (encoded by Glyma.11G108300) with two amino acid substitutions causing null (136Y) and active (248T) proteins. (A) Overall structure of F6H4 (Aokimame type). The protein structure was modeled using AlphaFold2 and the position of heme was predicted using the AlphaFill program. (B) Active-site pocket of the modeled structure. The position of the substrate was provided by AlphaFill (Figure S4). The substrate for F6H4, liquiriti- genin, was placed at the same position as the substrate (4-phenyl-1H-imidazole) in the modeled structure. The distances between 6C of the substrate and heme iron, and between 4′-OH of the sub- strate and 3-OH of threonine, were 4.5 and 3.5 Å, respectively. The heme, liquiritigenin, and amino acid side chains are shown as sticks. 4. Model of F6H4 Protein Structure 2.4. Model of F6H4 Protein Structure The protein structure was modeled using AlphaFold2 and the position of heme was predicted using the AlphaFill program. (B) Active-site pocket of the modeled structure. The position of the substrate was provided by AlphaFill (Figure S4). The substrate for F6H4, liquiritigenin, was placed at the same position as the substrate (4-phenyl-1H-imidazole) in the modeled structure. The distances between 6C of the substrate and heme iron, and between 4′-OH of the substrate and 3-OH of threonine, were 4.5 and 3.5 Å, respectively. The heme, liquiritigenin, and amino acid side chains are shown as sticks. The iron ions are shown as spheres. The carbon atoms of F6H4, Cys446, heme, and liquiritigenin are colored as aquamarine, green, gray, and orange, respectively. Amino acids Cys136 and Thr248 are colored in red. 4. Model of F6H4 Protein Structure 2.4. Model of F6H4 Protein Structure The iron ions are shown as spheres. The carbon atoms of F6H4, Cys446, heme, and liquiritigenin are colored as aquamarine, green, gray, and orange, respectively. Amino acids Cys136 and Thr248 are colored in red. 2.5. Expression Levels of F6H4 during Soybean Seed Development t Figure 4. Modeled protein structure of F6H4 (encoded by Glyma.11G108300) with two amino acid substitutions causing null (136Y) and active (248T) proteins. (A) Overall structure of F6H4 (Aokimame type). The protein structure was modeled using AlphaFold2 and the position of heme was predicted using the AlphaFill program. (B) Active-site pocket of the modeled structure. The position of the substrate was provided by AlphaFill (Figure S4). The substrate for F6H4, liquiritigenin, was placed at the same position as the substrate (4-phenyl-1H-imidazole) in the modeled structure. The distances between 6C of the substrate and heme iron, and between 4′-OH of the substrate and 3-OH of threonine, were 4.5 and 3.5 Å, respectively. The heme, liquiritigenin, and amino acid side chains are shown as sticks. The iron ions are shown as spheres. The carbon atoms of F6H4, Cys446, heme, and liquiritigenin are colored as aquamarine, green, gray, and orange, respectively. Amino acids Cys136 and Thr248 are colored in red. Figure 4. Modeled protein structure of F6H4 (encoded by Glyma.11G108300) with two amino acid substitutions causing null (136Y) and active (248T) proteins. (A) Overall structure of F6H4 (Aokimame type). The protein structure was modeled using AlphaFold2 and the position of heme was predicted using the AlphaFill program. (B) Active-site pocket of the modeled structure. The position of the substrate was provided by AlphaFill (Figure S4). The substrate for F6H4, liquiriti- genin, was placed at the same position as the substrate (4-phenyl-1H-imidazole) in the modeled structure. The distances between 6C of the substrate and heme iron, and between 4′-OH of the sub- strate and 3-OH of threonine, were 4.5 and 3.5 Å, respectively. The heme, liquiritigenin, and amino acid side chains are shown as sticks. The iron ions are shown as spheres. The carbon atoms of F6H4, Cys446, heme, and liquiritigenin are colored as aquamarine, green, gray, and orange, respectively. Amino acids Cys136 and Thr248 are colored in red. 2.5. Expression Levels of F6H4 during Soybean Seed Development Figure 4. Modeled protein structure of F6H4 (encoded by Glyma.11G108300) with two amino acid substitutions causing null (136Y) and active (248T) proteins. (A) Overall structure of F6H4 (Aokimame type). We compared the glycitein isoﬂavone accumulation patte Glyma 11G108300 (F6H4) in developing soybean seeds (Figur 2.5. Expression Levels of F6H4 during Soybean Seed Development Glyma.11G108300 (F6H4) in developing soybean seeds (Figure 5A). Glycitein isoﬂavone levels were very low in the cotyledons, and the expression of F6H4 in the cotyledons was also low at all sampling stages (I to VII; Figure 5B,C). In contrast, glycitein isoﬂavones accumulated in the hypocotyl (here, including the plumule, epicotyl, hypocotyl, and rad- icle) of soybean seeds during stages II to VI. The expression pattern of F6H4 corresponded to the accumulation pattern of glycitein isoﬂavones. The highest level of glycitein isoﬂa- vone appeared at stage VI, two stages (>10 days) after the peak of gene expression of F6H4. The expression of F6H4 increased, starting at stage II, peaked at stage IV, and then de- creased until stage VII (Figure 5C). The association between the accumulation pattern of glycitein isoﬂavones and the expression proﬁle of F6H4 suggests that this gene is involved in glycitein isoﬂavone synthesis. We compared the glycitein isoflavone accumulation pattern and expression profile of Glyma.11G108300 (F6H4) in developing soybean seeds (Figure 5A). Glycitein isoflavone levels were very low in the cotyledons, and the expression of F6H4 in the cotyledons was also low at all sampling stages (I to VII; Figure 5B,C). In contrast, glycitein isoflavones accumulated in the hypocotyl (here, including the plumule, epicotyl, hypocotyl, and radicle) of soybean seeds during stages II to VI. The expression pattern of F6H4 corresponded to the accumulation pattern of glycitein isoflavones. The highest level of glycitein isoflavone appeared at stage VI, two stages (>10 days) after the peak of gene expression of F6H4. The expression of F6H4 increased, starting at stage II, peaked at stage IV, and then decreased until stage VII (Figure 5C). The association between the accumulation pattern of glycitein isoflavones and the expression profile of F6H4 suggests that this gene is involved in glycitein isoflavone synthesis. Plants 2024, 13, 156 Plants 2024, 13, x FOR 7 of 19 7 of 19 Figure 5. Accumulation of glycitein isoﬂavones during soybean seed development and tissue-spe- ciﬁc expression of Glyma.11G108300. (A) Representative staging of seeds used for isoﬂavone extrac- tion and expression analysis. Hypocotyl parts (plumule, epicotyl, hypocotyl, and radicle) were ex- cised from the cotyledons, except at stage I, and analyzed separately. Separated hypocotyls are shown for stages VI and VII. (B) Isoﬂavone accumulation patterns in cotyledons and hypocotyls during soybean seed development. Each color indicates daidzein (white), genistein (gray), and glycitein isoﬂavones (black). We compared the glycitein isoﬂavone accumulation patte Glyma 11G108300 (F6H4) in developing soybean seeds (Figur 2.5. Expression Levels of F6H4 during Soybean Seed Development (C) Glyma.11G108300 expression proﬁle during soybean seed develop- ment. Relative expression of Glyma.11G108300/ELFa was evaluated with standard deviation. Figure 5. Accumulation of glycitein isoflavones during soybean seed development and tissue-specific expression of Glyma.11G108300. (A) Representative staging of seeds used for isoflavone extraction and expression analysis. Hypocotyl parts (plumule, epicotyl, hypocotyl, and radicle) were excised from the cotyledons, except at stage I, and analyzed separately. Separated hypocotyls are shown for stages VI and VII. (B) Isoflavone accumulation patterns in cotyledons and hypocotyls during soybean seed development. Each color indicates daidzein (white), genistein (gray), and glycitein isoflavones (black). (C) Glyma.11G108300 expression profile during soybean seed development. Relative expression of Glyma.11G108300/ELFa was evaluated with standard deviation. Figure 5. Accumulation of glycitein isoﬂavones during soybean seed development and tissue-spe- ciﬁc expression of Glyma.11G108300. (A) Representative staging of seeds used for isoﬂavone extrac- tion and expression analysis. Hypocotyl parts (plumule, epicotyl, hypocotyl, and radicle) were ex- cised from the cotyledons, except at stage I, and analyzed separately. Separated hypocotyls are shown for stages VI and VII. (B) Isoﬂavone accumulation patterns in cotyledons and hypocotyls during soybean seed development. Each color indicates daidzein (white), genistein (gray), and glycitein isoﬂavones (black). (C) Glyma.11G108300 expression proﬁle during soybean seed develop- ment. Relative expression of Glyma.11G108300/ELFa was evaluated with standard deviation. Figure 5. Accumulation of glycitein isoflavones during soybean seed development and tissue-specific expression of Glyma.11G108300. (A) Representative staging of seeds used for isoflavone extraction and expression analysis. Hypocotyl parts (plumule, epicotyl, hypocotyl, and radicle) were excised from the cotyledons, except at stage I, and analyzed separately. Separated hypocotyls are shown for stages VI and VII. (B) Isoflavone accumulation patterns in cotyledons and hypocotyls during soybean seed development. Each color indicates daidzein (white), genistein (gray), and glycitein isoflavones (black). (C) Glyma.11G108300 expression profile during soybean seed development. Relative expression of Glyma.11G108300/ELFa was evaluated with standard deviation. 2.7. Identiﬁcation of Novel Mutant Line for Glycitein Isoﬂavones 2.7. Identification of Novel Mutant Line for Glycitein Isoflavones During mutant line screening, we also identiﬁed a unique mutant line (K01) that showed a change in the HPLC retention time of glycitein isoﬂavones. This line showed a shift in the peak positions on the chromatogram: speciﬁcally, the glycitin and malonyl- glycitin peaks were replaced by peaks with an earlier retention time compared with the wild type (Figure 7). We mapped the mutated gene in K01 by using a population obtained from a cross between TOYO and K01. Selective genotyping analysis identiﬁed several DNA markers, located in the 1.0 to 4.0 Mbp region on Gm01, that were linked to the mu- tant phenotype. Further QTL analysis with all individuals in the population identiﬁed a QTL peak at the 0.43 Mbp position of Gm01 (Figure 8A). The marker genotypes at this position (13cM at P1_430457) coincided with the glycitein accumulation pattern in hypo- cotyl, and mutant (FUKU genetic background) homozygous plants showed alterations in content from high malonylglycitin to high malonylglycoside forms of 6-hydroxydaidzein (Figure 8B, Table S3). Glycitein biosynthesis from liquiritigenin is catalyzed by F6H, IFS, and IOMT via 6-hydroxyliquiritigenin and 6-hydroxydaidzein (Figure S1). Among these enzymes, IFS is essential for the synthesis of genistein and daidzein, and K01 shows no diﬀerence in genistein- (genistin and malonylgenistin) and daidzein- (daidzin and malo- nyldaidzin) related isoﬂavones content compared to FUKU. Therefore, we assumed that a mutation in IOMT was a candidate for the K01 mutated phenotype During mutant line screening, we also identified a unique mutant line (K01) that showed a change in the HPLC retention time of glycitein isoflavones. This line showed a shift in the peak positions on the chromatogram: specifically, the glycitin and malonyl- glycitin peaks were replaced by peaks with an earlier retention time compared with the wild type (Figure 7). We mapped the mutated gene in K01 by using a population obtained from a cross between TOYO and K01. Selective genotyping analysis identified several DNA markers, located in the 1.0 to 4.0 Mbp region on Gm01, that were linked to the mutant phenotype. Further QTL analysis with all individuals in the population identified a QTL peak at the 0.43 Mbp position of Gm01 (Figure 8A). The marker genotypes at this position (13cM at P1_430457) coincided with the glycitein accumulation pattern in hypocotyl, and mutant (FUKU genetic background) homozygous plants showed alterations in content from high malonylglycitin to high malonylglycoside forms of 6-hydroxydaidzein (Figure 8B, Table S3). 2.6. Phylogenetic Analysis of F6H Amino Acids Sequences Se e al F6H e e (F6H1 Gly a 18G080400 i W 8 2.6. Phylogenetic Analysis of F6H Amino Acids Sequences Phylogenetic tree of homologous amino acids sequences for ﬂavonoid 6-hydroxylase in soybean and two other legumes, Lotus japonicus and Medicago truncatula. The MEGA-X program with the neighbor-joining method were used for the analysis. Figure 6. Phylogenetic tree of homologous amino acids sequences for flavonoid 6-hydroxylase in soybean and two other legumes, Lotus japonicus and Medicago truncatula. The MEGA-X program with the neighbor-joining method were used for the analysis. 2.6. Phylogenetic Analysis of F6H Amino Acids Sequences Se e al F6H e e (F6H1 Gly a 18G080400 i W 8 2.6. Phylogenetic Analysis of F6H Amino Acids Sequences Several F6H genes (F6H1: Glyma.18G080400 in Wm82.a1.v2; F6H2: Glyma.18G080200; and F6H3: Glyma.08G326900) have been characterized [32]. We compared the predicted amino acid sequences of F6H4 and these other soybean F6H proteins. A phylogenetic tree of homologs from soybean, Lotus japonicus, and Medicago truncatula F6H amino acid se- quences showed F6H1 to F6H3 in one clade and F6H4 in another (Figure 6). The diﬀeren- tiation of two types of F6H proteins appeared to be conserved in legume plant species (Figure 6). The amino acid sequence identity and similarity between the predicted product of Glyma.11G108300 and that of the well-characterized F6H1 amino acids sequence (Glyma.18G080400) were 33% (168/505 aa) and 52% (264/505 aa). Within the soybean ge- nome, F6H1 has at least two homologs, F6H2 and F6H3, and Glyma.11G108300 (F6H4) also has at least two homologs, Glyma.10G203500 and Glyma.20G14800. These results indicate that at least two genes related to glycitein isoﬂavone biosynthesis evolved independently. Several F6H genes (F6H1: Glyma.18G080400 in Wm82.a1.v2; F6H2: Glyma.18G080200; and F6H3: Glyma.08G326900) have been characterized [32]. We compared the predicted amino acid sequences of F6H4 and these other soybean F6H proteins. A phylogenetic tree of homologs from soybean, Lotus japonicus, and Medicago truncatula F6H amino acid sequences showed F6H1 to F6H3 in one clade and F6H4 in another (Figure 6). The differ- entiation of two types of F6H proteins appeared to be conserved in legume plant species (Figure 6). The amino acid sequence identity and similarity between the predicted prod- uct of Glyma.11G108300 and that of the well-characterized F6H1 amino acids sequence (Glyma.18G080400) were 33% (168/505 aa) and 52% (264/505 aa). Within the soybean genome, F6H1 has at least two homologs, F6H2 and F6H3, and Glyma.11G108300 (F6H4) also has at least two homologs, Glyma.10G203500 and Glyma.20G14800. These results indicate that at least two genes related to glycitein isoflavone biosynthesis evolved independently. Plants 2024, 13, 156 Plants 2024, 13, x FOR 8 of 19 8 of 19 Figure 6. Phylogenetic tree of homologous amino acids sequences for ﬂavonoid 6-hydroxylase in soybean and two other legumes, Lotus japonicus and Medicago truncatula. The MEGA-X program with the neighbor-joining method were used for the analysis. Figure 6. Phylogenetic tree of homologous amino acids sequences for flavonoid 6-hydroxylase in soybean and two other legumes, Lotus japonicus and Medicago truncatula. The MEGA-X program with the neighbor-joining method were used for the analysis. Figure 6. 2.7. Identiﬁcation of Novel Mutant Line for Glycitein Isoﬂavones 2.7. Identification of Novel Mutant Line for Glycitein Isoflavones Glycitein biosynthesis from liquiritigenin is catalyzed by F6H, IFS, and IOMT via 6-hydroxyliquiritigenin and 6-hydroxydaidzein (Figure S1). Among these enzymes, IFS is essential for the synthesis of genistein and daidzein, and K01 shows no difference in genistein- (genistin and malonylgenistin) and daidzein- (daidzin and malonyldaidzin) related isoflavones content compared to FUKU. Therefore, we assumed that a mutation in IOMT was a candidate for the K01 mutated phenotype. Plants 2024, 13, 156 Plants 2024, 13, x FOR P 9 of 19 of 19 Figure 7. Identiﬁcation of a novel glycitein isoﬂavone peak-shift mutant. (A) Representative HPLC chromatograms of isoﬂavones extracted from hypocotyl tissue of the mutant line (K01) and wild type (Fukuyutaka). Peaks in common (white arrows) and diﬀering between the two lines (black arrows) are shown (B) Chemical structures of daidzein 6 hydroxydaidzein and glycitein Figure 7. Identification of a novel glycitein isoflavone peak-shift mutant. (A) Representative HPLC chromatograms of isoflavones extracted from hypocotyl tissue of the mutant line (K01) and wild type (Fukuyutaka). Peaks in common (white arrows) and differing between the two lines (black arrows) are shown. (B) Chemical structures of daidzein, 6-hydroxydaidzein, and glycitein. Figure 7. Identiﬁcation of a novel glycitein isoﬂavone peak-shift mutant. (A) Representative HPLC chromatograms of isoﬂavones extracted from hypocotyl tissue of the mutant line (K01) and wild type (Fukuyutaka). Peaks in common (white arrows) and diﬀering between the two lines (black arrows) are shown. (B) Chemical structures of daidzein, 6-hydroxydaidzein, and glycitein. Figure 7. Identiﬁcation of a novel glycitein isoﬂavone peak-shift mutant. (A) Representative HPLC chromatograms of isoﬂavones extracted from hypocotyl tissue of the mutant line (K01) and wild type (Fukuyutaka). Peaks in common (white arrows) and diﬀering between the two lines (black ) h (B) Ch l f d d 6 h d d d d l Figure 7. Identification of a novel glycitein isoflavone peak-shift mutant. (A) Representative HPLC chromatograms of isoflavones extracted from hypocotyl tissue of the mutant line (K01) and wild type (Fukuyutaka). Peaks in common (white arrows) and differing between the two lines (black arrows) are shown. (B) Chemical structures of daidzein, 6-hydroxydaidzein, and glycitein. Figure 7. Identiﬁcation of a novel glycitein isoﬂavone peak-shift mutant. (A) Representative HPLC chromatograms of isoﬂavones extracted from hypocotyl tissue of the mutant line (K01) and wild type (Fukuyutaka). Peaks in common (white arrows) and diﬀering between the two lines (black arrows) are shown. (B) Chemical structures of daidzein, 6-hydroxydaidzein, and glycitein. 2.7. Identiﬁcation of Novel Mutant Line for Glycitein Isoﬂavones 2.7. Identification of Novel Mutant Line for Glycitein Isoflavones a o ) a e o ( ) e i a u u e Figure 8. Mapping of the K01 mutated gene and location of premature stop codon. (A) Linkage map and QTL analysis of malonylglycitin content in a mapping population derived from Toyoshirome Figure 8. Mapping of the K01 mutated gene and location of premature stop codon. (A) Linkage map and QTL analysis of malonylglycitin content in a mapping population derived from Toyoshirome Figure 8. Mapping of the K01 mutated gene and location of premature stop codon. (A) Linkage map and QTL analysis of malonylglycitin content in a mapping population derived from Toyoshirome and K01. (B) Mapping experiments showing co-segregation between SNPs detected in Glyma.01G004200, malonylglycitin (black), and malonylglycoside forms of 6-hydroxydaidzein. (gray). (C) The predicted length of the peptide encoded by Glyma.01G004200 and the position of the truncation in K01. Figure 8. Mapping of the K01 mutated gene and location of premature stop codon. (A) Linkage map and QTL analysis of malonylglycitin content in a mapping population derived from Toyoshirome Figure 8. Mapping of the K01 mutated gene and location of premature stop codon. (A) Linkage map and QTL analysis of malonylglycitin content in a mapping population derived from Toyoshirome Figure 8. Mapping of the K01 mutated gene and location of premature stop codon. (A) Linkage map and QTL analysis of malonylglycitin content in a mapping population derived from Toyoshirome and K01. (B) Mapping experiments showing co-segregation between SNPs detected in Glyma.01G004200, malonylglycitin (black), and malonylglycoside forms of 6-hydroxydaidzein. (gray). (C) The predicted length of the peptide encoded by Glyma.01G004200 and the position of the truncation in K01. Plants 2024, 13, 156 10 of 19 zein. on of 10 of 19 zein. on of A survey of SNPs between K01 and FUKU with whole-genome NGS analysis revealed 16 SNPs in the 1.0 to 3.0 Mbp region on Gm01. Among these SNPs, an allele of Gm01_430457 caused a stop codon mutation in Glyma.01G004200 (238 aa; Figure 8C), annotated as methyltransferase in the soybean genome database (Wm82.a2.v1). In K01, the guanine nucleotide at position 554 was changed to adenine, changing the codon for tryptophan (TGG) to a stop codon (TAG) at the 185-aa position. We also analyzed the molecular weight of the K01-type aglycone after acid hydrolysis of isoflavones extracted from K01 seeds. 2.7. Identiﬁcation of Novel Mutant Line for Glycitein Isoﬂavones 2.7. Identification of Novel Mutant Line for Glycitein Isoflavones The retention time of the K01-type aglycone coincided with that of 6-hydroxydaidzein (6-HD, Figure S5), and mass-to-charge ratios (m/z) of glycoside and malonylglycoside forms of 6-hydroxydaidzein were identical to those of the shifted peaks in K01 (Figures S6 and S7). These results—a nonsense mutation in the candidate gene (IOMT) of K01 and 6-HD isoflavone accumulation in K01—indicate that the product of a novel IOMT (Glyma.01G004200) gene, hereafter designated IOMT3, is responsible for the conversion of 6-HD to glycitein. The expression profile of IOMT3 overlapped with that of F6H4. High expression was detected in hypocotyl, with the highest level at stage IV (Figure S8), as in F6H4 (Figure 5C). Plant OMT genes are categorized into two main groups: cation- independent (type I) and cation-dependent (type II) [37]. The amino acid sequences of representative OMT proteins [12] were compared to those of IOMT3 and its soybean homologs (Figure 9). IOMT3 was grouped into the type II OMT clade. The amino acid sequence identity and similarity between GmIOMT1 and IOMT3 were 126/228 (55%) and 162/228 (71%), respectively. A survey of SNPs between K01 and FUKU with whole-genome NGS analysis re- ealed 16 SNPs in the 1.0 to 3.0 Mbp region on Gm01. Among these SNPs, an allele of m01_430457 caused a stop codon mutation in Glyma.01G004200 (238 aa; Figure 8C), an- otated as methyltransferase in the soybean genome database (Wm82.a2.v1). In K01, the uanine nucleotide at position 554 was changed to adenine, changing the codon for tryp- ophan (TGG) to a stop codon (TAG) at the 185-aa position. We also analyzed the molec- lar weight of the K01-type aglycone after acid hydrolysis of isoﬂavones extracted from 01 seeds. The retention time of the K01-type aglycone coincided with that of 6-hy- roxydaidzein (6-HD, Figure S5), and mass-to-charge ratios (m/z) of glycoside and malo- ylglycoside forms of 6-hydroxydaidzein were identical to those of the shifted peaks in 01 (Figures S6 and S7). These results—a nonsense mutation in the candidate gene OMT) of K01 and 6-HD isoﬂavone accumulation in K01—indicate that the product of a ovel IOMT (Glyma.01G004200) gene, hereafter designated IOMT3, is responsible for the onversion of 6-HD to glycitein. The expression proﬁle of IOMT3 overlapped with that of 6H4. High expression was detected in hypocotyl, with the highest level at stage IV (Fig- re S8), as in F6H4 (Figure 5C). 2.7. Identiﬁcation of Novel Mutant Line for Glycitein Isoﬂavones 2.7. Identification of Novel Mutant Line for Glycitein Isoflavones Plant OMT genes are categorized into two main groups: ation-independent (type I) and cation-dependent (type II) [37]. The amino acid sequences f representative OMT proteins [12] were compared to those of IOMT3 and its soybean omologs (Figure 9). IOMT3 was grouped into the type II OMT clade. The amino acid equence identity and similarity between GmIOMT1 and IOMT3 were 126/228 (55%) and 62/228 (71%), respectively Figure 9. Phylogenetic tree of homologous amino acid sequences for isoﬂavone O-methyl transfer- ase in soybean and other legumes. The MEGA-X program with the neighbor-joining method were used for the analysis. Figure 9. Phylogenetic tree of homologous amino acid sequences for isoflavone O-methyl transferase in soybean and other legumes. The MEGA-X program with the neighbor-joining method were used for the analysis. gure 9. Phylogenetic tree of homologous amino acid sequences for isoﬂavone O-methyl transfer- e in soybean and other legumes. The MEGA-X program with the neighbor-joining method were ed for the analysis. Figure 9. Phylogenetic tree of homologous amino acid sequences for isoflavone O-methyl transferase in soybean and other legumes. The MEGA-X program with the neighbor-joining method were used for the analysis. gure 9. Phylogenetic tree of homologous amino acid sequences for isoﬂavone O-methyl transfer- e in soybean and other legumes. The MEGA-X program with the neighbor-joining method were ed for the analysis. Figure 9. Phylogenetic tree of homologous amino acid sequences for isoflavone O-methyl transferase in soybean and other legumes. The MEGA-X program with the neighbor-joining method were used for the analysis. changed peak positions on an HPLC chromatogram. Th types have so far not been the subject of any reports 3.1. Use of Mutant-Based Screening for Gene Identification yp j y p p g (Glyma.11G108300) and the other is IOMT3 (Glyma.01G004200). The location of Glyma.11G108300 coincided well with that of the qMgly‐11 region identiﬁed in previous studies [37], and the eﬀect of qMgly‐11 explained 56.5% of the phenotypic diversity of seed malonylglycitein content in soybean mini-core collections. In addition, GWAS analysis of over 1500 soybean germplasms with four repeated evaluations for malonyglycitin content identiﬁed the highest peak at the same positions [28]. The results of these studies indicate that qMgly‐11 is a major and stable QTL and would be a valuable locus for soybean breed- ers trying to improve soybean seed isoﬂavone components. Elicitor-inducible glycitein biosynthetic genes have been identiﬁed and characterized with transcript analysis using a inoculated or elicitor-treated soybean seedlings [11,12]; however, reports related to mu- tant-based screening of the isoﬂavone content in soybean seeds are limited. Our previous study identiﬁed a GmCHR5 mutant that showed a decrease in daidzein-related isoﬂa- vones compared with genistein derivatives [38]. Some authors have conducted GWAS In this study, we identified two genes responsible for glycitein biosynthesis from two mutant lines, one showing the absence of glycitein isoflavones and the other show- ing changed peak positions on an HPLC chromatogram. The mutant lines with such phenotypes have so far not been the subject of any reports. One responsible gene is F6H4 (Glyma.11G108300) and the other is IOMT3 (Glyma.01G004200). The location of Glyma.11G108300 coincided well with that of the qMgly-11 region identified in previous studies [37], and the effect of qMgly-11 explained 56.5% of the phenotypic diversity of seed malonylglycitein content in soybean mini-core collections. In addition, GWAS analysis of over 1500 soybean germplasms with four repeated evaluations for malonyglycitin con- tent identified the highest peak at the same positions [28]. The results of these studies indicate that qMgly-11 is a major and stable QTL and would be a valuable locus for soy- bean breeders trying to improve soybean seed isoflavone components. Elicitor-inducible glycitein biosynthetic genes have been identified and characterized with transcript analysis using a inoculated or elicitor-treated soybean seedlings [11,12]; however, reports related to mutant-based screening of the isoflavone content in soybean seeds are limited. Our previous study identified a GmCHR5 mutant that showed a decrease in daidzein-related isoflavones compared with genistein derivatives [38]. Some authors have conducted GWAS using many germplasm accessions and extensive SNP information [28,29]. 8. Transgenic Hairy Roots Expressing F6H4 and IOMT3 2.8. Transgenic Hairy Roots Expressing F6H4 and IOMT3 We further analyzed transgenic hairy roots of soybeans expressing F6H4 only, IOMT3 nly, and F6H4 and IOMT3 together, all under the control of the ubiquitin promoter (Fig- res S9 and 10). The transgenic hairy roots overexpressing F6H4 only showed accumula- on of malonylglycoside forms of 6-hydroxydaidzein (M_6HD) and a seven-fold (p < We further analyzed transgenic hairy roots of soybeans expressing F6H4 only, IOMT3 only, and F6H4 and IOMT3 together, all under the control of the ubiquitin promoter (Figure S9 and Figure 10). The transgenic hairy roots overexpressing F6H4 only showed accumulation of malonylglycoside forms of 6-hydroxydaidzein (M_6HD) and a seven-fold (p < 0.001) increase in total glycitein-related isoflavones (glycitin, malonylglycitin, and M_6HD) compared to those transformed with the empty vector. However, overexpression of IOMT3 barely affected the isoflavone accumulation pattern compared to the empty vector. Co-expression of F6H4 and IOMT3 significantly increased the level of glycitin and Plants 2024, 13, 156 11 of 19 ne was 11 of 19 ne was malonylglycitin but not that of M_6HD. In addition, the total amount of isoflavone was also increased by F6H4 and IOMT3 co-expression. glycitein biosynthesis in soybean seeds, especially within the “hypocotyl” (i.e., non-coty- ledon seed tissues). malonylglycitin but not that of M_6HD. In addition, the total amount of isoflavone w also increased by F6H4 and IOMT3 co-expression. g y y y , p y yp y ( , ledon seed tissues). Figure 10. Isoﬂavone accumulation in transgenic soybean hairy roots with four constructs. F-I, F6H4, and IOMT3; F6H and F6H4 only; IOMT and IOMT3 only; NC, empty vector. Ten transgenic hairy roots for each construct showing high isoﬂavone content were used for this analysis. M_6HD, malonylglycoside forms of 6-hydroxydaidzein; M_Daidzin, malonyldaidzin; M_Glycitin, malonyl- glycitin; TIC, total isoﬂavone content. Signiﬁcant diﬀerences with p-values by the Tukey–Kramer method are shown using lowercase letters; values marked with the same letter are not signiﬁcantly diﬀerent. N.S., no statistically signiﬁcant diﬀerences for the indicated isoﬂavone. Figure 10. Isoflavone accumulation in transgenic soybean hairy roots with four constructs. F-I, F6H4, and IOMT3; F6H and F6H4 only; IOMT and IOMT3 only; NC, empty vector. Ten transgenic hairy roots for each construct showing high isoflavone content were used for this analysis. M_6HD, malonylglycoside forms of 6-hydroxydaidzein; M_Daidzin, malonyldaidzin; M_Glycitin, malonyl- glycitin; TIC, total isoflavone content. 8. Transgenic Hairy Roots Expressing F6H4 and IOMT3 2.8. Transgenic Hairy Roots Expressing F6H4 and IOMT3 Significant differences with p-values by the Tukey–Kramer method are shown using lowercase letters; values marked with the same letter are not significantly different. N.S., no statistically significant differences for the indicated isoflavone. 3. Discussion 3.1. Use of Mutant‐Based Screening for Gene Identiﬁcation In this study, we identiﬁed two genes responsible for glycitein biosynthesis from two All results shown in this study indicate that F6H4 and IOMT3 are responsible for glycitein biosynthesis in soybean seeds, especially within the “hypocotyl” (i.e., non- cotyledon seed tissues). 3.2. Several Independent Pathways of Glycitein Biosynthesis in Soybeans A previous study [12] identified a unique type of IOMT, GmIOMT1 (Glyma.05G147000), from a fungus-inoculated soybean seedling by using metabolome and transcriptome analy- sis based on the “guilt-by-association” principle [12]. Analysis of transgenic hairy roots overexpressing F6H1 (Glyma.18G080400) and GmIOMT1 showed accumulation of glycitein isoflavones [12]. Phylogenetic analysis based on their amino acid sequences indicated that the differentiation between F6H1 (Glyma.18G080400) and F6H4 (Figure 6) and between GmIOMT1 (Glyma.05G147000) and IOMT3 (Figure 9) did not originate from genome du- plication during soybean evolution because, in each case, this differentiation came earlier in legume evolution. Given their evolutionary relationships, it is likely that there are two independent pathways related to isoflavone synthesis in soybean and other legumes: one triggered by inoculation with fungi and the other functioning during seed development. These pathways probably share the same CHS and IFS genes for genistein synthesis. How- ever, glycitein synthesis is controlled by different pathways, depending on the situation. In some cases, daidzein functions as a precursor for the phytoalexin glyceollin [40]. Induction of glyceollin through fungal inoculation also induced glycitein isoflavone synthesis [12]. One possibility is that the accumulation of glycitein isoflavone influences the amount of glyceollin in plant tissue. Further studies are necessary to determine the physiological sig- nificance of glycitein accumulation. In contrast, developing soybean seeds also accumulate isoflavones in their cotyledons and hypocotyls. The isoflavone accumulation patterns in these two tissues are also probably controlled by different mechanisms [31]. The correlation between the amount of glycosides and that of malonylglycosides of daidzin, genistin, and glycitein was very high (0.97 to 0.98) in the cotyledon and 0.89 to 0.96 in the hypocotyl within a wide sample of soybean varieties [38]. In contrast, the correlations between the amount of glycoside or malonylglycoside isoflavones between the cotyledon and hypocotyl were not significant (0.02 to 0.09 for glycoside and 0.03 to 0.14 for malonylglycoside). These phenomena indicate that the biosynthesis pathways of isoflavones in soybean are controlled by different mechanisms that depend on the type of plant tissue (root, leaves, cotyledon, and hypocotyl) and biotic (e.g., stimuli from the elicitor) or abiotic stresses, such as irrigation [41] or temperature during seed-filling periods [42]. Soybean roots also produce the aglycone form of isoflavones from isoflavones accumulated in vacuoles via demodification, such as deglycosylation [43]. 3.2. Several Independent Pathways of Glycitein Biosynthesis in Soybeans There are no data yet on isoflavone content for the mutant lines (MUT1246 and K01) at the whole-plant level, such as in roots or leaves, but it is likely that nodulation and other plant growth characteristics could be affected by mutations in F6H4 and IOMT3. Thus, further studies using these mutant lines will be useful for understanding the functional effects of these genes at the whole-plant level. changed peak positions on an HPLC chromatogram. Th types have so far not been the subject of any reports 3.1. Use of Mutant-Based Screening for Gene Identification However, the identification of genes responsible for regulating isoflavone content and its components remains limited in these studies. These results indicate that the wide genetic diversity used in GWAS analysis tends to mask the effects of single genes with small effects and the Plants 2024, 13, 156 12 of 19 effect of rare alleles, and it decreases the power to detect multiple alleles in germplasm collections [39]. Mutagenesis can also increase genetic diversity related to the regulation of isoflavone content and is useful for further soybean breeding programs that focus on the improvement in isoflavone components. Therefore, screening mutant lines for alterations in isoflavone content and components and identifying the genes responsible for these phenotypes are important for revealing the genetic pathways related to the synthesis and accumulation of isoflavones and the expansion of soybean genetic diversity for these traits. 3.4. The Possibility to Produce Novel Materials for Human Dietary Nutrition Isoflavones contribute to the taste of soybean seeds, particularly astringent tastes [44]. Among the three isoflavone aglycones, the bitterness order is glycitein > daidzein > genis- tein. Thus, the glycitein-absent null allele found in MUT1246 could become a good source for developing new soybean varieties with improved taste. In future, sensory testing using processed products made from soybean seeds defective in glycitein isoflavones will be necessary to evaluate whether the absence of glycitein has a positive effect on the taste of processed foods such as soymilk, tofu, or natto. p y As discussed above, the K01 mutant line accumulates the malonylglycoside forms of 6-hydroxydaidzein. Our preliminary screening using the Japanese and world soybean mini-core collection [33] identified no soybean accessions with the same phenotype as K01. Glycitein metabolism was analyzed by using rat and human liver microsomes, and human fecal flora were investigated [45]. In that study, 6-HD was detected among the metabolites obtained from glycitein digestion. In addition, 6-HD has antioxidant activity in cell culture models, and the antioxidant activity of dietary isoflavones may be due to the formation of antioxidant metabolites of 6-HD [46]. These results indicate that 6- HD has a nutritional function in the human diet. The combination of the AOKI-type qMgly-11 allele (threonine-248 type of F6H4) increasing F6H4 activity and the K01 mutant allele causing the accumulation of 6-HD malonylglycoside offers the possibility to develop “super” 6-HD-accumulating soybean seeds with high antioxidant effects. By stacking these alleles in future breeding programs, new soybean materials with novel nutritional features could be developed. 3.3. Effects of Enzyme Structure on Glycitein Biosynthesis Mapping experiments with MUT1246 and association mapping of the soybean mini- core collection revealed that the C136Y mutation eliminated glycitein production and that the T248A mutation reduced it. The amino acid position 136 formed a heme-binding pocket (Figure S4B). Thus, we propose that the mutation of cysteine 136 to tyrosine affects the heme-binding mode and induces the loss of mono-oxygenase activity because the side chain of tyrosine is quite bulky compared to that of cysteine (Figure S4B). In contrast, the T248A mutation preserved enzymatic activity. According to our predicted three-dimensional structure with liquiritigenin, the hydroxyl at its 4′ position is located at a hydrogen-bond Plants 2024, 13, 156 13 of 19 distance from the threonine side chain. This suggests that this bond plays an important role in retaining the substrate in the precise position and the proximity of carbon 6C of liquiritigenin to the heme iron (iron–oxo or iron–peroxo intermediates). Meanwhile, the T248A mutation preserves the capacity for substrate binding in the active site pocket, but the substrate would be perturbed, and this mutation reduces the activity because the side chain of alanine has less volume. Thus, we conclude that threonine 248 is important for the precise formation of a ternary complex (heme–liquiritigenin–enzyme). 4.2. Plant Materials The two mutant lines were crossed with Japanese soybean cultivar Toyoshirome (TOYO) (National Agriculture and Food Research Organization [NARO] Genebank; bred at the Kyushu Okinawa Agricultural Research Center, NARO), which has a different genetic background than FUKU. The F2 population obtained from a cross between MUT1246 and TOYO, containing 96 individuals, was used to confirm co-segregation of functional SNPs and their phenotypes. Another F2 population containing 96 plants was obtained from a cross between K01 and TOYO and used for mapping the mutated gene in the K01 line. The F2 seeds from each cross were used for HPLC and DNA analyses. 4. Materials and Methods 4.1. Mutant Library We screened our previously reported soybean mutant library in the Fukuyutaka (FUKU) genetic background [47] for lines showing drastically reduced or absent malonyl- glycitin. Among 2831 mutant lines, we found that the MUT1246 and K01 lines displayed different malonylglycitin contents than wild-type FUKU. We propagated the seeds of these mutant lines and used them as parental lines for mapping experiments. 4.3. NGS Analysis DNA used for whole-genome sequencing analysis was extracted from FUKU, Kumachi- 1, and the mutant line K01. Next-generation sequencing (NGS) analysis was outsourced to Novogene (Beijing, China). Sequences approximately 10 to 13 Gbp in length were obtained for each sample. Long-read sequence data from Kumachi-1 were also obtained using a Plants 2024, 13, 156 14 of 19 14 of 19 Minion sequencer (Oxford Nanopore Technologies, Oxford, UK) according to the manu- facturer’s instructions. A soybean reference sequence (Wm82.a2.v1) was obtained from a public database (https://phytozome-next.jgi.doe.gov/, accessed on 1 April 2021). To map the mutated gene in K01, FASTQ files containing sequence data were used to align short reads to a candidate region (Gm01 1.0 to 3.0 Mbp) in the reference sequence using the Bowtie 2 program (ver. 2.3.5.1 [48]). The SAMtools suite (ver. 0.1.18 [49]) was used to detect polymorphisms between FUKU and K01. We evaluated the functions of the detected SNPs by using the snpEFF program [50]. We also developed DNA markers to detect K01 and MUT1246 functional nucleotide polymorphisms using the nearest neighboring nucleotide substitution high-resolution melting (NNNs-HRM) technique and designed primers to detect SNPs, according to a previous study [51]. Primer sequences used in this study are summarized in Table S1. 4.4. Mapping Experiments DNA was extracted from powdered F2 seeds using an automated DNA extraction machine (GENE PREP STAR PI-480; Kurabo Industries Ltd., Osaka, Japan), according to the manufacturer’s instructions. DNA markers based on NNNS-HRM that detected poly- morphisms between FUKU (the genetic background of MUT1246 and K01) and TOYO were used for the mapping experiments, according to a previous study [38]. PCR experiments for all marker genotyping were conducted in a quantitative thermal cycler (LightCycler 96 System; Roche Diagnostics K.K., Minato, Tokyo, Japan). DNA marker information for mapping the K01 mutated locus and PCR conditions was provided in a previous study [51]. A selective genotyping technique [52] was used to identify candidate loci for the K01 mu- tated locus, with 10 individuals showing the K01-type isoflavone phenotype. Additional DNA markers for SNPs unique to each mutant line are summarized in Table S1. 4.6. Measurement of Isoflavone Content We extracted isoflavones from cotyledons, embryo tissues, and transgenic hairy roots. For analysis of the segregating populations, we removed the seed coat and separated seeds into cotyledons and “hypocotyls” (here, consisting of plumule, epicotyl, hypocotyl, and radicle) with a knife. Freeze-dried hypocotyl samples were weighed and then crushed in a Multi-Beads Shocker in a 3 mL tube with metal beads. Seed powder (100 mg) obtained from cotyledons or crushed powder of hypocotyls was suspended in 1 mL of extraction buffer [70% ethanol (v/v) and 0.1% acetic acid (v/v)]. The mixture was vortexed and sonicated (30 min at room temperature) using an ultrasonic cleaner (Bransonic 5800; Emerson Japan Ltd., Kanagawa, Japan). The mixture was briefly centrifuged, and the supernatant was transferred to a new tube. These extraction steps were repeated three times, and the total extract (approximately 3 mL) was filtered (0.45 µm membrane). Samples (3 µL) were analyzed by HPLC (Jasco Corp., Tokyo, Japan) on a Hydrosphere C18 column (YMC Co., Ltd., Kyoto, Japan). The details of the separation conditions (water-based two-solvent systems) have been described previously [31]. Isoflavones were detected at 254 nm using an ultraviolet (UV) detector. Standard curves for daidzein, daidzin, malonyldaidzin, glycitein, glycitin, malonyl- glycitin, genistein, genistin, malonylgenistin (Wako, Osaka, Japan), and 6-hydroxydaidzein (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were constructed. The retention times and areas of the isoflavone standards were used to calculate the amounts of isoflavones from the peak areas of the samples. The mass-to-charge ratios (m/z) of the K01-type isoflavones were measured using LCMS-2020 (SHIMADZU CORPORATION, Kyoto, Japan), which gave m/z 417 for daidzin, 433 for genistin and the glycosylated form of 6-hydroxydaidzein, and 447 for glycitein. The HPLC conditions were as described above. Acid hydrolysis of isoflavones was performed as follows: 10 hypocotyls obtained from K01 mutant seeds were crushed using a Multi-Beads Shocker. An isoflavone extract (3 mL) was prepared as described above. A total of 1 mL of this solution was mixed with 1 mL of 70% EtOH in HCl solution and heated for 2 h at 95 ◦C. NaOH solution (1.5 M) was added for neutralization (pH7 to 8). Litmus paper was used to determine the pH of the solutions. After neutralization, 2 mL of ethyl acetate was added, and the solution was vortexed and centrifuged briefly. 4.5. Construct Preparation for Ectopic Expression of Candidate Genes and Soybean Hairy Root Transformation Full-length coding sequences of F6H4 and IOMT3 were amplified from AOKI and FUKU developing seed cDNA, respectively, by PCR using KOD FX Neo polymerase (Toyobo, Osaka, Japan). Each PCR product was cloned into the pENTER/D-TOPO or pDONR221 entry vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. The cloned fragments were transferred into the destination vector pUB-GW-GFP [53] using the Gateway LR recombination reaction (Thermo Fisher Scientific). The resulting vectors were designated as the control (pUB-GW-GFP empty vector), F6H4 (containing Glyma.11G108300), and IOMT3 (containing Glyma.01G004200). y g y To produce a vector co-expressing F6H4 and IOMT3 (designated F6H4-IOMT3), the DNA sequence from the promoter to the terminator of F6H4 was amplified from the F6H4 vector using primers created for In-Fusion Cloning technology (Takara Bio, Kusatsu, Japan). The destination vector containing IOMT was digested with SacI, and a PCR frag- ment containing the promoter to terminator of F6H4 was inserted upstream of IOMT3 by homologous recombination. The primers used for cloning all of the genes are listed in Table S1. All of the newly constructed vectors were introduced into the Agrobacterium rhizogenes strain LBA1334 using the freeze–thaw method [54].i Transgenic hairy roots were generated as previously described [55] with slight modifi- cations [38]. Briefly, FUKU seeds were infected with LBA1334 harboring the above vectors, and transgenic hairy roots with strong green fluorescent protein (GFP) signals were selected, weighed, separated into two 3 mL plastic tubes with 5 mm metal beads (Yasuikikai, Osaka, Japan), frozen in liquid nitrogen, crushed with a Multi-Beads Shocker (Yasuikikai), and used for RNA extraction and isoflavone measurement. Plants 2024, 13, 156 15 of 19 15 of 19 4.6. Measurement of Isoflavone Content 4.6. Measurement of Isoflavone Content The supernatant (water phase) was removed, and the ethyl acetate phase was transferred into a new test tube and evaporated to dryness in a heating block (80 ◦C). Finally, the isoflavone hydrolysates were dissolved in 1 mL of extraction buffer (described above) and analyzed by HPLC. 4.8. Protein Structure Modeling With nearly 40,000 crystal structures of cytochrome P450s reported to date, including more than 400 crystal structures of F6Hs, the predicted structures of P450s are highly reliable. The structure of F6H4 (AOKI type) was modeled by using AlphaFold2_mmseq in ColabFold [57,58]. However, no co-factors such as heme were present in the structure modeled by AlphaFold2. To investigate the location of the heme in F6H4, we applied the predicted structure of apo-GmF6H to AlphaFill [59] 4.7. Expression Analysis The parental line of FUKU was grown in the field of Saga University (Honjo campus; lat 33.242, long 130.288; June to November 2019) under natural day-length conditions. Developing seeds were collected randomly from several plants and the developmental status of each stage was as follows: stage I, seeds with indistinguishable hypocotyl and cotyledons (~3 mm); II, seed length size 3–5 mm; III, seed size 5–7 mm; IV, seed size 7–8 mm; V, 40 days after flowering (DAF40), late seed enlargement stage, seed size over 8 mm; VI, 50 days after flowering (DAF50); and VII, 60 days after flowering (DAF60). Total RNA was isolated from the tissues (50–100 mg) using the Total RNA Extraction Kit Mini Plant (SciTrove, Tokyo, Japan) with additional rDNase I (Takara Bio Inc., Shiga, Japan) treatment. cDNA was synthesized from 1 µg total RNA using ReverTra Ace (Toyobo, Osaka, Japan). A 5 µL aliquot (approximately 25 ng) of cDNA was used as a template for PCR. Quantitative real-time PCR was conducted as follows in a LightCycler 96 System: 95 ◦C for 5 min for enzyme activation, followed by 95 ◦C for 15 s, 60 ◦C for 15 s, and 72 ◦C for 20 s for a total of 40 amplification cycles. EvaGreen Dye (20× in water; Biotium, Inc., Fremont, CA, USA) was used as the fluorescent dye, and dNTPs (Takara), homemade recombinant Taq polymerase, the PCR buffer mentioned in a previous study [51], and 1 pmol of each gene-specific primer were used to evaluate transcript levels of target genes. Expression relative to the internal Plants 2024, 13, 156 16 of 19 16 of 19 reference gene, GmELFa (Glyma.19G052400), was calculated using the 2−∆∆Ct method [56]. Data from three or four biological replicates were analyzed. Primers for expression analysis are listed in Table S1. reference gene, GmELFa (Glyma.19G052400), was calculated using the 2−∆∆Ct method [56]. Data from three or four biological replicates were analyzed. Primers for expression analysis are listed in Table S1. 4.9. Statistical Analysis We constructed a linkage map using AntMap Ver. 1.1 software [60] with default parameters. We identified QTLs by single-interval mapping using R/qtl software [61]. We used linear regression and analysis of variance (ANOVA) to confirm the effects of QTLs and to evaluate the association between the genotype of the closest DNA marker and isoflavone content. All analyses were performed using R software [62]. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants13020156/s1, Figure S1: Soybean isoflavone biosyn- thetic pathway; Figure S2: QTL analysis for the identification of qMgly-11 locus with two different pop- ulations; Figure S3: Association between glycitein isoflavone content and SNPs in Glyma.11G108300; Figure S4: Models of F6H4 protein; Figure S5: HPLC chromatogram of acid-hydrolyzed isoflavones obtained from K01 mutant line seeds; Figures S6 and S7: HPLC-MS analysis of Fukuyutaka (wild type) and K01 mutant line (peak-shift phenotype for glycitein isoflavones); Figure S8: Tissue-specific expression of Glyma.01G004200 (IOMT3); Figure S9: Configuration of vectors used in hairy root transformation tests; Table S1. Primers used in this study; Table S2: Co-segregation of F6H4 G407A (C136Y) mutant genotype and absence of glycitein isoflavones in a population obtained from a cross between MUT1246 and Toyoshirome; Table S3. Co-segregation of IOMT3 G554A (W185stop) mutant genotype and absence of glycitein isoflavones in a population obtained from a cross between K01 and Toyoshirome. Author Contributions: Conceptualization and writing, S.W., M.H., T.A., R.Y. and K.T. collected data for mutant screening and identification of the responsible genes. M.H. and A.M. evaluated the protein structure encoded by the identified genes. All authors have read and agreed to the published version of the manuscript. Funding: This work was the result of using research equipment shared in the MEXT Project for promoting public utilization of advanced research infrastructure (Program for supporting the intro- duction of the new sharing system), Grant Numbers JPMXS0422400020 and JPMXS0422400021. This research was partially funded by Grant-in-Aid for Scientific Research on Innovative Areas, IBmS: JSPS KAKENHI, Grant Number 22H04816. Data Availability Statement: Data are contained within the article. Data Availability Statement: Data are contained within the article. Acknowledgments: We are grateful to Tokiko Kitajima, Noriko Ryuda, and Reika Shinchi for their technical support. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. Physiol. Mol. Plant Pathol. 1996, 49, 1 20. [CrossRef] 2. Graham, T.L. Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates. Plant Physiol. 1991, 95, 594–603. [CrossRef] [PubMed]lil 1. Dakora, F.D.; Phillips, D.A. Diverse functions of isoflavonoids in legumes transcend anti-microbial definitions of phytoalexins. Physiol. Mol. 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Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W2087397784	https://link.springer.com/content/pdf/10.1007/s00236-015-0224-3.pdf	English	null	Two-way pebble transducers for partial functions and their composition	Acta informatica	2,015	cc-by	9,034	Acta Informatica (2015) 52:559–571 DOI 10.1007/s00236-015-0224-3 Acta Informatica (2015) 52:559–571 DOI 10.1007/s00236-015-0224-3 ORIGINAL ARTICLE J. Engelfriet (B) Leiden Institute of Advanced Computer Science, Leiden University, Leiden, The Netherlands e-mail: j.engelfriet@liacs.leidenuniv.nl Two-way pebble transducers for partial functions and their composition Joost Engelfriet Received: 8 August 2014 / Accepted: 13 February 2015 / Published online: 4 March 2015 © The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Two-way ﬁnite state transducers are considered that use a ﬁnite number of pebbles, of which the life times must be nested. For every nondeterministic transducer that realizes a partial function, an equivalent deterministic transducer can be constructed. The composition of two deterministic transducers can be realized by one such transducer with a minimal number of pebbles. 1 The mistake in the proof is explained in New Observation 5.10 of [10]. 1 Introduction Results that are known from the literature, or can easily be concluded from results in the literature, are stated as Propositions. 2 Deﬁnitions For binary relations R1 ⊆X × Y and R2 ⊆Y × Z, where X, Y, Z are sets, we denote by R1 ◦R2 their composition {(x, z) \| ∃y : (x, y) ∈R1, (y, z) ∈R2}. For classes of binary relations R1 and R2, we deﬁne R1 ◦R2 = {R1 ◦R2 \| R1 ∈R1, R2 ∈R2}. For a binary relation R ⊆X × Y, the domain of R is dom(R) = {x ∈X \| ∃y ∈Y : (x, y) ∈R}, and its range is ran(R) = {y ∈Y \| ∃x ∈X : (x, y) ∈R}. For a set Δ, the set of all strings over Δ (i.e., sequences of elements of Δ) is denoted Δ∗. For a string w ∈Δ∗, its length is denoted \|w\|, and w(i) denotes its ith element (for 1 ≤i ≤\|w\|). The empty string is denoted λ. For k ∈N = {0, 1, 2, . . .}, we deﬁne Δk = {w ∈Δ∗\| \|w\| = k} and Δ≤k = {w ∈Δ∗\| \|w\| ≤k}. 1 Introduction A pebble automaton is a two-way ﬁnite state automaton that uses a ﬁxed, ﬁnite number of pebbles that it can drop on, and lift from, the squares of its input tape. Pebble automata recog- nize regular languages only, provided the life times of the pebbles are nested (otherwise they recognize the logarithmic space languages). Automata with nested pebbles were introduced in [21], and in [11] for tree-walking automata. A tree transducer with nested pebbles was introduced in [25] as a model for XML-based query languages. In [15] some results were proved for the two-way ﬁnite state transducer with nested pebbles (pebble transducer, for short), which is the restriction of the tree transducer of [25] to monadic trees, i.e., to strings. One result of [15] is that every partial function that can be computed by a nondeterministic pebble transducer, can in fact be computed by a deterministic one, with the same number of pebbles. Unfortunately, the proof of this result in [15, Theorem 3] is wrong.1 In this paper the result is stated in Corollary 7, with a (hopefully) correct proof. Another result of [15] is that for every two deterministic pebble transducers M1 and M2, there is a deterministic pebble transducer M that computes the composition of the functions computed by M1 and M2. In the proof, the constructed transducer M has (k1 + 1)(k2 + 2) pebbles, where ki is the number of pebbles of Mi. It is conjectured in [15] that a transducer with (k1 + 1)(k2 + 1) −1 pebbles can do the same job (and it is easy to see that, in general, 123 560 J. Engelfriet it cannot be done with less pebbles). In this paper the conjecture is proved, and stated in Theorem 11. Our proofs are based on a reduction of pebble transducers to two-way ﬁnite state transduc- ers (without pebbles), which is a straightforward generalization of the reduction of pebble automata to two-way ﬁnite state automata in [20]. This allows us to use the well-known facts that a partial function computed by a nondeterministic two-way ﬁnite state transducer can also be computed by a deterministic one (see, e.g., Theorem 22 of [12]), and that the deterministic two-way ﬁnite state transductions are closed under composition ([6]). y p Other work on nested pebbles has appeared in, e.g., [2–5,13,14,16–19,22,26–28,30,3 All results stated in this paper are effective. 2.1 Pebble transducers If δ contains a transition (q, σ, b) →(q′, ϕ, w), then (q, i, π, v) ⊢u (q′, i′, π′, vw) if the following holds: if ϕ = right, then i ̸= \|u\| + 1, i′ = i + 1 and π′ = π, if ϕ = left, then i ̸= 0, i′ = i −1 and π′ = π, if ϕ = drop, then i′ = i, \|π\| < k and π′ = πi, if ϕ = lift, then i′ = i, π(\|π\|) = i and π = π′i. if ϕ = right, then i ̸= \|u\| + 1, i′ = i + 1 and π′ = π, if ϕ = left, then i ̸= 0, i′ = i −1 and π′ = π, if ϕ = drop, then i′ = i, \|π\| < k and π′ = πi, if ϕ = drop, then i′ = i, \|π\| < k and π′ = πi, if ϕ = lift, then i′ = i, π(\|π\|) = i and π = π′i. if ϕ = lift, then i′ = i, π(\|π\|) = i and π = π′i. The transduction computed by M, denoted τM, is the binary relation τM = {(u, v) ∈Σ∗× Δ∗\| ∃(q, i, π) : (q0, 0, λ, λ) ⊢∗ u (q, i, π, v), q ∈F}. The k-pebble transducer M is deterministic if δ does not contain two transitions with the same left-hand side. In that case, τM is a partial function from Σ∗to Δ∗. The k-pebble transducer M is deterministic if δ does not contain two transitions with the same left-hand side. In that case, τM is a partial function from Σ∗to Δ∗. The class of transductions computed by k-pebble transducers is denoted by PTk, and by DPTk for the deterministic transducers. Example 1 A deterministic 4-pebble transducer M can translate an input string u into an output string that is a concatenation of all strings v#w# where v and w are (occurrences of) nonempty substrings of u and vw is in a given regular language R. The transducer M moves pebble 1 from square 1 to square n of the input tape, where n = \|u\|. When pebble 1 is at square i, M moves pebble 2 from square i to square n. In this way M systematically considers all nonempty substrings v of u. 2.1 Pebble transducers A two-way ﬁnite state transducer (also called two-way generalized sequential machine, or 2gsm) is a ﬁnite state automaton with a two-way input tape, delimited by the endmarkers ◁and ▷, and a one-way output tape. A k-pebble transducer is a two-way ﬁnite state transducer that additionally carries k pebbles, each with a unique name, say, 1, . . . , k. Initially there are no pebbles on the input tape, but during its computation the transducer can drop pebbles on the squares of the input tape, lift them, drop them again, etc. In one step of its computation, the transducer can determine which pebbles are lying on the current square of the input tape, lift one of these pebbles, or drop a new pebble. However, the life times of the pebbles should be nested. This means that at each moment of time, pebbles 1 to ℓ(for some 0 ≤ℓ≤k) are on the input tape, and at such a moment the only pebble that can be dropped on the current square is pebble ℓ+ 1, whereas the only pebble that can be lifted from the current square (if it is on that square) is pebble ℓ. Formally, a k-pebble transducer (with k ≥0) is a system M = (Σ, Δ, Q, q0, F, δ) where Σ and Δ are the input and output alphabet, respectively, Q is the ﬁnite set of states, q0 ∈Q is the initial state, F ⊆Q is the set of ﬁnal states, and δ is the ﬁnite set of transitions. Each transition is of the form (q, σ, b) →(q′, ϕ, w) with q ∈Q −F, σ ∈Σ ∪{◁, ▷}, b ∈{0, 1}k, q′ ∈Q, ϕ ∈{right, left, drop, lift}, and w ∈Δ∗. 2.1 Pebble transducers For a ﬁxed position of pebbles 1 and 2, M systematically considers all nonempty substrings w of u, using pebbles 3 and 4 for that purpose. For each position of all 4 pebbles, M walks from pebble 1 to pebble 2 and then from pebble 3 to pebble 4, simulating a ﬁnite automaton that recognizes the regular language R. If vw ∈R, then M repeats that walk and outputs the string v#w#. ⊓⊔ ⊓⊔ 2.2 Counting transducers 2.1 Pebble transducers Intuitively, such a transition means that if M is in state q, σ is the symbol on the current square, and, for every 1 ≤m ≤k, b(m) = 1 if and only if pebble m is on the current square, then M can go into state q′, output the string w, and execute the instruction ϕ, i.e., move the reading head one square to the right or left, drop pebble ℓ+ 1 (the “next” pebble), or lift pebble ℓ(the “last” pebble); note that these 123 123 123 Pebble transducers for partial functions 561 instructions are undeﬁned if, respectively, σ = ▷, σ = ◁, all k pebbles are on the input tape (i.e., ℓ= k), or pebble ℓis not on the current square (i.e., either ℓ= 0 or b(ℓ) = 0). For a given input string u ∈Σ∗, the input tape of M contains the string ◁u ▷. The squares of this tape are numbered 0, 1, . . . , \|u\|, \|u\|+1. Accordingly, a pebble conﬁguration of M on ◁u ▷is a string π ∈{0, . . . , \|u\|+1}≤k, where π(m) = j means that pebble m is currently on square j (for 1 ≤m ≤\|π\| and j ∈{0, . . . , \|u\|+1}), and \|π\| < m ≤k means that pebble m is currently not on the input tape. A conﬁguration of M on ◁u ▷is a triple (q, i, π) with q ∈Q, the current state of M, i ∈{0, . . . , \|u\| + 1}, the current position of the reading head, and π ∈{0, . . . , \|u\| + 1}≤k, the current pebble conﬁguration. The one step computation relation ⊢u is deﬁned in the obvious way on 4-tuples (q, i, π, v) where (q, i, π) is a conﬁguration and v ∈Δ∗is the current content of the output, as follows. Let σ be the content of the current input square, i.e., σ = ◁if i = 0, σ = u(i) if 1 ≤i ≤\|u\|, and σ = ▷if i = \|u\| + 1. Let b ∈{0, 1}k indicate which pebbles are placed on this square, i.e., for every 1 ≤m ≤k, b(m) = 1 if and only if π(m) = i. 2.2 Counting transducers Our ﬁrst result (in the next section) will be the equivalence of the k-pebble transducer with a related type of two-way pebble transducer that uses its k pebbles in a very restricted, nonstandardway.Ratherthanmanipulatingpebblesbydroppingandliftingthem,atransducer M of this new type has all its k pebbles on the input tape at all times. To explain how M can move the pebbles, we ﬁrst note that a pebble conﬁguration π ∈{0, . . . , \|u\|+1}k on ◁u ▷(with all pebbles on the input tape) can be viewed as usual as a number num(π) in the (\|u\|+2)-ary number system, with 0 ≤num(π) ≤(\|u\| + 2)k −1. As an example, for k = 3 and \|u\| = 8, the pebble conﬁguration 097 (which means that pebble 1 is on ◁, pebble 2 is on ▷, and pebble 562 J. Engelfriet 3 is on the 7th symbol of u) corresponds to the (decimal) number 97; in this case the pebble conﬁgurations on u of length k represent all numbers between 0 (all pebbles on ◁) and 999 (all pebbles on ▷). For pebble conﬁgurations π1 and π2 such that num(π1)+1 = num(π2), we say that π2 is the next pebble conﬁguration of π1, and that π1 is the previous pebble conﬁguration of π2. It is well known that a k-pebble transducer (as deﬁned in Sect. 2.1) can count from 1 to (\|u\| + 2)k by systematically constructing all pebble conﬁgurations of length k, going from one to the next, starting with the ﬁrst pebble conﬁguration of length k (representing 0, with all pebbles on ◁) and ending with the last one (representing (\|u\| + 2)k −1, with all pebbles on ▷). The transducer M of the new type only has the instructions right and left, which are now also deﬁned when the current symbol under the reading head is ▷or ◁, respectively (provided the current pebble conﬁguration is not the last or the ﬁrst, respectively). The effect of moving right from ▷, is that the reading head jumps to ◁and the pebble conﬁguration is changed to the next one. Similarly, the effect of moving left from ◁, is that the reading head jumps to ▷and the pebble conﬁguration is changed to the previous one. We note here that (as in [25]) the reading head can be viewed as an additional pebble, viz. 2.2 Counting transducers pebble k + 1, and so a pair (i, π) consisting of the current position of the reading head and the current pebble conﬁguration, can be viewed as the pebble conﬁguration πi ∈{0, . . . , \|u\| + 1}k+1 on ◁u ▷. Viewed in this way, the instruction right changes the pebble conﬁguration πi to the next one, and the instruction left changes it to the previous one. This shows the naturalness of the new interpretation of right and left. We will call the new type of transducer a k-counting transducer. Formally, a k-counting transducer (with k ≥0) is a system M = (Σ, Δ, Q, q0, F, δ), deﬁned in the same way as a k-pebble transducer except that it does not have instructions drop and lift, i.e., ϕ ∈{right, left} in every transition (q, σ, b) →(q′, ϕ, w). A pebble conﬁguration of M on ◁u ▷(with u ∈Σ∗) is a string π ∈{0, . . . , \|u\| + 1}k, and a conﬁguration of M on ◁u ▷is of the form (q, i, π), as for the k-pebble transducer (but with the pebble conﬁguration of length k). The one step relation ⊢u is deﬁned as for the k-pebble transducer on 4-tuples (q, i, π, v), except that now: if ϕ = right then either i ̸= \|u\| + 1, i′ = i + 1 and π′ = π, if ϕ = right then either i ̸= \|u\| + 1, i′ = i + 1 and π′ = π, or i = \|u\| + 1, i′ = 0, and π′ is the next pebble conﬁguration of π, if ϕ = left then either i ̸= 0, i′ = i −1 and π′ = π, if ϕ = left then either i ̸= 0, i′ = i −1 and π′ = π, or i = 0, i′ = \|u\| + 1, and π′ is the previous pebble conﬁguration of π. or i = 0, i′ = \|u\| + 1, and π′ is the previous pebble conﬁguration of π. Finally, the transduction computed by M, denoted τM, is Finally, the transduction computed by M, denoted τM, is Finally, the transduction computed by M, denoted τM, is τM = {(u, v) ∈Σ∗× Δ∗\| ∃(q, i, π) : (q0, 0, 0k, λ) ⊢∗ u (q, i, π, v), q ∈F}. 2.2 Counting transducers Note that 0k denotes a string of k 0’s; it is the ﬁrst pebble conﬁguration. Determinism of M is deﬁned as for the k-pebble transducer. Note that 0k denotes a string of k 0’s; it is the ﬁrst pebble conﬁguration. Determinism of M is deﬁned as for the k-pebble transducer. Note that 0k denotes a string of k 0’s; it is the ﬁrst pebble conﬁguration. Determinism of M is deﬁned as for the k-pebble transducer. The class of transductions computed by k-counting transducers is denoted by CTk, and by DCTk for the deterministic transducers. The class of transductions computed by k-counting transducers is denoted by CTk, and by DCTk for the deterministic transducers. Example 2 A 4-counting transducer M′ that computes the same transduction as the 4-pebble transducer M of Example 1, systematically considers all possible pebble conﬁgurations by repeatedly moving to the right. For each such pebble conﬁguration it checks that the pebbles are not on an endmarker, and it checks that pebble 2 is not to the left of pebble 1, and pebble 4 is not to the left of pebble 3. If so, pebbles 1 and 2 determine a nonempty substring v of the input string u, and pebbles 3 and 4 determine a nonempty substring w. Then M′ operates in the same way as M. ⊓⊔ 123 3 Equivalence of pebble and counting transducers This section contains a basic result, underlying the proofs of our main results. We ﬁrst show that the two types of two-way pebble transducer, deﬁned in the previous section, are equivalent, and then we use this to characterize the k-pebble transductions in terms of 0-pebble transductions. One direction of the characterization was shown for k = 1 in the proof of Lemma 1 of [15]. For arbitrary k the characterization was presented for transducers with empty output alphabet, i.e., for automata, in [20] (and rediscovered by this author). Lemma 3 For every k ∈N, PTk = CTk and DPTk = DCTk. Lemma 3 For every k ∈N, PTk = CTk and DPTk = DCTk. Lemma 3 For every k ∈N, PTk = CTk and DPTk = DCTk. Proof We ﬁrst show the obvious fact that every k-counting transducer M can be simulated by a k-pebble transducer M′. The transducer M′ initializes the simulation by dropping all its pebbles on ◁, and then stepwise simulates M. The instruction right of M is simulated by the same instruction of M′ when the reading head is not on ▷. Now assume that the reading head is on ▷. If all pebbles are on ▷, then M′ aborts. Otherwise, M′ must construct the next pebble conﬁguration of M. To do this, M′ ﬁrst lifts all pebbles m + 1 to k, where m is the largest number such that pebble m is not on ▷. Then M′ walks to the left, ﬁnds pebble m and moves it one square to the right. Finally, M′ walks to the left until it is on ◁, and drops pebbles m +1 to k. The instruction left of M is simulated in a symmetrical way. Note that the initialization and the simulation of each step are deterministic subroutines. Thus, CTk ⊆PTk and DCTk ⊆DPTk. k k We now show that every k-pebble transducer M can be simulated by a k-counting trans- ducer M′. Again, M′ stepwise simulates M. A pebble conﬁguration of M with pebbles 1 to ℓon the input tape is simulated by the pebble conﬁguration of M′ where pebbles 1 to ℓare on the same squares as those of M, and pebbles ℓ+ 1 to k are all on the same square as the reading head. The number ℓis kept in the ﬁnite state of M′. 123 563 Pebble transducers for partial functions Whenever we construct a k-pebble or k-counting transducer, we can also use transitions containing an identity instruction ϕ = stay, with the semantics i′ = i and π′ = π. Such an instruction can easily be simulated in two steps, ﬁrst moving right and then moving left (or vice versa if the reading head is on ▷). pebbk,Σ(u) = code(◁u ▷, π0) · · · code(◁u ▷, πs) where s = (\|u\|+2)k −1 and num(π j) = j for every 0 ≤j ≤s. In other words, pebbk,Σ(u) is theconcatenationofallconsecutive(encodingsof)k-pebbleconﬁgurationson◁u ▷.Todeﬁne our intended speciﬁc k-counting transductions, we note that the ﬁrst symbol of pebbk,Σ(u) is (◁, 1k) and its last symbol is (▷, 1k), where 1k denotes a string of k 1’s; moreover, these symbols do not occur anywhere else in pebbk,Σ(u). where s = (\|u\|+2)k −1 and num(π j) = j for every 0 ≤j ≤s. In other words, pebbk,Σ(u) is theconcatenationofallconsecutive(encodingsof)k-pebbleconﬁgurationson◁u ▷.Todeﬁne our intended speciﬁc k-counting transductions, we note that the ﬁrst symbol of pebbk,Σ(u) is (◁, 1k) and its last symbol is (▷, 1k), where 1k denotes a string of k 1’s; moreover, these symbols do not occur anywhere else in pebbk,Σ(u). k, We now deﬁne the mapping pebk,Σ (with one b!) such that for u ∈Σ∗, pebbk,Σ(u) = (◁, 1k) · pebk,Σ(u) · (▷, 1k). Thus, pebk,Σ(u) is the result of removing the ﬁrst and last symbol from pebbk,Σ(u). In the proof of the next theorem (Theorem 4), a transducer that receives pebk,Σ(u) as input, will view ◁as (◁, 1k) and ▷as (▷, 1k); in this way, the transducer views the content of its input tape as pebbk,Σ(u). Thus, pebk,Σ(u) is the result of removing the ﬁrst and last symbol from pebbk,Σ(u). In the proof of the next theorem (Theorem 4), a transducer that receives pebk,Σ(u) as input, will view ◁as (◁, 1k) and ▷as (▷, 1k); in this way, the transducer views the content of its input tape as pebbk,Σ(u). , For k ≥0, we denote by PEBk the class of all mappings pebk,Σ, where Σ is a ranked alphabet. It is easy to see that pebbk,Σ is in DCTk: by repeatedly moving right, a deterministic k-counting transducer M can consecutively go through the k-pebble conﬁgurations π0 to πs, and for each 0 ≤j ≤s output the string code(◁u ▷, π j). To be precise, M has initial state q and ﬁnal state q′, and it has the transitions (q, σ, b) →(q, right, (σ, b)) for all (σ, b) ̸= (▷, 1k), plus the transition (q, ▷, 1k) →(q′, left, (▷, 1k)). It should be clear that therefore also pebk,Σ is in DCTk, just suppressing the ﬁrst and last symbol of the output of M. Consequently PEBk ⊆DPTk, by Lemma 3. 3 Equivalence of pebble and counting transducers Note that M′ already starts in the correct conﬁguration (with ℓ= 0 in its ﬁnite state). The simulation of the instructions drop and lift is easy: ℓ:= ℓ+ 1 and ℓ:= ℓ−1, respectively, in the ﬁnite state. To simulate an instruction right, M′ ﬁrst checks that the reading head is not on ▷. Then M′ has to move its reading head and all pebbles ℓ+1 to k one square to the right. To do this, M′ repeatedly moves to the right (i.e., executes the instruction right with the semantics of the counting transducer) until the reading head and all pebbles ℓ+ 1 to k are again on the same square. Note that each time M′ moves to the right from ▷, the next pebble conﬁguration is realized. For instance, for \|u\| = 8, k = 4 and ℓ= 2, if num(π) = 7444 for the current pebble conﬁguration π and the reading head is on square 4, then, after the simulation of right, num(π′) = 7455 for the new pebble conﬁguration π′, and the reading head is on square 5; note that 74555 is the ﬁrst number after 74444 for which the three least signiﬁcant digits are equal. The simulation of the instruction left is symmetrical. Again, the simulation of each step is a deterministic subroutine, and so PTk ⊆CTk and DPTk ⊆DCTk. ⊓⊔ ⊓⊔ In the remainder of this section we show that the k-counting transductions, and hence the k-pebble transductions, can be characterized as the compositions of a very speciﬁc kind of deterministic k-counting transductions with the 0-counting transductions (i.e., 2gsm’s). Let 123 564 J. Engelfriet us now deﬁne these speciﬁc k-counting transductions. There is one for each input alphabet Σ, called pebk,Σ; it is the mapping Pk in [20]. us now deﬁne these speciﬁc k-counting transductions. There is one for each input alphabet Σ, called pebk,Σ; it is the mapping Pk in [20]. , For u ∈Σ∗, a string π ∈{0, . . . , \|u\|+1}k will be called a k-pebble conﬁguration on ◁u ▷; it is a pebble conﬁguration of a k-counting transducer with input alphabet Σ. We will need an obvious encoding of the pair (◁u ▷, π) as a string over the alphabet (Σ ∪{◁, ▷})×{0, 1}k. 3 Equivalence of pebble and counting transducers If u = σ1 · · · σn with n ≥0 and σi ∈Σ, then code(◁u ▷, π) = (◁, b0)(σ1, b1) · · · (σn, bn)(▷, bn+1) where bi(m) = 1 if and only if π(m) = i, for every 0 ≤i ≤n + 1 and 1 ≤m ≤k. We deﬁne the mapping pebbk,Σ (with two b’s!) such that for u ∈Σ∗, pebbk,Σ(u) = code(◁u ▷, π0) · · · code(◁u ▷, πs) Proposition 5 Every transduction in PT0 has a uniformizer in DPT0. Proposition 5 Every transduction in PT0 has a uniformizer in DPT0. Proof A writing 0-pebble transducer is a 0-pebble transducer that, in addition, can write on its input tape. For this purpose, it has an additional “work alphabet” Ω that contains the input alphabet and the endmarkers. Its transitions are of the form (q, σ, b) →(q′, σ ′, ϕ, w) with σ, σ ′ ∈Ω, meaning that σ is overwritten by σ ′ in the current square. A Hennie machine is a writing 0-pebble transducer M for which there is a number k ∈N such that for each (u, v) ∈τM there is a k-visiting computation of M on input u with output v, which means that each square of the input tape is visited at most k times. For more details see Section 7 of [12]. The Hennie machine was introduced as an accepting device in [23], where it was shown that, due to the k-visiting restriction, it accepts regular languages only. It was shown in [12] that the Hennie machine computes the MSO deﬁnable string transductions, i.e., the string transductions that are deﬁnable in monadic second-order logic. It is easy to see that for every 0-pebble transducer M there is a Hennie machine M′ such that τM′ is a uniformizer of τM. In fact, if (u, v) ∈τM then M has a computation on input u (with some output v′) that never enters the same state twice at the same square (because if, during some computation, M enters state q at square j on time t1 and on time t2 > t1, then the subcomputation between t1 and t2 can be skipped). Such a computation is k-visiting, where k is the number of states of M. A Hennie machine M′ can easily simulate the k-visiting computations of M using the work alphabet (Σ ∪{◁, ▷}) × {0, 1, . . . , k} (where Σ is the input alphabet) and counting the visits to each square in the symbol at that square. To be precise, each transition (q, σ, b) →(q′, ϕ, w) of M is replaced by all transitions (q, (σ, i), b) →(q′, (σ, i +1), ϕ, w) of M′ with 0 ≤i < k, where we identify (σ, 0) with σ. pebbk,Σ(u) = code(◁u ▷, π0) · · · code(◁u ▷, πs) We now state the characterization of the k-pebble transductions, of which the proof is intuitively obvious from Lemma 3. Theorem 4 For every k ≥0, PTk = PEBk ◦PT0 and DPTk = PEBk ◦DPT0. Proof Using Lemma 3, we prove that CTk = PEBk ◦CT0 and DCTk = PEBk ◦DCT0. For a k-counting transducer M = (Σ, Δ, Q, q0, F, δ) we deﬁne the associated 0-counting transducer M′ = (Σ′, Δ, Q, q0, F, δ′) where Σ′ = (Σ ∪{◁, ▷}) × {0, 1}k and δ′ is deﬁned as follows. If (q, σ, b) →(q′, ϕ, w) is a transition in δ, then δ′ contains the transition (q, (σ, b), λ) →(q′, ϕ, w), where (◁, 1k) is identiﬁed with ◁and (▷, 1k) with ▷. Note that M′ is deterministic if and only if M is deterministic. It should be clear that pebk,Σ ◦τM′ = τM. Indeed, if M moves right from ▷, then M′ moves right from some (▷, b) that is not identiﬁed with ▷, and so M′ moves from the last symbol of code(◁u ▷, π j), where u is the input string and π j is the current pebble conﬁguration of M, to the ﬁrst symbol of code(◁u ▷, π j+1). And similarly when M moves left from ◁. As long as M does not move right from ▷or 123 Pebble transducers for partial functions 565 move left from ◁, M′ stays on the substring code(◁u ▷, π j) of pebbk,Σ(u) = ◁pebk,Σ(u) ▷, simulating M. move left from ◁, M′ stays on the substring code(◁u ▷, π j) of pebbk,Σ(u) = ◁pebk,Σ(u) ▷, simulating M. This shows that CTk ⊆PEBk ◦CT0 and DCTk ⊆PEBk ◦DCT0. The reverse inclusions hold because, obviously, every 0-counting transducer with input alphabet (Σ ∪{◁, ▷}) × {0, 1}k is the associated transducer of a k-counting transducer with input alphabet Σ. ⊓⊔ The constructions involved in this theorem, for the case that the output alphabet is empty, are used in [20] to reduce the descriptional complexity of two-way k-pebble automata to that of two-way (0-pebble) automata: if nondeterministic two-way automata can be simulated by deterministic two-way automata with a polynomial number of states (which is not known, cf. [29]), then the same is true for k-pebble automata. We will use Theorem 4 in the following sections in a similar way, to transfer results for 2gsm’s to k-pebble transducers. 4 Uniformizers In this section we use Theorem 4 to prove our ﬁrst main result: that every partial function in PTk is in DPTk. In fact, we will prove the stronger result that every transduction in PTk has a uniformizer in DPTk. A uniformizer of a binary relation R is a binary relation R′ such that R′ ⊆R and dom(R′) = dom(R), see, e.g., [1,8]. Note that the only uniformizer of a partial function f is f itself. We ﬁrst prove the result for k = 0, i.e., for 2gsm’s. A proof was already sketched in the proof of Theorem 4.9 of [9]. Here we give a proof using the results of [12]. Proposition 5 Every transduction in PT0 has a uniformizer in DPT0. By Theorem 25 of [12], every transduction computed by a Hennie machine is a nondeter- ministic MSO deﬁnable string transduction (deﬁned in Section 6.1 of [12]). By the proof of Theorem 21 of [12], for every nondeterministic MSO deﬁnable string transduction τ ′ = τM′ By Theorem 25 of [12], every transduction computed by a Hennie machine is a nondeter- ministic MSO deﬁnable string transduction (deﬁned in Section 6.1 of [12]). By the proof of Theorem 21 of [12], for every nondeterministic MSO deﬁnable string transduction τ ′ = τM′ By Theorem 25 of [12], every transduction computed by a Hennie machine is a nondeter- ministic MSO deﬁnable string transduction (deﬁned in Section 6.1 of [12]). By the proof of Theorem 21 of [12], for every nondeterministic MSO deﬁnable string transduction τ ′ = τM′ By Theorem 25 of [12], every transduction computed by a Hennie machine is a nondeter- ministic MSO deﬁnable string transduction (deﬁned in Section 6.1 of [12]). By the proof of Theorem 21 of [12], for every nondeterministic MSO deﬁnable string transduction τ ′ = τM′ 123 123 566 J. Engelfriet thereisadeterministic MSOdeﬁnablestringtransductionτ ′′ (deﬁnedinSection4of[12])that is a uniformizer of τ ′ and hence a uniformizer of τM. By Theorem 13 of [12], τ ′′ can be com- puted by a deterministic 0-pebble transducer M′′, and so τM′′ = τ ′′ is a uniformizer of τM. ⊓⊔ thereisadeterministic MSOdeﬁnablestringtransductionτ ′′ (deﬁnedinSection4of[12])that is a uniformizer of τ ′ and hence a uniformizer of τM. By Theorem 13 of [12], τ ′′ can be com- puted by a deterministic 0-pebble transducer M′′, and so τM′′ = τ ′′ is a uniformizer of τM. ⊓⊔ We now prove our ﬁrst main result. Theorem 6 For every k ≥0, every transduction in PTk has a uniformizer in DPTk. Theorem 6 For every k ≥0, every transduction in PTk has a uniformizer in DPTk Proof By Theorem 4 every transduction in PTk is of the form pebΣ,k ◦τ with τ ∈PT0. By Proposition 5, τ has a uniformizer τ ′ ∈DPT0. This clearly implies that pebΣ,k ◦τ ′ is a uniformizer of pebΣ,k ◦τ, and pebΣ,k ◦τ ′ is in DPTk by Theorem 4. ⊓⊔ Corollary 7 For every k ≥0, if τ ∈PTk is a partial function, then τ ∈DPTk. 5 Composition In this section we prove that the composition of the transductions of two deterministic pebble transducers with k and m pebbles respectively, can be computed by a deterministic pebble transducer with (k + 1)(m + 1) −1 = km + k + m pebbles. For k = m = 0, i.e., for deterministic 2gsm’s, this was proved in [6] (see also Theorems 8.10 and 7.14 of [7]). Corollary 9 For every k ≥0, DPTk = PEBBk ◦DPT0. Corollary 9 For every k ≥0, DPTk = PEBBk ◦DPT0. From Proposition 8 and Corollary 9 (or Theorem 4) we immediately obtain the inclusion DPTk ◦DPT0 ⊆DPTk. In the next lemma we prove a special case of the symmetric inclusion DPT0 ◦DPTk ⊆DPTk. Lemma 10 For every k ≥0, DPT0 ◦PEBBk ⊆DPTk. Proof Let τ ∈DPT0 where τ is a partial function from Σ∗to Δ∗. We will construct a deterministic k-pebble transducer N such that τN = τ ◦pebbΔ,k. Proof Let τ ∈DPT0 where τ is a partial function from Σ∗to Δ∗. We will construct a deterministic k-pebble transducer N such that τN = τ ◦pebbΔ,k. , Let M = (Σ, Δ ∪{◁, ▷}, Q, q0, F, δ) be a deterministic 0-pebble transducer such that τM(u) = ◁τ(u) ▷for every u ∈Σ∗. A conﬁguration (q, i, λ) of M (where λ is the empty pebble conﬁguration) will be denoted by (q, i). Without loss of generality, we assume that at each computation step M outputs at most one symbol. Consider an input string u ∈dom(τM). Each occurrence of an output symbol in τM(u) is produced by M in a certain conﬁguration (q, i) during the next computation step, where q is a state and i is a square of the input tape ◁u ▷. By the above assumption, this conﬁguration is unique, because if two such occurrences would be produced by M in the same conﬁguration, then the computation of M on input u would loop, contradicting the fact that u ∈dom(τM). We may assume that ◁is produced in conﬁguration (q◁, 0) and ▷in conﬁguration (q▷, 0) for certain states q◁and q▷(probably the initial state and a ﬁnal state of M); thus, M produces output ◁and ▷when it reads ◁. Proposition 8 DPT0 ◦DPT0 ⊆DPT0. Proposition 8 DPT0 ◦DPT0 ⊆DPT0. Let PEBBk denote the class of all mappings pebbk,Σ, where Σ is a ranked alphabet. It should be clear that PEBBk ⊆PEBk ◦DPT0 and PEBk ⊆PEBBk ◦DPT0 (where the 0-pebble transducer adds or removes the ﬁrst and last symbol, respectively). This, together with Proposition 8 and Theorem 4, gives the following corollary. 123 123 567 Pebble transducers for partial functions In the computations of N, a k-pebble conﬁguration π ∈{0, 1, . . . , \|v\|, \|v\| + 1}k on the output string τM(u) = ◁v ▷, where v = τ(u), is uniquely represented by a pair (α,π) where α ∈Qk associates a state with each pebble and π ∈{0, 1, . . . , \|u\|, \|u\| + 1}k is a pebble conﬁguration of N on ◁u ▷, such that, for every 1 ≤m ≤k, the pair (α(m),π(m)) is the conﬁguration in which M produces (the symbol in) square π(m) of the output tape. The association α is kept in the ﬁnite state of N. For ﬁxed (α,π), the string code(◁v ▷, π) can be produced by N as output, by simulating the computation of M on input u. When M, in conﬁguration (q, i), outputs a symbol a ∈ Δ ∪{◁, ▷} during the next computation step, N instead outputs the symbol (a, b) with b ∈{0, 1}k such that, for every 1 ≤m ≤k, b(m) = 1 if and only if (α(m),π(m)) = (q, i), i.e., pebble m is in the current square and is associated with the current state of M. Next we explain how N can realize the representation of the next k-pebble conﬁguration of π, for given (α,π). First N walks to ◁and determines the largest number m, 1 ≤m ≤k, such that (α(m),π(m)) ̸= (q▷, 0) (which means that pebble m is not on the last symbol of ◁v ▷). If there is no such m, then N halts in a ﬁnal state. If m exists, then N lifts the pebbles m + 1 to k. After that, N must move pebble m one square to the right on ◁v ▷. To do that, it again simulates the computation of M on input u. On the moment that M is in conﬁguration (α(m),π(m)), N lifts pebble m and continues the simulation of M until the next output symbol is produced. Then N drops pebble m on the current square, associates it with the current state of M, and stops the simulation. Finally, N walks to ◁, drops the pebbles m + 1 to k, and associates them with q◁. Initially, N checks that the input string u is in the domain of τM. Theorem 11 For every k, m ∈N, DPTk ◦DPTm ⊆DPTkm+k+m. Proof We ﬁrst observe that it sufﬁces to show that 123 Since the domain of τM is regular (see, e.g., [31]), N can simulate a ﬁnite automaton recognizing it. If u ∈dom(τM), then N drops all its pebbles on ◁and associates them with q◁. ⊓⊔ We now prove our second main result. PEBk ◦PEBBm ⊆PEBBkm+k+m ◦DPT0. Indeed, DPTk ◦DPTm = PEBk ◦DPT0 ◦DPTm = PEBk ◦DPT0 ◦PEBBm ◦DPT0 ⊆PEBk ◦ DPTm ◦DPT0 = PEBk ◦PEBBm ◦DPT0 ◦DPT0 ⊆PEBk ◦PEBBm ◦DPT0 by Theorem 4, Corollary 9, Lemma 10, Corollary 9, and Proposition 8, respectively. If the above inclusion holds, then this is included in PEBBkm+k+m ◦DPT0 ◦DPT0 ⊆PEBBkm+k+m ◦DPT0 = DPTkm+k+m by Proposition 8 and Corollary 9. Let r = km + k + m. Let Σ be an alphabet, let Δ = (Σ ∪{◁, ▷}) × {0, 1}k, let Γ = (Σ ∪{◁, ▷}) × {0, 1}r, and let Ω = (Δ ∪{◁, ▷}) × {0, 1}m. We will deﬁne a deterministic 0-pebble transducer M with input alphabet Γ and output alphabet Ω, such that τM(pebbr,Σ(u)) = pebbm,Δ(pebk,Σ(u)) for every u ∈Σ∗. In what follows we identify (◁, 1k) with ◁and (▷, 1k) with ▷, in the alphabet Δ. Thus Δ∪{◁, ▷} = Δ, and so the symbols in Ω are of the form ((σ, b1), b2) with σ ∈Σ ∪{◁, ▷}, b1 ∈{0, 1}k and b2 ∈{0, 1}m. for every u ∈Σ∗. In what follows we identify (◁, 1k) with ◁and (▷, 1k) with ▷, in the alphabet Δ. Thus Δ∪{◁, ▷} = Δ, and so the symbols in Ω are of the form ((σ, b1), b2) with σ ∈Σ ∪{◁, ▷}, b1 ∈{0, 1}k and b2 ∈{0, 1}m. For u ∈Σ∗, we recall that pebbk,Σ(u) = code(◁u ▷, π0) · · · code(◁u ▷, πs) where π0, . . . , πs is the sequence of consecutive k-pebble conﬁgurations on ◁u ▷. Due to the above identiﬁcation, 123 123 J. Engelfriet 568 pebbm,Δ(pebk,Σ(u)) = code(pebbk,Σ(u), ρ0) · · · code(pebbk,Σ(u), ρt) where ρ0, . . . , ρt is the sequence of consecutive m-pebble conﬁgurations on pebbk,Σ(u). We also have that pebbr,Σ(u) = code(◁u ▷, η0) · · · code(◁u ▷, ηz) where η0, . . . , ηz is the sequence of consecutive r-pebble conﬁgurations on ◁u ▷. Note that for n = \| ◁u ▷\|, we have \|pebbk,Σ(u)\| = nk+1, and hence \|pebbm,Δ(pebk,Σ(u))\| = \|pebbr,Σ(u)\| = n(k+1)(m+1). We now give meaningful names to the r = km + k + m = m(k + 1) + k pebbles involved in pebbr,Σ. From 1 to r, they receive the names ⟨1, 1⟩, . . . , ⟨1, k⟩, ⟨1, k + 1⟩, ⟨2, 1⟩, . . . PEBk ◦PEBBm ⊆PEBBkm+k+m ◦DPT0. , ⟨2, k⟩, ⟨2, k + 1⟩, ⟨m, 1⟩, . . . , ⟨m, k⟩, ⟨m, k + 1⟩, ⟨m + 1, 1⟩, . . . , ⟨m + 1, k⟩. The pebbles in the last row, ⟨m + 1, 1⟩, . . . , ⟨m + 1, k⟩, correspond to the pebbles 1, . . . , k that determine the k-pebble conﬁgurations π0, . . . , πs of pebbk,Σ on ◁u ▷. The pebbles in the ﬁrst m rows determine the m pebble conﬁgurations ρ ρ of pebb on pebb (u) The pebbles in the last row, ⟨m + 1, 1⟩, . . . , ⟨m + 1, k⟩, correspond to the pebbles 1, . . . , k that determine the k-pebble conﬁgurations π0, . . . , πs of pebbk,Σ on ◁u ▷. The pebbles in the ﬁrst m rows determine the m-pebble conﬁgurations ρ0, . . . , ρt of pebbm,Δ on pebbk,Σ(u). The pebbles in the jth row, 1 ≤j ≤m, determine the position of pebble j on pebbk,Σ(u). To be precise, the pebbles ⟨j, 1⟩, . . . , ⟨j, k⟩determine the k-pebble conﬁguration πℓsuch The pebbles in the last row, ⟨m + 1, 1⟩, . . . , ⟨m + 1, k⟩, correspond to the pebbles 1, . . . , k that determine the k-pebble conﬁgurations π0, . . . , πs of pebbk,Σ on ◁u ▷. The pebbles in the ﬁrst m rows determine the m-pebble conﬁgurations ρ0, . . . , ρt of pebbm,Δ on pebbk,Σ(u). The pebbles in the jth row, 1 ≤j ≤m, determine the position of pebble j on pebbk,Σ(u). To be precise, the pebbles ⟨j, 1⟩, . . . , ⟨j, k⟩determine the k-pebble conﬁguration πℓsuch that pebble j occurs in the substring code(◁u ▷, πℓ) of pebbk,Σ(u), and the pebble ⟨j, k +1⟩ determines the position of pebble j in that substring. The transducer M is very simple: it computes a symbol-to-symbol string homomorphism from Γ to Ω. By Theorem 11, DPT = k∈N DPTk is closed under composition (as already proved in [15]). This is not true for PT = k∈N PTk. , , In the same way as Proposition 8 it can be shown that DPT0 ◦PT0 ⊆PT0. From this it follows in the same way as above that DPTk ◦PTm ⊆PTkm+k+m for every k, m ∈N. PEBk ◦PEBBm ⊆PEBBkm+k+m ◦DPT0. Walking from left to right through the input tape containing pebbr,Σ(u), it changes each symbol (σ, b), with Σ ∪{◁, ▷} and b ∈{0, 1}r, into the symbol ((σ, b1), b2) such that • b1(i) = b(⟨m + 1, i⟩) for every 1 ≤i ≤k, and • for every 1 ≤j ≤m, b2( j) = 1 if and only if – b(⟨j, i⟩) = b(⟨m + 1, i⟩) for every 1 ≤i ≤k, and – b(⟨j, k + 1⟩) = 1. • b1(i) = b(⟨m + 1, i⟩) for every 1 ≤i ≤k, and • for every 1 ≤j ≤m, b2( j) = 1 if and only if – b(⟨j, i⟩) = b(⟨m + 1, i⟩) for every 1 ≤i ≤k, and – b(⟨j, k + 1⟩) = 1. – b(⟨j, i⟩) = b(⟨m + 1, i⟩) for every 1 ≤i ≤k, and – b(⟨j, k + 1⟩) = 1. It should be clear, after some thinking, that M indeed translates each input string pebbr,Σ(u) into pebbm,Δ(pebk,Σ(u)). ⊓⊔ It should be clear, after some thinking, that M indeed translates each input string pebbr,Σ(u) into pebbm,Δ(pebk,Σ(u)). ⊓⊔ ⊓⊔ It is well known (and easy to see) that for every transduction τ ∈DPTk and every string u in itsdomain,\|τ(u)\| = O(\|u\|k+1).Infact,if M isadeterministick-pebbletransducercomputing τ, then the number of conﬁgurations of M on ◁u ▷is O(\|u\|k+1) and each conﬁguration occurs at most once in the computation of M on input u. As observed in the proof of Theorem 11, \|pebbm,Δ(pebk,Σ(u))\| = (\|u\| + 2)(k+1)(m+1). Hence pebk,Σ ◦pebbm,Δ cannot be computed by a deterministic pebble transducer with less than (k + 1)(m + 1) −1 pebbles. Since pebk,Σ ∈DPTk and pebbm,Δ ∈DPTm, this shows that Theorem 11 is optimal. , , In the same way as Proposition 8 it can be shown that DPT0 ◦PT0 ⊆PT0. From this it follows in the same way as above that DPTk ◦PTm ⊆PTkm+k+m for every k, m ∈N. , , In the same way as Proposition 8 it can be shown that DPT0 ◦PT0 ⊆PT0. From this it follows in the same way as above that DPTk ◦PTm ⊆PTkm+k+m for every k, m ∈N. y y By Theorem 11, DPT = k∈N DPTk is closed under composition (as already proved in [15]). This is not true for PT = k∈N PTk. 123 Pebble transducers for partial functions 569 Theorem 13 PT∗has uniformizers in DPT. Proof Consider a transduction τ1 ◦· · · ◦τn with τi ∈PT for 1 ≤i ≤n. Since dom(τi) is regular (see, for instance, [15]), we may assume that the range of τi−1 is contained in the domain of τi for 2 ≤i ≤n (when computing τi−1, simulate a ﬁnite automaton for dom(τi) on the output). By Theorem 6, τi has a uniformizer τ ′ i in DPT. By the above assumption, τ ′ 1 ◦· · · ◦τ ′ n is a uniformizer of τ1 ◦· · · ◦τn, and τ ′ 1 ◦· · · ◦τ ′ n is in DPT by Theorem 11. ⊓⊔ ⊓⊔ Proposition 12 PT is not closed under composition. Proof The proof is entirely similar to the proof of Lemma 15 of [12], as follows. Let τ be the transduction {(an, w#w \| n ≥0, w ∈{a, b}∗, \|w\| = n}. Obviously, τ ∈PT0 ◦DPT0 (ﬁrst translate an into all strings w ∈{a, b}∗of length n, then translate w into w#w). Proof The proof is entirely similar to the proof of Lemma 15 of [12], as follows. Let τ be the transduction {(an, w#w \| n ≥0, w ∈{a, b}∗, \|w\| = n}. Obviously, τ ∈PT0 ◦DPT0 (ﬁrst translate an into all strings w ∈{a, b}∗of length n, then translate w into w#w). Assume that τ is computed by a k-pebble transducer M with m states, that produces at most one output symbol at each computation step. The number of conﬁgurations of M on ◁an ▷is m ·(n +2)k+1. Choose n such that 2n > m ·(n +2)k+1. Consider the behavior of M on input an, and consider in particular the conﬁguration of M when it has just produced the symbol # of the output string w#w. Since there are 2n output strings w#w for an, there exist two strings w1 and w2 for which this conﬁguration is the same. Then the computation of output w1#w1 can be switched halfway to the computation of w2#w2, resulting in a computation of output w1#w2 with w1 ̸= w2. ⊓⊔ ⊓⊔ We ﬁnally show that the composition closure PT∗of PT does not contain more partial functions than PT: all partial functions in PT∗are already in DPT. We deﬁne PT1 = PT, PTn+1 = PTn ◦PT, and PT∗= n≥1 PTn. Theorem 13 PT∗has uniformizers in DPT. 6 Conclusion By Proposition 12, PT ⊊PT2. It is open whether PTn ⊊PTn+1 for every n. A variant of pebble handling is to allow a pebble to be lifted also when the reading head is not at the square where the pebble was dropped. In the literature these are called “strong” pebbles, as opposed to the “weak” pebbles in this paper. It is shown in [2] that strong k-pebble tree automata have the same expressive power as weak ones. It is not difﬁcult to show that deterministic strong 1-pebble transducers have the same power as weak ones, but apart from that it is open whether or not strong k-pebble transducers have more power than weak ones. It is also open whether the results of this paper are also valid for strong pebble transducers. We ﬁnally mention that it is open whether the equivalence problem for deterministic k-pebble transducers is decidable. By an argument similar to the one in the proof of Theorem 4 of [15], it can be shown that every deterministic k-pebble transduction is a composition of k deterministic macro tree transductions. For an overview on the equivalence problem for deterministic macro tree transducers see [24]. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. 123 123 J. Engelfriet 570 References 1. 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